U.S. patent application number 10/626671 was filed with the patent office on 2004-05-27 for sulfohydrolases, corresponding amino acid and nucleotide sequences, sulfohydrolase preparations, processes, and products thereof.
This patent application is currently assigned to Centre National De La Recherche Scientifique (CNRS). Invention is credited to De Ruiter, Gerhard, Genicot, Sabine, Kloareg, Bernard, Penninkhof, Bea, Potin, Phillipe, Richard, Odile, Rudolph, Brian.
Application Number | 20040101942 10/626671 |
Document ID | / |
Family ID | 22458322 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040101942 |
Kind Code |
A1 |
Genicot, Sabine ; et
al. |
May 27, 2004 |
Sulfohydrolases, corresponding amino acid and nucleotide sequences,
sulfohydrolase preparations, processes, and products thereof
Abstract
A purified sulfohydrolase having a purity level based on total
amount of protein of at least about 40 wt %. Isolated nucleic acid
sequence and amino acid sequences. A process for purifying at least
one sulfohydrolase, including subjecting an extract from seaweed to
fractionation to obtain fractions; and subjecting at least one of
the fractions to phenyl sepharose chromatography to obtain
sepharose fractions containing at least one sulfohydrolase. An
enzymatically modified compound which has been modified by an
isolated sulfohydrolase having a purity level based on total amount
of protein of at least about 40 wt %. A process of enzymatically
modifying a sulfated compound, including combining at least one
sulfohydrolase, having a purity level based on total amount of
protein of at least about 40 wt %, with a sulfated compound form a
reaction mixture; and incubating the reaction mixture to remove
sulfate groups from the sulfated compound to form an enzymatically
modified compound. A process of enzymatically modifying a sulfated
compound, including incubating a first sulfohydrolase with a
sulfated compound to remove sulfate groups from the sulfated
compound to form an intermediate compound; and subsequently
incubating the intermediate compound with a second sulfohydrolase
to remove sulfate groups to form an enzymatically modified
compound. A method for extracting one of nu- and mu-carrageenan
from seaweed, including dispersing seaweed in a salt solution
including K.sub.2CO.sub.3 to form a dispersion; filtering the
dispersion to obtain a liquid; ultrafiltering the dispersion to
remove salts; concentrating the liquid; adjusting the pH of the
liquid to about 8 to 8.5; and precipitating one of nu- and
mu-carrageenan from the liquid.
Inventors: |
Genicot, Sabine; (Saint Pol
de Leon, FR) ; Kloareg, Bernard; (Plouenan, FR)
; Potin, Phillipe; (Roscoff, FR) ; Rudolph,
Brian; (Koge, DK) ; De Ruiter, Gerhard; (Ede,
NL) ; Penninkhof, Bea; (Epse, NL) ; Richard,
Odile; (Teille, FR) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
Centre National De La Recherche
Scientifique (CNRS)
Paris
FR
|
Family ID: |
22458322 |
Appl. No.: |
10/626671 |
Filed: |
July 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10626671 |
Jul 25, 2003 |
|
|
|
09567003 |
May 9, 2000 |
|
|
|
6620604 |
|
|
|
|
60133376 |
May 10, 1999 |
|
|
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Current U.S.
Class: |
435/196 |
Current CPC
Class: |
C08B 37/0042 20130101;
C12N 9/14 20130101; C08B 37/0036 20130101 |
Class at
Publication: |
435/196 |
International
Class: |
C12N 009/16 |
Claims
What is claimed is:
1. An isolated protein comprising an amino acid sequence comprising
SEQ ID NO: 22.
2. An isolated protein comprising an amino acid sequence which has
at least about 35% homology with SEQ ID NO: 22.
3. The isolated protein of claim 2, wherein the amino acid sequence
has at least about 50% homology with SEQ ID NO: 22.
4. The isolated protein of claim 2, wherein the amino acid sequence
has at least about 80% homology with SEQ ID NO: 22.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 09/567,003 filed May 9, 2000 which
claims the priority under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Application No. 60/133,376, filed May 10, 1999, the
disclosures of which are expressly incorporated by reference herein
in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to sulfohydrolases, such as
galactan sulfohydrolases, such as nu- and mu-carrageenan
sulfohydrolases. The present invention is directed to the amino
acid and nucleotide sequences of sulfohydrolases. The present
invention is further directed to enzymatic modification of sulfated
compounds, such as galactans. For example, the enzymatic
modification may involve tailoring of the properties of sulfated
galactans, such as gelling properties, such as by removal of
sulfate groups and creation of a bridge between ring positions in a
saccharide structure of the galactan. The present invention is
further directed to processes of extracting nu-carrageenan from
seaweed. The present invention is also directed to enzymatically
modified compounds.
[0004] 2. Discussion of Background
[0005] Hydrocolloids, which may be broadly defined as substances
that yield a gel in the presence of water, are used in part for
their rheological properties, and may also provide benefits in
stability.
[0006] There are several classes of hydrocolloids. One
categorization approach breaks these classes into exudates, such as
gum arabic, ghatti, karaya, talha, and tragacanth; extracts, such
as alginate from brown seaweeds, agar, carrageenan, and furcelleran
from red seaweeds, and konjak (glucomannan), guar, pectin and
arabinogalactan from land plants; biopolymers, such as xanthan;
chemically modified hydrocolloids, such as the cellulosics,
including carboxymethyl cellulose, hydroxypropyl cellulose, and
carboxymethylhydroxymethyl cellulose; and intermediate forms, such
as microcrystalline cellulose.
[0007] Red seaweeds are known sources of industrial gelling and
thickening cell-wall sulfated galactans referred to as agar and
carrageenans. They consist of a linear backbone of galactopyranose
residues linked by alternating alpha(1.fwdarw.3) and
beta(1.fwdarw.4) linkages. While all .beta.-linked residues are in
the D-configuration, the alpha(1.fwdarw.4)-linked galactose units
are in the L-configuration in agars and in the D-configuration in
carrageenans.
[0008] Agar is extracted from dried algae by more or less hot
alkaline solutions (100-120.degree. C.). After filtration, agar
solutions are allowed to gel, de-watered by pressing, dried, and
ground, as disclosed in ARMISEN et al., "Production, Properties and
Uses of Agar", Production and Utilization of Products from
Commercial Seaweeds, FAO Fisheries Technical Paper, 288, pp. 1-57
(1987), the disclosure of which is herein incorporated by reference
in its entirety. Seaweed sources for agar extraction include the
genera Gelidium, Pterocladia, Gelidiella, and Gracilaria, as
disclosed in STANLEY, "Production, Properties and Uses of
Carrageenan", Production and Utilization of Products from
Commercial Seaweeds, FAO Fisheries Technical Paper, 288, pp.
116-146 (1987), the disclosure of which is herein incorporated by
reference in its entirety.
[0009] Carrageenan is itself a generic name for a family of natural
water-soluble sulfated galactans isolated from red seaweeds. The
thickening and gelation properties exhibited by carrageenans are
useful in food and cosmetic formulations, as disclosed in
THERKELSEN, "Carrageenan", Industrial Gums: Polysaccharides and
their Derivatives, 3rd ed., pp. 145-180, (1993), and DeRUITER et
al., "Carrageenan Biotechnology", Trends in Food Science &
Technology, Vol. 8, pp. 389-395 (1997), both of which are herein
incorporated by reference in their entireties.
[0010] Seaweed sources for carrageenan include the genera Eucheuma
(such as E. spinosum, E. cottonii (=Kappaphycus alvarezii), and E.
denticulatum), Chondrus (such as C. crispus), Calliblepharis (such
as C. jubata), and Gigartina (such as G. radula and G.
stellata).
[0011] Carrageenans are linear, partially sulfated galactans mainly
composed of repeating dimers of an alpha(1-4)-linked
D-galactopyranose or 3,6-anhydro-D-galactopyranose residue and a
beta(1-3)-linked D-galactopyranose residue. As noted above, agars
are likewise linear, partially sulfated galactans of similar
structure, except that the alpha(1-4)-linked galactopyranose
residue is in the L-form. Carrageenan occurs in several structures
that differ primarily in the number and placement of sulfate groups
on the dimer backbone, and in whether the individual residues of
the dimer are present in the left hand (.sup.4C.sub.1) or
right-hand (.sup.1C.sub.4) `chair` configuration. These structures
include kappa (.kappa.), iota (.iota.), lambda (.lambda.), theta
(.theta.), mu (.mu.), and nu (.nu.), as shown below. The iota-,
kappa-, and theta-carrageenans contain 3,6-anhydro bridges, whereas
the nu-, mu-, and lambda-carrageenans do not have this bridge.
Furthermore, the conformation of the alpha-linked unit of nu-, mu-,
and lambda-carrageenans is different from the
anhydrobridge-containing carrageenans, preventing sufficient helix
aggregation. Helix aggregation is important because helices
facilitate formation of gels. In this regard, kappa-carrageenan
forms firm, brittle gels, whereas iota-carrageenan forms elastic,
soft gels, and whereas lambda-carrageenan is a non-gelling
thickening agent. 1
[0012] The amount of SO.sub.3.sup.- in carrageenans can be
considerable and vary between 0 and 41% (w/w), resulting in highly
negatively charged polymers. Ideal kappa-, iota-, and
lambda-carrageenan dimers respectively have 1, 2, and 3 sulfate
esters groups, resulting in typical sulfate contents of
respectively 22%, 32%, and 38% (w/w). However, large variations in
sulfate can occur in commercial extracts due to differences in
seaweed species or batches. The sulfate ester linkages are
chemically very stable, and there are no apparent or practical
chemical methods to modify the sulfate level or distribution
without also lowering the molecular weight of the polymer, except
for the removal of 6-O-sulfate from precursor carrageenans as is
done during alkaline treatment.
[0013] Carrageenan is typically extracted commercially from red
seaweeds by boiling in aqueous solution, sometimes under alkaline
conditions, followed by filtration, concentration, precipitation,
and drying. Precipitation may either be by alcohol addition, or by
gelling with salts followed by pressing of the gel, as discussed in
STANLEY, "Production, Properties and Uses of Carrageenan",
Production and Utilization of Products from Commercial Seaweeds,
FAO Fisheries Technical Paper, 288, pp. 116-146 (1987), the
disclosure of which is herein incorporated by reference in its
entirety. Semi-refined carrageenans are also produced by treating
seaweeds with alkali followed by thorough rinsing with water. These
treatments improve the gelling characteristics of the carrageenan
preparation and remove most of the proteins, pigments and small
metabolites; such preparations also contain other polymers such as
cellulosic materials, as discussed in HOFFMANN et al., "Effect of
Isolation Procedures on the Molecular Composition and Physical
Properties of Eucheuma Cottonii Carrageenan", Food Hydrocolloids,
9, pp. 281-289 (1995), the disclosure of which is herein
incorporated by reference in its entirety.
[0014] The non-gelling mu- and nu-carrageenans are the natural
precursors present in seaweed of, respectively, kappa- and
iota-carrageenans, and have a sulfate ester group at the C-6
position of the alpha(1-4)-linked D-galactopyranose residue of the
dimeric unit. It has been generally assumed that elimination of the
sulfate from the C-6 sulfate ester of the precursors, and formation
of the 3,6-anhydro bridge, occur concomitantly during the strong
alkaline treatment.
[0015] The functional properties (including helix formation,
Theological properties, and applications) of the different
carrageenans are determined by (1) molecular weight of the polymer
(2) the number of sulfate ester groups and their place of
substitution of the carbon backbone, and (3) the number of
3,6-anhydro-galactose residues, as discussed in THERKELSEN,
"Carrageenan", Industrial Gums: Polysaccharides and their
Derivatives, 3rd edition, pp. 145-180 (1993); VIEBKE et al.,
"Characterization of Kappa- and Iota-Carrageenan Coils and Helices
by MALLS/GPC", Carbohydr. Polym., Vol. 27, pp. 145-154 (1995); and
Le QUESTEL et al., "Computer Modelling of Sulfated Carbohydrates:
Applications to Carrageenans", Int. J. Biol. Macromol., Vol. 17,
pp. 161-174 (1995), the disclosures of which are herein
incorporated by reference in their entireties.
[0016] Alkali treatments allow some control over the ratio of
carrageenan forms in the final product. However, it is often
difficult to predict and obtain the desired final product.
[0017] It has been reported that the red seaweed Porphyra
umbilicalis, contains a "sulfohydrolase" which catalyzes the
release of sulfate from porphyran, the major polysaccharide from
Porphyra spp. related to agar in that it contains about 10% (w/w)
of 3,6-anhydro-L-galactose. It also contains L-galactose-6-sulfate.
These latter units can be converted into 3,6-anhydro-L-galactose by
the action of an enzyme partially purified from an extract of the
parent seaweed as discussed in REES, "Enzymic Synthesis of
3:6-Anhydro-L-Galactose within Porphyran from L-Galactose
6-Sulphate Units", Biochem. J., 81, pp. 347-352 (1961); and REES,
"Enzymatic Desulphation of Porphyran", Biochem. J., 80, pp. 449-453
(1961), the disclosures of which are both incorporated herein by
reference in their entireties.
[0018] It has also been reported that carrageenan-producing red
seaweeds contain a "sulfohydrolase" which catalyzes the release of
sulfate from carrageenan precursors. The sulfohydrolase in Chondrus
crispus is discussed in WONG et al., "Sulfohydrolase Activity and
Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)", Plant
Physiology, Vol. 61, pp. 663-666 (1978), the disclosure of which is
herein incorporated by reference in its entirety. The
sulfohydrolase in Calliblepharis jubata is discussed in ZINOUN et
al., "Evidence of Sulfohydrolase Activity in the Red Alga
Calliblepharis jubata", Botanica Marina, Vol. 40, pp. 49-53 (1997),
the disclosure of which is herein incorporated by reference in its
entirety. The sulfohydrolase in Gigartina stellata is discussed in
both LAWSON et al., "An Enzyme for the Metabolic Control of
Polysaccharide Conformation and Function", Nature, Vol. 227, pp.
392-93 (Jul. 25, 1970) and WONG et al., "Sulfohydrolase Activity
and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)",
Plant Physiology, Vol. 61, pp. 663-666 (1978), the disclosures of
which are herein incorporated by reference in their entireties.
Such enzymes have not been previously purified to homogeneity and
no electrophoresis data has been provided in previous reports.
[0019] The mode of action and degree of specificity of galactan
sulfohydrolases have only been described in general terms in the
literature. As discussed in CRAIGIE et al., "Carrageenan
Biosynthesis", Proc. Int. Seaweed Symp., pp. 369-377 (1979), the
disclosure of which is herein incorporated by reference, it is
unclear how many sulfohydrolases with different specificity are
present in C. crispus. In general, there is little discussion in
the literature of the chemical structure of the sulfohydrolase
substrates, the end-products of enzymatic action, and the extent of
action of the sulfohydrolase on the galactan precursor.
[0020] Thus, there is a need for a variety of sulfohydrolases with
different specificity which can be used to tailor the properties of
sulfated compounds, such as galactans.
SUMMARY OF THE INVENTION
[0021] The present invention is directed to providing purified
sulfohydrolases having various specificities.
[0022] The present invention is also directed to amino acid and
nucleotide sequences of sulfohydrolases.
[0023] The present invention is directed to methods of
enzymatically modifying sulfated compounds.
[0024] The present invention is additionally directed to methods of
extracting carrageenan from seaweed.
[0025] The present invention is further directed to sulfated
compounds, such as galactans, which have been modified with
sulfohydrolases, such as to modify gelling properties.
[0026] In accordance with one aspect, the present invention is
directed to sulfohydrolase having a purity level based on total
amount of protein of at least about 40 wt %. The purity level may
also be at least about 70 wt %, at least about 90 wt %, or at least
about 95 wt %.
[0027] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
of SEQ ID NO: 17.
[0028] In accordance with yet another aspect, the present invention
is directed to an isolated nucleic acid sequence comprising a
sequence having at least about 25% homology with SEQ ID NO: 17. The
homology with SEQ ID NO: 17 may also be at least about 50%, at
least about 80%, or at least about 90%.
[0029] In accordance with still another aspect, the present
invention is directed to an isolated nucleic acid sequence which
will hybridize under hybridization conditions with a nucleic acid
of SEQ ID NO: 17.
[0030] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
of SEQ ID NO: 18.
[0031] In accordance with yet another aspect, the present invention
is directed to an isolated nucleic acid sequence comprising a
sequence of SEQ ID NO: 19.
[0032] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
of SEQ ID NO: 20.
[0033] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
of SEQ ID NO: 21.
[0034] In accordance with yet another aspect, the present invention
is directed to an isolated nucleic acid sequence comprising a
sequence having at least about 25% homology with SEQ ID NO: 18. The
homology with SEQ ID NO: 18 may also be at least about 50%, at
least about 80%, or at least about 90%.
[0035] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
having at least about 90% homology with SEQ ID NO: 19.
[0036] In accordance with still another aspect, the present
invention is directed to an isolated nucleic acid sequence
comprising a sequence having at least about 90% homology with SEQ
ID NO: 20.
[0037] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence comprising a sequence
having at least about 90% homology with SEQ ID NO: 21.
[0038] In accordance with yet another aspect, the present invention
is directed to an isolated nucleic acid sequence which will
hybridize under hybridization conditions with a nucleic acid of SEQ
ID NO: 18.
[0039] In accordance with another aspect, the present invention is
directed to an isolated nucleic acid sequence encoding for an amino
acid sequence corresponding to SEQ ID NO: 22.
[0040] In accordance with yet another aspect, the present invention
is directed to an isolated protein comprising an amino acid
sequence comprising SEQ ID NO: 22.
[0041] In accordance with still another aspect, the present
invention is directed to an isolated protein comprising an amino
acid sequence which has at least about 35% homology with SEQ ID NO:
22. The homology with SEQ ID NO: 22 may also be at least about 50%
or at least about 80%.
[0042] In accordance with yet another aspect, the present invention
is directed to an isolated nucleic acid sequence encoding for an
amino acid sequence corresponding to SEQ ID NO: 23.
[0043] In accordance with another aspect, the present invention is
directed to an isolated protein comprising an amino acid sequence
comprising SEQ ID NO: 23.
[0044] In accordance with yet another aspect, the present invention
is directed to an isolated protein comprising an amino acid
sequence which has at least about 20% homology with SEQ ID NO: 23.
The homology with SEQ ID NO: 23 may also be at least about 50% or
at least about 80%.
[0045] In accordance with a further aspect, the present invention
is directed to a process for purifying at least one sulfohydrolase,
comprising: subjecting an extract from seaweed to fractionation to
obtain fractions; and subjecting at least one of the fractions to
phenyl sepharose chromatography to obtain sepharose fractions
containing at least one sulfohydrolase.
[0046] In accordance with still another aspect, the present
invention is directed to an enzymatically modified compound which
has been modified by an isolated sulfohydrolase having a purity
level based on total amount of protein of at least about 40 wt %.
The purity of the isolated sulfohydrolase may be at least about 70
wt %, at least about 90 wt %, or at least about 95 wt %.
[0047] In accordance with a further aspect, the present invention
is directed to a process of enzymatically modifying a sulfated
compound, comprising: combining at least one sulfohydrolase, having
a purity level based on total amount of protein of at least about
40 wt %, with a sulfated compound to form a reaction mixture; and
incubating the reaction mixture to remove sulfate groups from the
sulfated compound to form an enzymatically modified compound.
[0048] In accordance with still another aspect, the present
invention is directed to a process of enzymatically modifying a
sulfated compound, comprising: incubating a first sulfohydrolase
with a sulfated compound to remove sulfate groups from the sulfated
compound to form an intermediate compound; and subsequently
incubating the intermediate compound with a second sulfohydrolase
to remove sulfate groups to form an enzymatically modified
compound.
[0049] In accordance with another aspect, the present invention is
directed to a product made by incubating a sulfated compound with a
solution having a protein content consisting essentially of a
sulfohydrolase which removes sulfate groups processively.
[0050] In accordance with still another aspect, the present
invention is directed to a product made by incubating a sulfated
compound with a solution having a protein content consisting
essentially of a sulfohydrolase which removes sulfate groups
randomly.
[0051] In accordance with yet another aspect, the present invention
is directed to a method for extracting one of nu- and
mu-carrageenan from seaweed, comprising: dispersing seaweed in a
salt solution comprising K.sub.2CO.sub.3 to form a dispersion;
filtering the dispersion to obtain a liquid; ultrafiltering the
dispersion to remove salts; concentrating the liquid; adjusting the
pH of the liquid to about 8 to 8.5; and precipitating the one of
nu- and mu-carrageenan from the liquid.
[0052] In one aspect, the sulfohydrolase is capable of removing
sulfate from hydrocolloid. The hydrocolloid may be one of
glycosaminoglycan, fucan, and galactan. In other words, the
sulfohydrolase hydrolyzes ester-sulfate bonds present in
hydrocolloids.
[0053] In another aspect, the sulfohydrolase is capable of removing
sulfate from galactan.
[0054] In another aspect, the galactan comprises carrageenan. The
carrageenan may comprise mu-carrageenan, e.g., comprising at least
about 50 mol % mu-carrageenan. The carrageenan may comprise
nu-carrageenan, e.g., comprising at least about 50 mol %
nu-carrageenan.
[0055] In another aspect, the galactan comprises agar.
[0056] In yet another aspect, the sulfohydrolase comprises
6-O-sulfohydrolase.
[0057] In still another aspect, the sulfohydrolase is capable of
converting nu-carrageenan into iota-carrageenan.
[0058] In another aspect, the sulfohydrolase is capable of
converting mu-carrageenan into kappa-carrageenan.
[0059] In another aspect, the sulfohydrolase is capable of removing
6-O-sulfate to induce anhydrobridge formation.
[0060] In another aspect, the fractionation comprises ammonium
sulfate fractionation.
[0061] In still another aspect, the method further comprises
subjecting at least one of the phenyl sepharose fractions to DEAE
sepharose chromatography to obtain DEAE sepharose fractions. At
least one of the DEAE sepharose fractions may be subjected to
heparin sepharose chromatography.
[0062] In another aspect, the incubation is at a temperature of
about 0 to 60.degree. C.
[0063] In still another aspect, the incubation is at a pH of about
5.5 to 9.5.
[0064] In yet another aspect, the at least one sulfohydrolase to be
added to the reaction mixture is contained within a solution having
a concentration of the at least one sulfohydrolase of at least
about 2 to 85 .mu.g/ml.
[0065] In another aspect, the sulfated compound is at a
concentration of about 0.012 to 2% (w/v) in the reaction
mixture.
[0066] In yet another aspect, the modified compound comprises
iota-carrageenan.
[0067] In still another aspect, the modified compound comprises
kappa-carrageenan.
[0068] In another aspect, the enzymatically modified compound
comprises precursor enriched kappa-carrageenan.
[0069] In another aspect, the enzymatically modified compound
comprises precursor enriched iota-carrageenan.
[0070] In another aspect, the removal of sulfate comprises removing
6-sulfate group from a galactan.
[0071] In still another aspect, the removal of sulfate comprises
converting nu-carrageenan to iota-carrageenan to form a product
that does not gel under conditions wherein the product has a
concentration of about 0.7% (w/v) in an aqueous solution having 0.1
M of potassium at a temperature of 40.degree. C. and a pH of 7.
[0072] In another aspect, the first sulfohydrolase removes sulfate
randomly.
[0073] In yet another aspect, the second sulfohydrolase removes
sulfate processively.
[0074] In another aspect, the seaweed comprises Spinosum.
[0075] In still another aspect, the seaweed is freeze-dried and
milled prior to being dispersed in the salt solution.
[0076] In yet another aspect, the salt solution further comprises
KCl. The KCl may have a concentration of about 1 to 1.5 M.
[0077] In another aspect, the dispersion is allowed to sit for at
least about 18 hours prior to filtering.
[0078] In another aspect, the concentrating is by a factor of about
2 to 3.
[0079] In still another aspect, the adjusting of the pH of the
liquid is by adding base.
[0080] In another aspect, the precipitating is by adding isopropyl
alcohol.
[0081] In yet another aspect, the dialyzing is conducted until the
conductivity of the water is stabilized at less than about 2
.mu.S/cm.
[0082] In another aspect, after the precipitating of the dialyzed
one of nu- and mu-carrageenan, the one of nu- and mu-carrageenan is
freeze-dried and milled.
[0083] In another aspect, the precipitated dialyzed one of nu- and
mu-carrageenan has a one of nu- and mu-content of about 18 to 35
mol %.
[0084] In another aspect, the precipitated dialyzed one of nu- and
mu-carrageenan has a molecular weight of about 700 to 800 kDa.
DETAILED DESCRIPTION OF THE INVENTION
[0085] The particulars shown herein are by way of example and for
purposes of illustrative discussion of the various embodiments of
the present invention only and are presented in the cause of
providing what is believed to be the most useful and readily
understood description of the principles and conceptual aspects of
the invention. In this regard, no attempt is made to show details
of the invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0086] All percent measurements in this application, unless
otherwise stated, are measured by weight/volume based upon grams
per milliliter. Thus, for example, 30% represents 30 grams out of
every 100 milliliters of the sample.
[0087] Unless otherwise stated, a reference to a compound or
component, includes the compound or component by itself, as well as
in combination with other compounds or components, such as mixtures
of compounds.
[0088] Before further discussion, a definition of the following
terms will aid in the understanding of the present invention.
[0089] "Galactan": a polysaccharide composed of galactose units and
additional units; such as agars and carrageenans. Galactans have at
least 50 mol % of galactose units.
[0090] "Hydrocolloid": a hydrophilic polysaccharide or derivative,
e.g., plant polysaccharide, that swells to produce a viscous
dispersion or solution when added to water.
[0091] "Hybridization conditions": nucleic acid of interest is
transferred onto Nylon membranes, available from Amersham Pharmacia
Biotech AB, as described in SAMBROOK et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring
Harbor, N.Y. (1989), the disclosure of which is herein incorporated
by reference in its entirety. The membranes are hybridized
overnight in 6.times.SSC, 5.times. Denhar't, 0.1% SDS and 100
.mu.g/ml of salmon sperm DNA at 42.degree. C. In this regard, SSC
is an aqueous solution of 3 M sodium chloride and 0.3 M sodium
citrate made in accordance with the procedure described in SAMBROOK
et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH
Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure
of which is herein incorporated by reference in its entirety. After
hybridization, filters were washed at 42.degree. C. for 15 minutes
in the following solutions, 2.times.SSC, 0.1% SDS; 1.times.SSC,
0.1% SDS and exposed to photostimulated screen, available from
Molecular Dynamics, Uppsala, Sweden, scanned using Storm, also
available from Molecular Dynamics, Uppsala, Sweden.
[0092] To determine the percent identity or percent homology of two
amino acid sequences or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of a first amino acid or nucleic
acid sequence for optimal alignment with a second amino or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions (e.g., overlapping positions).times. 100). Preferably,
the two sequences are the same length.
[0093] The determination of percent homology between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of KARLIN et al.,
Proc. Natl. Acad. Sci. USA, 87, pp. 2264-2268 (1990), the
disclosure of which is herein incorporated by reference in its
entirety, modified as in KARLIN et al., Proc. Natl. Acad. Sci. USA,
90, pp. 5873-5877 (1993), the disclosure of which is herein
incorporated by reference in its entirety. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of ALTSCHUL et
al., J. Mol. Biol., 215, pp. 403-410 (1990), the disclosure of
which is herein incorporated by reference in its entirety. BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to sulfohydrolase nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
sulfohydrolase protein molecules of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in ALTSCHUL et al., Nucleic Acids Res., 25, pp. 3389-3402
(1997), the disclosure of which is herein incorporated by reference
in its entirety. Alternatively, PSI-Blast can be used to perform an
iterated search which detects distant relationships between
molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of MYERS
et al., CABIOS, 4, pp. 11-17 (1988), the disclosure of which is
herein incorporated by reference in its entirety. Such an algorithm
is incorporated into the ALIGN program (version 2.0) which is part
of the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used.
[0094] As an overview, the present invention relates to the
isolation and purification of sulfohydrolases. For instance, the
sulfohydrolases may be purified from seaweed. The sulfohydrolases
are preferably those which are able to remove sulfate from sulfated
compounds, such as carrageenan. The sulfohydrolases may also be
able to form 3,6-anhydro bridges on the sulfated compounds, e.g.,
carrageenan. The present invention also relates to methods of
enzymatic modification of sulfated compounds. The present invention
is further directed to modified sulfated compounds, such as
modified carrageenans, such as nu-carrageenan which has been
modified to at least have portions which correspond to
iota-carrageenan, or such as mu-carrageenan which has been modified
to at least have portions which correspond to kappa-carrageenan.
The present invention is also directed to methods of extracting
carrageenan from seaweed.
[0095] Given the guidance in the present application and the
current state of the art of: (1) protein purification and (2)
partial amino acid sequence determination; (3) construction and use
of probes to locate corresponding DNA sequencing; and (4) cloning
and sequencing, the discussion which follows makes it possible to
(1) isolate the sulfohydrolases; (2) identify their amino acid
sequences; (3) construct probes for the corresponding DNA
sequences; and (4) identify, isolate, purify, and determine the DNA
sequences encoding the enzymes. See, for example, SAMBROOK et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory
Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is
herein incorporated by reference in its entirety.
[0096] The compounds to be enzymatically modified by the present
invention include sulfated compounds. The sulfated compounds
include galactans, glycosaminoglycans, and fucans.
[0097] Fucans are highly heterogeneous sulfated polysaccharides
composed of different sugar residues, such as fucose, galactose,
mannose, and uronic acid. The number of fucan groups and their
compositional patterns differ considerably. Fucans are produced in
brown algae and in Echinoderm. In this regard, fucans may be from
the matricial phase of the cell walls of brown algae, which
primarily contain L-fucose. L-fucose residues are often linked by
alpha(1.fwdarw.3) linkages and sulfated on the C4 position.
Enzymatic modification of fucan may affect the anti-coagulating
properties of fucans.
[0098] Glycosaminoglycans (GAGs) is a family of sulfated
polysaccharides from the extracellular matrix of animals, which
encompasses, heparin, heparin sulfate, chondroitin sulfate,
dermatan sulfate, and keratan sulfate. GAGs consist of disaccharide
repeating units containing hexuronic acid and hexosamine. Heparin
and heparin sulfate are variably sulfated glucosaminoglycans that
consists primarily of alternating alpha(1.fwdarw.4)-linked residues
of D-iduronate-2-sulfate or D-glucuronate-2-sulfate and
N-sulfo-D-glucosamine-6-sulfate. Enzymatic modification of heparin
may affect the anti-clotting properties of heparin. Chondroitin
sulfate and dermatan sulfate consist primarily of alternating
alpha(1-4)-linked residues of D-iduronate or D-glucuronate and
N-sulfo-D-galactosamine-6-sulfate. Usually the C2 or C4 position of
the N-acetylgalactosamine is sulfated and the C2 position of
iduronic acid in dermatan sulfate is also frequently sulfated.
Keratan sulfate disaccharide consists of galactose and
N-acetylglucosamine. The C6 position of either the galactose or the
N-acetylglucosamine can be sulfated.
[0099] Galactans may be obtained from many different sources. For
example, galactans may be obtained from seaweed. Examples of
galactans include carrageenan and agar.
[0100] As an example, the extraction of agar from seaweed is known.
For instance, the extraction of agar from seaweeds is described in
SELBY et al., "Agar", Industrial Gums: Polysaccharides and their
Derivatives, 3rd ed., (1993), the disclosure of which is herein
incorporated by reference in its entirety.
[0101] The extraction of carrageenan from seaweeds is known. For
instance, the extraction of carrageenan from seaweeds is described
in THERKELSEN, "Carrageenan", Industrial Gums: Polysaccharides and
their Derivatives, 3rd ed., (1993), the disclosure of which is
herein incorporated by reference in its entirety.
[0102] The present invention is also directed to a method for
extracting higher than natural levels of nu- and mu-carrageenan
with high molecular weight, i.e., above about 500 kDa. Depending
upon the carrageenan to be extracted, the source for carrageenan
may be a seaweed, e.g., Spinosum, Cottonii, and Gigartina
radula.
[0103] The seaweed may be freeze-dried, milled, and dispersed in an
aqueous KCl solution having a concentration of preferably about
0.05 to 0.3 M, more preferably about 0.1 to 0.15 M, and most
preferably about 0.125 M KCl. The dispersion also contains
K.sub.2CO.sub.3 at a concentration of preferably about 0.0005 to
0.003 M, more preferably about 0.001 to 0.0015 M, and most
preferably about 0.00125 M to maintain an alkaline environment,
i.e., a pH of preferably about 7 to 9, more preferably about 7.5 to
8.5, and most preferably about 7.8 to 8.3. The corresponding
calcium salts, i.e., chlorides and carbonates, may also be used,
but since the solubility of these is relatively poor, potassium
salts are preferred.
[0104] The dispersion is allowed to sit for a period of time,
preferably about 1 to 72 hours, more preferably about 16 to 32
hours, and most preferably about 24 hours, at a temperature of
about 5 to 55.degree. C., more preferably about 20 to 50.degree.
C., and most preferably about room temperature.
[0105] After this period of time, the dispersion is filtered. For
example, filtration may be conducted with a pressurized
kieselguhr-filter.
[0106] After filtration, the liquid is preferably ultrafiltered
and/or diafiltered, e.g., with an MWCO-membrane of 30 kDa, to
remove excess salts. Late in the ultrafiltration, washing of the
extract is preferably done by addition of tap water at room
temperature. The washing is preferably done until conductivity is
below 2 mS/cm and stable. The liquid is preferably concentrated on
the ultrafiltration to 25 to 30%. The pH is preferably adjusted to
about 8 to 8.5 with, e.g., 0.1 M NaOH.
[0107] The liquid is then evaporated to preferably about 10 to 90%,
more preferably about 30 to 70%, and most preferably about 50%. For
instance, the liquid may be evaporated on a vacuum evaporator.
[0108] After evaporation, the pH is adjusted to preferably about 7
to 9, more preferably about 7.5 to 8.5, and most preferably about
8. For instance, the pH may be adjusted to about 8 by adding 1 M
NaOH. The carrageenan is then precipitated using isopropyl alcohol
(IPA), e.g., 100% (v/v) IPA, available from BP Chemicals Ltd., UK
and freeze-dried.
[0109] The resulting extract has an enriched nu- or mu-content of
preferably about 15 to 40 mol %, more preferably about 17 to 30 mol
%, and most preferably about 19 to 24 mol %. The resulting extract
also has a high molecular weight of preferably about 500 to 1100
kDa, more preferably about 600 to 900 kDa, and most preferably
about 700 to 800 kDa.
[0110] The sulfohydrolases of the present invention may be isolated
from seaweeds producing sulfated galactans, such as the families of
Solieriaceae (such as Eucheuma, Kappaphycus), Gigartinaceae (such
as Chondrus, Gigartina), Furcellariaceae, Hypneaceae,
Phyllophoraceae, Cystocloniaceae (such as Calliblepharis), and
Bangiaceae (such as Porphyra). For example, a non-exhaustive list
of seaweed sources and potential seaweed sources for
sulfohydrolases include Eucheuma spinosum, Eucheuma cottonii
(=Kappaphycus alvarezii), Eucheuma denticulatum, Chondrus crispus,
Calliblepharis jubata, Gigartina radula, Gigartina stellata, and
Porphyra umbilicalis.
[0111] The following paragraphs describe techniques for isolating
the enzyme. From the guidance and examples of purification
techniques described in the present application, a skilled artisan
would be able to develop additional techniques for isolating the
sulfohydrolases.
[0112] To isolate sulfohydrolases in accordance with the present
invention, it is often preferred that potential enzyme sources,
e.g., seaweeds, be screened for enzyme activity to ensure that an
enzyme of interest is present in the potential enzyme source. As
discussed in more detail below, the screening typically involves
obtaining a crude extract from the potential enzyme source. The
crude extract would then be used to treat a variety of substrates
under a variety of conditions to generally determine the activity
of any enzymes present in the crude extract. With this information,
a skilled artisan would be able to generally determine whether it
might be worthwhile to subject the potential enzyme source to more
thorough purification, as discussed below, and to generally
determine conditions under which an enzyme is stable and
active.
[0113] As an example of a crude extraction technique, to isolate
enzymes from C. crispus, gametophyte plants of C. crispus may be
frozen in liquid nitrogen and ground to a powder. Although the
powder may be used immediately, to maintain the integrity of
enzymes in the powder, the powder may be kept at a low temperature,
such as -20.degree. C. to -80.degree. C. until further use.
[0114] This powder may then be extracted with a buffer, e.g., 4
volumes of 50 mM Tris-HCl buffer (pH 9.5) containing 10 mM
2-mercaptoethanol and 500 mM KCl. After centrifugation, the
supernatant may then be fractionated with cold (e.g., -20.degree.
C.) acetone, e.g., in a two step process: (1) from 0 to 30%
saturation; and (2) from 30 to 60% saturation of acetone. After
each fractionation step, a centrifugation is preferably performed,
e.g., at 4.degree. C. for 30 min. at 10,000.times.g. The pellets
may be dissolved in a buffer, e.g., 2 ml of 50 mM Tris-HCl buffer
(pH 7.1) containing 10 mM 2-mercaptoethanol. The pellets as well as
an aliquot of the supernatants may then be dialyzed, e.g.,
overnight at 4.degree. C. against 3.times.3 liters of 50 mM
Tris-HCl buffer (pH 7.1) containing 10 mM 2-mercaptoethanol using a
Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum
Laboratories, Inc., Rancho Dominguez, Calif., USA. The pellets and
the supernatants may be used to search for sulfohydrolase
activity.
[0115] As another example of a crude extraction technique, to
isolate enzyme from E. cottonii (=Kappaphycus alvarezii), Cottonii
may be frozen in liquid nitrogen. The cottonii may be used
immediately or kept at low temperature, e.g., -80.degree. C., until
further use. Prior to extraction the Cottonii may be ground to a
fine powder.
[0116] All of the following steps may be performed at 4.degree. C.,
unless otherwise noted. The powder may then be subjected to
extraction using a buffer, e.g., 50 mM Tris-HCl (pH 9.5)+500 mM KCl
and 10 mM 2-mercaptoethanol. The resulting suspension may be
stirred, e.g., overnight, and then centrifuged, e.g., at
10,000.times.g for 75 min. using a Beckman J2-21, Rotor JA20
available from Beckman Instruments, Inc., Fullerton, Calif., USA.
The supernatant may be fractionated with cold acetone as described
previously. After centrifugation, supernatant and pellets are
collected. Pellets, as well as an aliquot of the supernatant may
then be dialyzed, e.g., using an MWCO 6-8000 or 3500 dialysis
membrane available from Spectrum Laboratories, Inc., Rancho
Dominguez, Calif., USA, for 20 hours against 4.times.1 liter of 50
mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol. After dialysis, the
supernatant and the redissolved pellets may be used to search for
sulfohydrolase.
[0117] Once a crude extract is obtained, to determine whether an
extract has any sulfohydrolase activity, several assays are
possible. For instance, sulfohydrolases remove sulfate groups from
carrageenan such that measuring the level of free sulfate is an
indication of sulfohydrolase activity. 6-O-sulfohydrolases, which
are able to remove 6-O-sulfate, also tend to cause the formation of
3,6-anhydrogalactose bridges and to induce, therefore, an increase
of viscosity. Therefore, measuring the level of
3,6-anhydrogalactose bridges is another measure of sulfohydrolase
activity. Additionally, 6-O-sulfohydrolase activity corresponds
with an increase in viscosity such that viscosity may be measured
to indicate sulfohydrolase activity.
[0118] Regarding the measuring of free sulfate, the activity of the
sulfohydrolases on carrageenans is measured in the present
application by a new assay for measuring the free sulfate. Several
assays for the determination of free sulfate have been described in
the literature, but these assays are either time consuming or not
sensitive enough. See WONG et al., "Sulfohydrolase Activity and
Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)", Plant
Physiology, Vol. 61, pp. 663-666 (1978); REES, "Enzymatic
Desulphation of Porphyran", Biochem. J., 80, pp. 449-453 (1961);
and ZINOUN et al., "Evidence of Sulfohydrolase Activity in the Red
Alga Calliblepharis jubata", Botanica Marina, Vol. 40, pp. 49-53
(1997), the disclosures of which are herein incorporated by
reference in their entireties.
[0119] The new assay for determining free sulfate levels is based
on the determination of the free sulfate by high performance anion
exchange chromatography using auto suppressed conductivity
detection within 8 minutes. Besides being fast and fully automated,
this method has the advantage of being reproducible and sensitive
since as few as 10 ppm of free sulfate can be easily detected.
[0120] For instance, sulfohydrolase activity may be assayed by
measuring the amount of sulfate released upon incubation of the
enzyme extract on carrageenan. The reaction mixture may contain 100
.mu.l of enzyme sample in 50 mM Tris-HCl (pH 7.1)/10 mM
2-mercaptoethanol and 100 .mu.l of 1.4% (w/v) carrageenan. In this
regard, the carrageenan is either in the same buffer or in MilliQ
water, available from Millipore Corporation, Bedford, Mass., USA.
It should be noted that the enzyme sample may be obtained by any
appropriate technique, such as the crude extraction techniques
described above. The sample, however, may be any other sample for
which a measure of the activity is desired. Unless otherwise noted,
a reference mixture is made using enzyme extract boiled for 10
minutes prior to use. After 6 to 15 hours incubation at 48.degree.
C., carrageenan is removed from the reaction mixture by
centrifugation at 3320.times.g for 1 hr at 30.degree. C. in a
Microcon-10 unit, available from Amicon Bioseparations, Millipore
Corporation, Bedford, Mass., USA.
[0121] The amount of free sulfate present in the filtrate from the
Microcon-10 unit is then analyzed by HPAEC (high performance anion
exchange chromatography) using a Dionex DX 500 chromatography
system equipped with a GP40 gradient pump and an ED40
electrochemical detector, all available from Dionex Corporation,
Sunnyvale, Calif., USA. The column, an IonPac AS12 A anion exchange
column (4.times.200 mm, also available from Dionex Corporation) was
mounted on an AG12 Guard column (4.times.50 mm, also available from
Dionex Corporation). The eluent is 9.5 mM Na.sub.2CO.sub.3/0.5 mM
NaHCO.sub.3 at a flow rate of 1.5 ml/min. Detection of anion is
performed by ASRS conductivity using an anion self regenerating
suppressor ASRS-1 (4 mm, also available from Dionex Corporation)
with an SRS (Self Regenerating Suppressor) current of 50 mA.
[0122] Regarding measuring 3,6 anhydrogalactose bridges, another
assay may be used. The amount of 3,6 anhydrogalactose bridge
produced during the desulfatation reaction is measured in this
application using the technique described in JOL et al., "A Novel
High-Performance Anion-Exchange Chromatographic Method for the
Analysis of Carrageenans and Agars Containing
3,6-Anhydrogalactose", Analytical Biochemistry, 268, pp. 213-222
(1999), the disclosure of which is herein incorporated by reference
in its entirety.
[0123] Regarding measuring viscosity levels, viscometric
measurements may be carried out on the reaction mixtures. The
viscosity of the reaction mixture is directly measured using a
programmable Brookfield rheometer model DV III, available from
Brookfield Engineering Laboratories, Stoughton, Mass., USA,
thermostated at 48.degree. C. Measurements are performed with a
CP52 spindle for 10 minutes using a shear rate of 120 rpm.
[0124] Once a sample has been identified as a potential source for
enzymes, e.g., by the above described screening, the
sulfohydrolases may be purified to homogeneity in accordance with
the present invention. From the guidance and purification
techniques described in the present application, a skilled artisan
would be able to develop additional purification techniques.
[0125] A typical example of the purification of the enzymes from C.
crispus is shown and discussed on the following pages; all steps
are performed at 4.degree. C. unless otherwise noted:
1 2
[0126] As noted above, all steps of fractionation and purification
in the above purification process are performed at 4.degree. C.
unless otherwise noted.
[0127] Gametophyte plants of Chondrus crispus may be frozen in
liquid nitrogen and ground. For example, the frozen C. crispus may
be automatically ground to pieces which are less than 1 mm long by
using a Forplex miller, available from Forplex Industrie, Boulogne
Billancourt, France. This "powder" may be used immediately or may
be kept at low temperature, e.g., -20.degree. C. to -80.degree. C.,
until further use.
[0128] The ground C. crispus may then be subjected to extraction.
For example, this frozen ground C. crispus in the amount of 650 g
may be allowed to thaw overnight in 1.5 volumes (v/w) of 4.degree.
C. extracting buffer (50 mM Tris-HCl, pH 9.5/500 mM KCl/10 mM
2-Mercaptoethanol). In other words, the 650 g of C. crispus may be
allowed to thaw in 975 ml of the buffer. The suspension may be
stirred overnight and then centrifuged, e.g., at 10,000.times.g for
75 min.
[0129] The supernatant from extraction may then be subjected to
fractionation. For instance, the supernatant may be brought to 30%
(NH.sub.4).sub.2SO.sub.4 saturation (16.4 g ammonium sulfate/100 ml
of sample) by adding ammonium sulfate. In this regard, this
percentage refers to the percent of saturation in ammonium sulfate
which ammonium sulfate solutions are saturated at 3.9 M at
0.degree. C. When all the ammonium sulfate is dissolved, the
mixture may be allowed to stand for about 30 min. and then
centrifuged, e.g., at 24,700.times.g for 60 min.
[0130] Sulfohydrolase activity usually cannot be detected in the
crude extract and sometimes not in the ammonium sulfate
supernatant. This property may be the result of interferences with
polysaccharides or proteins that are removed during the phenyl
sepharose chromatography which is described below. An important
purpose of the phenyl sepharose chromatography is to remove
phycoerytrin, a hydrophilic protein, which constitutes the major
component in the Chondrus extract.
[0131] After centrifugation of the ammonium sulfate mixture, the
supernatant may be collected and loaded on a phenyl sepharose
column, e.g., a Phenyl Sepharose 6 fast flow (2.0.times.24 cm)
column, available from Amersham Pharmacia Biotech AB, Uppsala,
Sweden, previously equilibrated in 50 mM Tris-HCl buffer (pH 8.7),
30% (NH.sub.4).sub.2SO.sub.4 saturation, 500 mM KCl and 10 mM
2-Mercaptoethanol (buffer D). The column may be washed with this
buffer up to negligible absorbance at 280 nm in the effluent. The
bound proteins may then be eluted with a linear decreasing gradient
of (NH.sub.4).sub.2SO.sub.4 made of 360 ml of buffer D and 360 ml
of the same buffer without (NH.sub.4).sub.2SO.sub.4 (buffer C). At
the end of the gradient, phycoerythrin as well as some other
proteins are often eluted with buffer C alone. As an example, the
elution may be conducted under the following conditions:
2 Buffer D: 50 mM Tris-HCl buffer, pH 8.7, containing 30% of
saturation of (NH.sub.4).sub.2SO.sub.4, 500 mM KCl, and 10 mM
2-mercaptoethanol. Buffer C: 50 mM Tris-HCl buffer, pH 8.7,
containing 500 mM KCl, and 10 mM 2-mercaptoethanol. Flow Rate: 1
ml/min Fraction Size: 8 ml Time (min) % Buffer D % Buffer C 0 100 0
720 0 100
[0132] The above described extraction and phenyl sepharose
chromatography are preferably performed at least twice in order to
have enough material for subsequent steps.
[0133] Fractions of interest, i.e., fractions that are later found
to have sulfohydrolase activity, from the phenyl sepharose
chromatography may then be dialyzed. For instance, active fractions
may be pooled and dialyzed for 36 hr against 5.times.10 l of 50 mM
Tris-HCl buffer (pH 7.1) containing 10 mM 2-Mercaptoethanol (buffer
A), using a Spectra/por membrane MWCO 6-8000 or 3500, available
from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA.
After dialysis, the pH and the conductivity of the sample may be
checked and adjusted to those of buffer A (i.e., pH 7.1 and
conductivity of about 1.7 mS/cm) if necessary.
[0134] After dialysis, the sample may then be subjected to DEAE
Sepharose chromatography. For instance, the sample may be applied
at a flow rate of 1 ml/min to a DEAE Sepharose column (2.0.times.22
cm), available from Amersham Pharmacia Biotech AB, Uppsala, Sweden,
previously equilibrated with buffer A. The column may be washed
with buffer A until the absorption at 280 nm is negligible. Then
the adsorbed proteins may be eluted, e.g., at a flow rate of 1
ml/min with a linear NaCl (from 0 to 1 M) gradient in buffer A. As
an example, the elution may be performed as follows:
3 Buffer A: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM
2-mercaptoethanol Buffer B: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM
2-mercaptoethanol + 1 M NaCl Fraction Size: 7.5 ml Flow Rate: 1
ml/min Time (min) % Buffer A % Buffer B 0 100 0 390 35 65 450 35 65
630 0 100
[0135] SDS PAGE analysis of DEAE Sepharose chromatography fractions
eluting between 650 and 800 mM NaCl normally reveal the presence of
one single band at 34.9 kDa. This band corresponds to an enzyme
denoted sulfohydrolase II which is discussed in more detail
below.
[0136] In addition to sulfohydrolase II, sulfohydrolase I elutes
between 300 and 600 mM NaCl during the DEAE chromatography. These
fractions, however, usually include impurities as well. To isolate
sulfohydrolase I, further purification, such as dialysis and
semi-affinity and cation-exchange chromatography, e.g., heparin
chromatography, is required.
[0137] As an example of heparin chromatography, half of a fraction
eluting with 580 mM NaCl may be dialyzed against buffer A using a
Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum
Laboratories, Inc., Rancho Dominguez, Calif., USA. The fraction may
then be loaded (flow rate 1.5 ml/min) on top of a HiTrap heparin
column, available from Amersham Pharmacia Biotech AB, Uppsala,
Sweden, previously equilibrated with buffer A. The column may then
be washed with this buffer at a flow rate of 1.5 ml/min until the
absorbance at 280 nm is negligible. Bound proteins may then be
eluted using a three step increasing NaCl gradient (from 0 to 1200
mM). In particular, elution may be conducted using the following
gradient:
4 Buffer A: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM
2-mercaptoethanol Buffer B': 50 mM Tris-HCl buffer, pH 7.1 and 10
mM 2-mercaptoethanol + 1.2 M NaCl Fraction size: 1.5 ml Flow rate:
1.5 ml/min Time (min) % Buffer A % Buffer B' 0 100 0 10 71 29 15 71
29 25 34 66 30 34 66 35 0 100 45 0 100
[0138] SDS-PAGE analysis of fractions eluting with 1200 mM NaCl
should reveal a single faint band characterized by a molecular
weight of 62.1 kDa. This band corresponds to an enzyme denoted
sulfohydrolase I.
[0139] The above-described purification procedure preferably starts
with 2.times.600-700 g of fresh seaweed, such as the above-noted
650 g. With this amount of material, both enzymes separate well
after DEAE chromatography, but the sulfohydrolase I is still not
pure. The above-described chromatography on semi-affinity and
cation exchange chromatography is necessary to purify this enzyme
to homogeneity.
[0140] Cottonii sulfohydrolases may also be purified using a
procedure which is similar to the above procedure for purifying C.
crispus sulfohydrolases I and II.
[0141] For instance, Cottonii may be frozen in liquid nitrogen and
kept at -80.degree. C. until further use. Prior to extraction the
frozen Cottonii may be ground, e.g., manually to a fine powder in a
mortar, using liquid nitrogen which was poured with a baker into
the mortar to keep the Cottonii frozen during the grinding.
[0142] To partially purify the Cottonii sulfohydrolase, the
following steps may be performed at 4.degree. C. Ground Cottonii
may be extracted in buffer, e.g., 25 g of ground Cottonii in 40 ml
of 50 mM Tris-HCl (pH 9.5)+500 mM KCl and 10 mM 2-mercaptoethanol.
The resulting suspension may be stirred, e.g., with a magnetic
stirrer overnight, and then centrifuged, e.g., at 10,000.times.g
for 75 min using a Beckman J2-21, Rotor JA20 available from Beckman
Instruments, Inc., Fullerton, Calif., USA. The supernatant may be
dialyzed, e.g., using a MWCO 6-8000 or 3500 dialysis membrane
available from Spectrum Laboratories, Inc., Rancho Dominguez,
Calif., USA, for 20 hours against 4.times.1 liter of 50 mM Tris-HCl
(pH 8.5)+10 mM 2-mercaptoethanol. After dialysis, the supernatant
may be loaded on a heparin column, e.g., at a flow rate of 1 ml/min
on top of a Heparin agarose type II-S column (27.5.times.1 cm
column, 10.times.0.5 cm gel size), available from Millipore,
Stonehouse, England, with the heparin type II-S agarose gel being
available from Sigma Chemical, St. Louis, Mo., USA, previously
equilibrated in the same buffer. The column may be washed with this
buffer until the absorption at 280 nm is negligible. Then, the
adsorbed proteins may be eluted, e.g., for 40 minutes with a linear
NaCl (from 0 to 1 M) gradient. In particular, elution may be
conducted using the following gradient:
5 Buffer A: 50 mM Tris-HCl (pH 8.5) + 10 mM 2-mercaptoethanol
Buffer B: 50 mM Tris-HCl (pH 8.5) + 10 mM 2-mercaptoethanol + 1 M
NaCl Fraction size: 2 ml Flow rate: 1 ml/min Time (min) % of Buffer
A % of Buffer B 0 100 0 40 0 100
[0143] Although most of the proteins in the extract typically do
not bind to the heparin agarose, the sulfohydrolase binds to the
gel as most of the activity, in terms of sulfate released and/or
viscosity increase, is found in later fractions. The fractions from
the heparin chromatography may be concentrated, e.g., by about
13.times. by centrifugation at 3320.times.g for 1 hr at 4.degree.
C. in a Microcon-10 unit, available from Amicon Bioseparations,
Millipore Corporation, Bedford, Mass., USA, prior to SDS-PAGE
analysis and silver nitrate staining, according to MERRIL et al.,
"Ultrasensitive Stain for Proteins on Polyacrylamide Gels Shows
Regional Variation in Cerebrospinal Fluid Proteins", Science, 211,
pp. 1437-1438 (1981), the disclosure of which is herein
incorporated by reference in its entirety.
[0144] The different active fractions are all characterized by the
presence of two common proteins: one which is characterized by a
molecular weight of about 65 kDa and another which is characterized
by a molecular weight of about 55 kDa.
[0145] In accordance with the present invention, based on total
weight of protein in the sample, sulfohydrolases may have high
purity. The sulfohydrolase is preferably purified to at least about
40 wt %, more preferably at least about 50 wt %, even more
preferably at least about 60 wt %, even more preferably at least
about 70 wt %, even more preferably at least about 80 wt %, even
more preferably at least about 90 wt %, even more preferably at
least about 95 wt %, even more preferably at least about 99 wt %,
and most preferably 100 wt %, based on total weight of protein. As
discussed in more detail below, this high level of purity allows
tailoring of the properties of the sulfated compounds to be
modified.
[0146] Once the sulfohydrolases have been purified, the amino acid
sequences may be determined. The peptide sequence of these proteins
may be determined by conventional techniques, such as Edman's
degradation.
[0147] In order to determine the N-terminal amino acid sequence of
both sulfohydrolases, the sulfohydrolases were purified generally
in accordance with the above-described purification protocol
involving extraction, ammonium sulfate fractionation, phenyl
sepharose chromatography, DEAE Sepharose chromatography, etc. In
particular, fractions 60 and 61 from the DEAE Sepharose
chromatography were concentrated 20.times., by centrifugation at
3320.times.g for 1 hr at 4.degree. C. in a Microcon-10 unit,
available from Amicon Bioseparations, Millipore Corporation,
Bedford, Mass., USA, prior to SDS PAGE. Fractions 38 to 47 from the
DEAE chromatography were pooled and dialyzed for 6 hours at
4.degree. C. against 3.times.3 liters of 50 mM Tris-HCl (pH 7.1)+10
mM 2-mercaptoethanol by using a MWCO 6-8000 or 3500 dialysis
membrane available from Spectrum Laboratories, Inc., Rancho
Dominguez, Calif., USA. After dialysis, the conductivity of the
sample was adjusted to that of the buffer by dilution with milliQ
water, available from Millipore Corporation, Bedford, Mass., USA.
Half of the sample (57 ml) was then loaded (flow rate 1.5 ml/min)
on top of a HiTrap heparin column available from Pharmacia Biotech
AB, Uppsala, Sweden, previously equilibrated with 50 mM Tris-HCl
(pH 7.1)+10 mM 2-mercaptoethanol. The column was washed with this
buffer until the absorbance at 280 nm was negligible. Then, elution
of proteins was performed using an increasing gradient (from 0 to
1.2 M) of NaCl. Fractions 24, 25, 26, and 28, which constitute the
peak of elution for absorbance at 280 nm, were concentrated about
13.times., by centrifugation at 3320.times.g for 1 hr at 4.degree.
C. in a Microcon-10 unit, available from Amicon Bioseparations,
Millipore Corporation, Bedford, Mass., USA, before being loaded on
a SDS gel for SDS PAGE.
[0148] Accordingly, two fractions from the DEAE chromatography
(fractions 60 and 61) as well as four fractions from HiTrap Heparin
chromatography (fractions 24, 25, 26, and 28), were subjected to
SDS PAGE (12% total monomer
(acrylamide+N,N'-methylenebisacrylamide) in grams per 100 ml gels)
in accordance with the technique described in LAEMMLI, Nature, 227,
pp. 680-685 (1970), the disclosure of which is herein incorporated
by reference in its entirety, to separate the enzymes.
[0149] After SDS PAGE, the proteins were transferred onto a
"Hybond-P" membrane (polyvinylidene difluoride (PVDF) membrane),
available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. The
transfer was performed for 2 hrs at 250 mA in 50 mM Tris-HCl buffer
(pH 8.25) containing 50 mM borate. After transfer, the proteins
were then fixed for few seconds in 100% (w/v) methanol and the
membrane was then stained for 1 minute with a solution of 0.1%
(w/v) Coomassie Blue R250, available from Bio-Rad Laboratories,
Hercules, Calif., USA, in 50% (v/v) methanol and 10% (v/v) acetic
acid. Destaining is carried out by soaking the membrane for 4-5
minutes in a 50% (v/v) methanol and 10% (v/v) acetic acid solution.
After rinsing for 1-2 minutes with MilliQ water, available from
Millipore Corporation, Bedford, Mass., USA, the bands corresponding
to sulfohydrolases I and II were cut and sent to the Institute
Pasteur, Paris, France, for N-terminal amino acid sequence
determination by Edman's degradation. From this experiment, it
appears that sulfohydrolase II has a blocked amino-terminal end
and, therefore, its amino-terminal sequence could not be
determined. The Edman's degradation of the sulfohydrolase I
resulted in a determination that the N-terminal had the amino acid
sequence of SEQ ID NO: 1.
[0150] A similar strategy was used to determine the amino acid
sequence of some internal peptides. In this case, however, SDS PAGE
(12% (total monomer (acrylamide+N,N'-methylenebisacrylamide) in
grams per 100 ml gel) gels) in accordance with the technique
described in LAEMMLI, Nature, 227, pp. 680-685 (1970), the
disclosure of which is herein incorporated by reference in its
entirety, was not followed by a transfer. In particular,
immediately after electrophoresis, the gel was stained overnight
with a 0.0033% (w/v) amido black solution in 50% (v/v) methanol and
10% (v/v) acetic acid. The gel was then briefly rinsed (4.times.10
min.) with MilliQ water, available from Millipore Corporation,
Bedford, Mass., USA, in order to remove traces of methanol and
acetic acid. Then, the bands corresponding to sulfohydrolases I and
II were excised and dried for about 30 min. at 30.degree. C. in a
centrifuged evaporator RC 10.10, Jouan, France, equipped with a
refrigerated trap RCT 90, Jouan, France, before being sent to the
Institute Pasteur, Paris, France, for proteolytic digestions and
microsequence analysis, as discussed below.
[0151] Slices of acrylamide, which contained about 15-30 .mu.g of
sulfohydrolase I, were submitted to proteolysis with 0.4 .mu.g
endoprotease-LysC in 350 .mu.l of 0.05 Tris-HCl (pH 8.6)/0.03%
(w/v) SDS for 18 h at 37.degree. C. The resulting peptides were
then submitted to reverse-phase HPLC on a 250 mm.times.2.1 mm DEAE
C18 column, available from CIL CLUZEAU, Paris, France. Elution,
carried out with an acetonitrile gradient from 2 to 45% (v/v) in
0.1% (v/v) trifluoroacetic acid at a flow rate of 0.2 ml/min,
allowed many peptides to be separated. Among them, peptides 11, 12,
18, 22, and 25 were significant and were collected for sequencing.
These peptides were analyzed at the Institute Pasteur, Paris,
France, by Edman's degradation using an Applied Biosystems 473A
automated gas phase amino acid sequencer, available from PE Applied
Biosystems, Foster City, Calif., USA, in accordance with standard
procedures. As a result of this analysis, sulfohydrolase I was
determined to include SEQ ID NOS: 2-5.
[0152] Acrylamide slices containing about 10 .mu.g of SDS PAGE
purified sulfohydrolase II were subjected to proteolysis in 600
.mu.l of 0.05 M Tris-HCl (pH 8.6)/0.01% (v/v) "Tween 20"
polyoxyethylene sorbitan monolaurate, available from USB,
Cleveland, Ohio, USA, and 4% (w/v) of bovine trypsin. Tryptic
digestion was carried out for 18 h at 37.degree. C. The resulting
tryptic peptides were purified by reverse-phase HPLC on a 250
mm.times.2.1 mm DEAE C18 column, available from CIL CLUZEAU, Paris,
France. Elution was performed with an acetonitrile gradient from 2
to 45% (v/v) for 60 minutes in 0.1% (v/v) trifluoroacetic acid at a
flow rate of 0.2 ml/min. Among the isolated peptides, peptides 13,
14, 24, and 25 were collected because they were the most
significant, and were analyzed at the Institute Pasteur, Paris,
France, by Edman's degradation using an Applied Biosystems 473A
automated gas phase amino acid sequencer, available from PE Applied
Biosystems, Foster City, Calif., USA, in accordance with standard
procedures. As a result of this analysis, sulfohydrolase I was
determined to include SEQ ID NOS: 6-11.
[0153] On the basis of the above indicated amino acid sequences,
sequence probing processes were carried out for the corresponding
cDNA to determine the nucleotide sequence for sulfohydrolase I and
II. As described in more detail below, mRNA was isolated from C.
crispus, from the mRNA a cDNA library was synthesized, the cDNA
library was probed with PCR fragments obtained using
oligonucleotides based on the above-described amino acid sequences
for sulfohydrolase I and II, and positive cDNAs were sequenced.
[0154] Total RNA was prepared from gametophytes of Chondrus crispus
as described by APT et al., "The Gene Family Encoding the
Fucoxanthin Chlorophyll Proteins from the Brown Alga Macrocystis
pyrifera", Mol. Gen. Genet., 246, pp. 455-464 (1995), the
disclosure of which is herein incorporated by reference.
Purification of the mRNA was then performed using the PolyA T tract
mRNA Isolation system IV kit, available from Promega, Madison,
Wis., USA, according to the instructions of the manufacturer.
[0155] Once the mRNA was isolated, the cDNA library for C. crispus
was constructed. The cDNA synthesis was performed using a lambda
ZAP.RTM.II vector cDNA synthesis kit, available from Stratagene, La
Jolla, Calif., USA, according to the instructions of the
manufacturer. Double stranded cDNA were fractionated through a
sepharose CL-2B column, available from Amersham Pharmacia Biotech
AB, Uppsala, Sweden. The fractions with an average size of 600 to
1500 pb, as estimated on acrylamide gel (5%), were selected for the
preparation of the library. Ligation of cDNA into lambda ZapII
vector as well as transformation of the host strain XL1-Blue MRF'
were performed according to the instructions of the manufacturer
(Stratagene, La Jolla, Calif., USA).
[0156] The probe design and screening protocol for screening the
cDNA library was as follows. From SEQ ID NOS: 2 and 10,
respectively, degenerated oligonucleotides for sulfohydrolase I and
II were designed which correspond to SEQ ID NO: 12 for
sulfohydrolase I and SEQ ID NOS: 13-14 for sulfohydrolase II.
Degenerated oligonucleotide, SEQ ID NO: 12 was used with vector
primer SEQ ID NO: 16 (5'AATACGACTCACTATAG3') to amplify by PCR DNA
fragments corresponding to sulfohydrolase I, in the C. crispus cDNA
library.
[0157] Degenerated oligonucleotides, SEQ ID NOS: 13 and 14 were
used with vector primer SEQ ID NO: 15 (5'ATTAACCCTCACTAAAG3') to
amplify by PCR DNA fragments corresponding to sulfohydrolase II in
the C. crispus cDNA library as follows. A first PCR using
oligonucleotides SEQ ID NO: 14 and SEQ ID NO: 15 gave DNA
fragments. Using this first PCR product and oligonucleotides SEQ ID
NO: 15 and SEQ ID NO: 13, a DNA fragment corresponding to
sulfohydrolase II was amplified.
[0158] The cloned PCR fragments corresponding to the sulfohydrolase
I gene and the sulfohydrolase II gene were labelled by random
priming using the Megaprime kit, available from Amersham Pharmacia
Biotech AB, Uppsala, Sweden, and 1850 kBq of -.sup.32P dCTP,
available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. The
resulting probes were purified using Sephacryl SR 200, available
from Amersham Pharmacia Biotech AB, Uppsala, Sweden, and used to
screen the C. crispus gametophytes cDNA library.
[0159] For both sulfohydrolase I and II screenings, plaques were
transferred onto Nylon membranes, available from Amersham Pharmacia
Biotech AB, as described in SAMBROOK et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring
Harbor, N.Y. (1989), the disclosure of which is herein incorporated
by reference in its entirety. The membranes were hybridized
overnight in 6.times.SSC, 5.times. Denhar't, 0.1% SDS and 100
.mu.g/ml of salmon sperm DNA at 65.degree. C. In this regard, SSC
is an aqueous solution of 3 M sodium chloride and 0.3 M sodium
citrate made in accordance with the procedure described in SAMBROOK
et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH
Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure
of which is herein incorporated by reference in its entirety. After
hybridization, filters were washed 15 minutes in the following
solutions, 2.times.SSC, 0.1% SDS; 1.times.SSC, 0.1% SDS and
0.4.times.SSC, 0.1% SDS and exposed to photostimulated screen,
available from Molecular Dynamics, Uppsala, Sweden, scanned using
Storm, also available from Molecular Dynamics.
[0160] For sulfohydrolase I, using the screening conditions
described above, the above-described labelled probes for
sulfohydrolase I were used in three rounds of screening to screen
300,000 phages from the cDNA library for the presence of
sulfohydrolase I cDNA. A total of 408 positive phages (1.4% no %)
were positive from which 30 were selected. In a second round, 10 of
these positives were screened from which 7 were positive. From
these, 6 were found positive in a third round of screening.
[0161] For sulfohydrolase II, using the screening conditions
described above, the above-described labelled probes for
sulfohydrolase II were used in three rounds of screening to screen
600,000 phages from the cDNA library for the presence of
sulfohydrolase II cDNA. Twelve positive phages (1/50,000) were
found from which 10 were selected. In a second round these
positives were screened from which 9 were positive. From these 9, 9
were found positive in a third round of screening. The 5' and 3'
extremities of the 9 positive cDNAs were sequenced and sequencing
revealed that only 4 of the 9 corresponded to the sulfohydrolase II
cDNAs. Thus, the sulfohydrolase II gene seems to be weakly
expressed in C. crispus gametophyte (1/150,000 cDNAs).
[0162] For both sulfohydrolase I and II, in vivo excision was
achieved according to the instruction of the manufacturer
(packaging kit, available from Stratagene, La Jolla, Calif., USA),
and the 5' and 3' termini of each cDNA were sequenced, enabling
their identification. In particular, the sequences were determined
using the Thermo-Sequenase core sequencing kit with 7-deaza-dGTP
from Vistra TM. Sequence reactions were run on a Vistra DNA
automated sequencer 725, available from Molecular Dynamics,
Uppsala, Sweden.
[0163] Using the above method, the sequence of the sulfohydrolase I
cDNA was determined to be SEQ ID NO 17. In particular, one cDNA was
fully sequenced and the sequence termini were determined for the
others. Sequencing revealed that they are identical cDNAs, five are
full length cDNAs and one is a partial length cDNA.
[0164] The sequence for sulfohydrolase I contains the ATG start
codon, the signal peptide (possibly for secretion from the ER to
the cell wall) with the probable amino acid cleavage between
Alanine 20 and Lysine 21, the stop codon and the 3' UTR
(untranslated region).
[0165] The above method suggests that the nucleotide sequences for
sulfohydrolase II cDNAs are SEQ ID NOS: 18, 19, 20, and 21. In this
regard, it is apparent that these 4 cDNAs are different. Three (3)
cDNAs are full-length (SEQ ID NOS: 19, 20, and 21) and one may be a
partial length cDNA (SEQ ID NO: 18).
[0166] From the nucleotide sequences of both sulfohydrolase I and
II, the corresponding amino acid sequences were deduced. In
particular, the amino acid sequence of sulfohydrolase I was found
to be SEQ ID NO: 22. The amino acid sequence of sulfohydrolase II
was found to be SEQ ID NO 23.
[0167] Regarding the amino acid sequence for sulfohydrolase I, all
protein microsequences determined by the Edman's degradation were
found in the sequence. The amino acid sequence of sulfohydrolase I
has a 20 amino acid long signal peptide. The cleavage site is
between Ala 20 and Lys 21. Therefore, the mature enzyme comprises
594 amino acids and contains one potential N-glycosylation site
(NFTI; AA 72-75) and has some similarity with the FAD binding
domain.
[0168] Sulfohydrolase I shares at its C-terminus, 22% of sequence
identity over 300 amino acids with the L-amino acid oxidase of
Chlamydomonas reinhardtii, which was described in VALLON et al.,
"cDNA Sequence of M(Alpha), the Catalytic Subunit of the
Chlamydomonas reinhardtii L-Amino Acid Oxidase (Accession No.
U78797): a New Sequence Motif Shared by a Wide Variety of
Flavoproteins", Plant Physiol., 115, pp. 1729-1731 (1997), the
disclosure of which is herein incorporated by reference in its
entirety. In this regard, L-amino-acid oxidases have been found in
a variety of organisms. They catalyze the reaction:
H.sub.2N--CHR--COOH+O.s-
ub.2+H.sub.2O.fwdarw.O.dbd.CR--COOH+NH.sub.3+H.sub.2O.sub.2. In C.
reinhardtii, it has been shown that this 65 kDa enzyme is
periplasmic, gametespecific and induced by ammonium
deprivation.
[0169] Sulfohydrolase II is 279 amino acids long and contains one
glycosylation site. Analysis of sulfohydrolase II sequences
indicated that no significant similarity was found with known
sequences in the databases available at NCBI
(http://www.ncbi.nlm.nih.gov/BLAST/) using BLAST software.
[0170] The determination of the amino acid sequences of
sulfohydrolase I and II also allows an accurate determination of
the molecular weights of these enzymes. In this regard, as
estimated by SDS PAGE, the molecular weights of sulfohydrolase I
and II are 62.1 and 34.9 kDa, respectively. In the case of the of
sulfohydrolase I, this MW is slightly lower than the one deduced
from the amino acid sequence which gave a molecular weight of 66.8
kDa. In contrast, relative to the SDS PAGE estimate, sulfohydrolase
II has a slightly lower molecular weight of 30.9 kDa when
calculated from the amino acid sequence.
[0171] From both sequences a theoretical isoelectric point (pI) was
calculated using the program Mac Molly translate. Sulfohydrolase I
and II are characterized by a pI of 8.4 and 9.3 respectively.
[0172] From the amino acid and nucleotide sequences of
sulfohydrolase I and II, analogs or homologs of these
sulfohydrolases may be detected and isolated using known techniques
based upon sequence homology to the sulfohydrolases enzymes
disclosed herein. Thus, all or part of the known enzyme coding
sequence may be used to construct a probe which will hybridize
selectively to identical or highly similar enzyme coding sequences
present in genomic or cDNA libraries from a particular source. In
particular, nucleotide sequences having a % homology of at least
about 25%, more preferably at least about 50%, and most preferably
at least about 80%, relative to the specifically identified
sequences of the present invention, are in accordance with the
present invention. Similarly, amino acid sequences having a %
homology of at least 25%, more preferably at least 50%, and most
preferably at least 80%, relative to the specifically identified
sequences of the present invention, are in accordance with the
present invention.
[0173] Hybridization techniques include hybridization screening of
plated cDNA libraries, and amplification by polymerase chain
reaction (PCR) using oligonucleotide primers that correspond to
conserved sequence domains. See INNIS et al., PCR Protocols, a
Guide to Methods and Applications, Academic Press (1990), the
disclosure of which is herein incorporated by reference in its
entirety.
[0174] Further, references regarding the above techniques include
AUSUBEL et al., Current Protocols in Molecular Biology, Green
Publishing Company Assoc. and John Wiley Interscience, (1992); and,
BERGER et al., Guide to Molecular Cloning Techniques, Academic
Press (1987), the disclosures of which are herein incorporated by
reference in their entireties.
[0175] Homologous sulfohydrolases may be found, for example, by
extracting DNA and RNA from the source of interest and conducting
Southern hybridization, genomic and cDNA library screening, using a
complete or partial sequence from one of the known
sulfohydrolases.
[0176] For instance, total DNA may be extracted in accordance with
the method disclosed in APT et al., "The Gene Family Encoding the
Fucoxanthin Chlorophyll Proteins from the Brown Alga Macrocystis
pyrifera", Mol. Gen. Genet., 246, pp. 455-464 (1995), the
disclosure of which is herein incorporated by reference. In this
regard, it should be noted that it is often not necessary to remove
polysaccharides in the manner described by APT et al.
[0177] Southern hybridization may be conducted by, for example, by
digesting 7 .mu.g of total DNA of Chondrus crispus, Eucheuma
cottonii, Eucheuma spinosum, and Gracilaria gracilis for 4 hours at
37.degree. C. with 60 U of HindIII, available from Amersham
Pharmacia Biotech AB, Uppsala, Sweden, and 80 U of EcoRI, available
from Biolabs, Beverly, Mass., USA. After precipitation, the
digested DNA may be redissolved in 40 .mu.l of 100 mM Tris (pH
7.5)+10 mM EDTA. The fragments may then be fractionated on a 0.8%
agarose gel (migration overnight at 35 V) before being transferred
under vacuum for 3 hours on a Hybond-N+ membrane, available from
Amersham Pharmacia Biotech AB, Uppsala, Sweden, using a "Trans DNA
express vacuum blotter" apparatus, available from Appligene,
Gaithersburg, Md., USA, under the conditions given by the
manufacturer.
[0178] Prehybridation and hybridization may be performed at
60.degree. C. for Chondrus crispus and at 42.degree. C. for the
other algae in the conditions given by SAMBROOK et al., Molecular
Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold
Spring Harbor, N.Y. (1989), the disclosure of which is herein
incorporated by reference in its entirety. After hybridization, the
membranes were washed as follows:
[0179] For all the algae except Chondrus crispus
[0180] 2.times.SSC+0.1% SDS for 2.times.20 min; and
[0181] 1.times.SSC+0.1% SDS for 2.times.15 min.
[0182] For Chondrus crispus
[0183] Same as above with an additional wash in 0.5.times.SSC+0.1%
SDS for 15 min.
[0184] Southern hybridizations were conducted by using the partial
length cDNA encoding for sulfohydrolase I. After digestion of the
cDNA with EcoRI and XhoI, both available from Biolabs, Beverly,
Mass., 2 fragments were generated: one of 400 bp and the other one
of 1600 bp. This latter one was then collected and labelled using
the ECF Random-Prime Labelling and detection System, available from
Amersham Pharmacia Biotech AB, Uppsala, Sweden. Labelling,
hybridization and detection were performed as recommended by the
manufacturer.
[0185] Southern hybridization may also be done by using the full
length cDNA encoding for the sulfohydrolase II. This Southern
hybridization reveals one major band of about 2.5 kB as well as 3
other bands (which size is between 4 an 1.2 kB) in Chondrus
crispus. However, even after 60 hours of exposition, 3 faint
visible bands could be detected in Eucheuma spinosum and cottonii
but nothing could be detected in Gracilaria gracilis. Similar
results were obtained using the full length cDNA encoding for
sulfohydrolase I as a probe.
[0186] The results are summarized below:
6 Seaweed sulfohydrolase I cDNA sulfohydrolase II cDNA Chondrus
crispus 1 major band + 2-3 faint 1 major band + bands 3 faint bands
Gigartina 1 band + 3-4 faint bands undetermined skottsbergii
Gracilaria gracilis NA no bands Eucheuma cottonii 1 band + faint
bands 3 faint bands Eucheuma spinosum faint bands 3 faint bands
[0187] All these results indicate therefore that homologous
sulfohydrolases I exist in E. spinosum, E. cottonii and in
Gigartina skottsbergii and homologous sulfohydrolases II exist in
E. spinosum and in E. cottonii, but homology with the respective
Chondrus enzymes is low.
[0188] Almost pure sulfohydrolase II, which in this case had been
purified to 80-90% (w/w) homogeneity, based on the total amount of
protein in the sample, was incubated with the iota-precursor giving
a gel, whereas the boiled enzyme did not. The enzyme was also
incubated in 4 different concentrations with the iota-precursor.
Low amounts of enzyme created a soft gel whereas higher amounts
gave a stronger gel.
[0189] After incubation of nu-carrageenan with sulfohydrolase I,
sulfate release was detected but there was no increase in viscosity
of the carrageenan sample. In contrast, incubation with
sulfohydrolase II resulted in sulfate release and an increase in
viscosity. NMR analysis showed that all of the isolated enzyme
fractions acted to convert nu-carrageenan to iota-carrageenan.
[0190] Because of the observed modifications, the previous theory
advanced in U.S. Provisional Application No. 60/133,376 of a
sulfohydrolase and an anhydrolase has now been abandoned. This
previous theory described the enzymes as possessing two different
functions: (1) one enzyme to remove the C-6 sulfate on the nu
precursor; and (2) one enzyme to form the 3,6-anhydro-galactose
bridge, thereby converting the structure to iota carrageenan. It
should be noted, however, that although U.S. Provisional
Application No. 60/133,376 apparently failed to accurately describe
the mode of action for the enzyme, this provisional application
disclosed how to purify and use these enzymes.
[0191] While not wishing to be bound by theory, it is now believed
that sulfohydrolase I is removing the sulfate groups randomly. In
contrast, sulfohydrolase II might act progressively by sliding
along the molecule to thereby create stretches of iota, which can
aggregate with other similar molecules to create a gel.
[0192] When incubating the iota-precursor with first the random
enzyme followed by inactivation and thereafter incubation with the
processive enzyme, a higher viscosity was obtained when compared to
using the processive enzyme alone. It seems that the random enzyme
is preparing the polymer in such a way that it is easier for the
processive enzyme to slide along the molecule.
[0193] Taking into consideration the above, the high purity
sulfohydrolases of the present invention allow the tailoring of
properties of sulfated compounds.
[0194] An advantage of using an enzyme is that it can work at
relatively low temperatures, such as 0 to 60.degree. C., more
preferably 20 to 55.degree. C., and most preferably 37 to
48.degree. C. Accordingly, the enzyme does not need strong heating
to make the carrageenan gel.
[0195] The incubation is preferably conducted at a pH of about 5.5
to 9.5, more preferably about 6 to 9.5, and most preferably about 7
to 9. In this regard, incubation with the C. crispus
sulfohydrolases is preferably conducted at a pH of about 6.5 to
7.5. Incubation with the Cottonii sulfohydrolase is preferably
conducted at a pH of about 8 to 9.5.
[0196] The concentration of total proteins of the samples
containing the sulfohydrolases is preferably at least about 2 to 85
.mu.g/ml, more preferably about 10 to 80 .mu.g/ml, and most
preferably about 25 to 70 .mu.g/ml. In the case of almost pure
sulfohydrolase II, the concentration is preferably 1 to 10
.mu.g/ml, more preferably about 2.5 to 10 .mu.g/ml, and most
preferably about 2.5 to 5 .mu.g/ml.
[0197] The concentration of the substrate, e.g., carrageenan, in
the reaction mixture is preferably about 0.012 to 2% (w/v), more
preferably about 0.12 to 2% (w/v), and most preferably about 0.7 to
1.75% (w/v).
[0198] For incubation with Cottonii extract, the optimal conditions
in which the desulfatation reaction should be performed are as
follows:
[0199] Substrate: mu-carrageenan at a final concentration of about
1.5% (w/v)
[0200] Activity/stability as a function of temperature: 9 hours
incubation at 48.degree. C. releases as much free sulfate as
incubation for 16 hours at 37.degree. C. or 48 hours at 20.degree.
C.
[0201] Activity/stability as a function of pH: Tris-HCl 100 mM (pH
9.0)+2-mercaptoethanol 10 mM or Bis-tris 100 mM (pH 8.5)+10 mM
2-mercaptoethanol seem to be the most appropriate buffers for the
sulfohydrolase of Cottonii
[0202] Amount of proteins: 5 .mu.g of total proteins is the minimum
amount of material required to get a significant signal on the
Dionex.
[0203] The purified sulfohydrolases of the present invention are
effective in modifying sulfated compounds, e.g., carrageenan. For
instance, 1 mg of enzyme extract in accordance with the present
invention (in this case 0-30% acetone fraction) is able to release
400 nmoles of sulfate per hour.
[0204] Sulfohydrolase I and II may be used separately, or in
combination, to modify substrates, and in particular to
specifically tailor the number and distribution of sulfate groups
and/or anhydro-bridges in carrageenan. The high purity
sulfohydrolases of the present invention are especially useful in
tailoring properties.
[0205] Possibly, the most direct application of this discovery is
substitution of enzymatic modification for existing chemical
modification processes where comparable (or better) functionality,
e.g., gelling properties, viscosity, texture in food, can be
achieved with enzymatic modification.
[0206] For example, a carrageenan that contains precursor and
therefore does not gel, can be induced to gel enzymatically.
[0207] As another example, a nu-carrageenan may be converted into
an iota-carrageenan to form a product that does not gel under
conditions wherein the product has a concentration of about 0.7%
(w/v) in an aqueous solution having 0.1 M of potassium at a
temperature of 65.degree. C. and a pH of 7.
[0208] In addition, and by way of non-limiting example only, the
functionality of these enzymes may have the following applications:
selective control of the induction of 3,6-anhydrogalactose
residues; tailoring those properties of carrageenans which are
related to the number and distribution of both the sulfate groups
and 3,6-anhydrogalactose residues; inducing modification and
gelation of iota-precursor in aqueous systems (including cold
gelation of cold-soluble carrageenan solutions and pastes, with
representative use including food, pharmaceutical, textile, and
personal hygiene products, further including special uses such as
dairy products, especially powder formulations, toothpaste, and
other paste or slurry products); control of the production of iota
carrageenan for gelatin replacement (as the enzyme is expected to
give an incomplete modification in comparison with the current
alkaline process it will allow better control of the gelatin-like
properties which depend on such incomplete modification);
modification of only iota-precursor in kappa-iota-precursor
mixtures, as for example neutral extracts of cold water seaweed
specifies, which may give lower gel strength but increased
water-binding capacity as well as other new characteristics; and
use with other galactan structures having 6-sulfate groups,
including agaroids, as well as with homologous nucleic acids and
proteins in carrageenophytes and agarophytes.
[0209] The precursor-containing carrageenan may be used by
injection into or to coat uncooked meat, e.g., chicken, salmon. If
such enzymes are included in the mixture, they will make the
carrageenan gel, so that when the meat is cooked, juices are
prevented from leaking out of the meat.
[0210] Further possible uses include the following:
[0211] preparation of high molecular weight carrageenan with high
viscosity and high gel strength by using mild conditions for
extraction of the carrageenan, followed by use of the enzymes for
modification of the carrageenan; and
[0212] gelation at or near ambient temperature (cold gelation),
reducing or eliminating the need for heating and/or cooling steps
in processes, such as yogurt production, that require the
production of a gel.
[0213] The present invention will be further illustrated by way of
the following Examples. These examples are non-limiting and do not
restrict the scope of the invention.
[0214] Unless stated otherwise, all percentages, parts, etc.
presented in the examples are by weight.
EXAMPLES 1-4
[0215] These Examples are directed to studying the specificity of
C. crispus sulfohydrolase extracts on different types of
carrageenans.
[0216] In order to study the specificity of the Chondrus
sulfohydrolases, nu-, mu-, iota- and kappa-carrageenans were used
as substrates. The iota- and kappa-carrageenans were isolated from
their seaweed sources by alkaline extraction giving complete
3,6-anhydrogalactose formation. The precursor-rich carrageenans,
containing mu-, nu-, or lambda-carrageenan, were isolated by
neutral extraction with water. The lambda-carrageenan was extracted
from manually sorted tetrasporophytes. All carrageenans were
recovered by precipitation in isopropyl alcohol. These
carrageenans, as listed in Table 1 below, were provided by Hercules
Incorporated, Lille Skensved, Denmark.
7TABLE 1 Seaweed Source Seaweed Trade Sulfate Content Carrageenan
Botanical Name Name (% (w/w)) Nu Eucheuma denticulatum Spinosum 33
Mu Kappaphycus alvarezii Cottonii 23 Iota Eucheuma denticulatum
Spinosum 33 Kappa Kappaphycus alvarezii Cottonii 20
[0217] Before being used, the carrageenans were dialyzed overnight
against 10 liters of 50 mM Tris-HCl (pH 7.1) buffer containing 10
mM 2-mercaptoethanol, using a Spectra/por membrane MWCO 6-8000 or
3500, available from Spectrum Laboratories, Inc., Rancho Dominguez,
Calif., USA. The final concentration of the carrageenans, after
dialysis was about 1.25% (w/v).
[0218] Gametophyte plants of C. crispus were frozen in liquid
nitrogen and ground automatically to pieces which were less than 1
mm long using a Forplex miller, available from Forplex Industrie,
Boulogne Billancourt, France. This "powder" was used immediately or
kept at -20.degree. C. or -80.degree. C. until further use.
[0219] C. crispus extracts were prepared in the following manner.
Fifteen (15) g of the milled C. crispus were extracted with 4
volumes (i.e., 60 ml) of 50 mM Tris-HCl buffer (pH 9.5) containing
10 mM 2-mercaptoethanol and 500 mM KCl. The supernatant was then
fractionated with cold (-20.degree. C.) acetone: 2 steps of
fractionation were performed: from 0 to 30% saturation and from 30
to 60% saturation of acetone. In this regard, the volume of acetone
to add to z ml of sample, when going from A % to B % of cold
acetone saturation is determined as follows: z (B-A)/(100-B).
[0220] After each fractionation step, a centrifugation was
performed at 4.degree. C. for 30 min. at 10,000.times.g. The
pellets were dissolved in 2 ml of 50 mM Tris-HCl buffer (pH 7.1)
containing 10 mM 2-mercaptoethanol. The pellets as well as an
aliquot of each of the supernatants were then dialyzed overnight at
4.degree. C. against 3.times.3 liters of 50 mM Tris-HCl buffer (pH
7.1) containing 10 mM 2-mercaptoethanol, using a Spectra/por
membrane MWCO 6-8000 or 3500, available from Spectrum Laboratories,
Inc., Rancho Dominguez, Calif., USA.
[0221] From these extracts, reaction mixtures for screening of
activity were made to have the compositions shown in Table 2
below.
8 TABLE 2 Component Control Sample Carrageenan, as listed in Table
3 (1.25% 500 .mu.l 500 .mu.l (w/v) in 50 mM Tris-HCl (pH 7.1) + 10
mM 2-mercaptoethanol) 50 mM Tris-HCl (pH 7.1) + 10 mM 2- 500 .mu.l
250 .mu.l mercaptoethanol Chondrus extract 0 .mu.l 250 .mu.l
[0222] As summarized in Table 3 below, the various carrageenans
described above were incubated for 40 hours at room temperature at
a pH of 7.1 with the Chondrus extracts obtained in the manner
described above. The amount of free sulfate for each Sample was
measured using the free sulfate assay method described in the
specification. Similarly, the amount of free sulfate was measured
by the same assay method for each corresponding Control which
included the same carrageenan but which did not include Chondrus
extract as shown in Table 2. The difference in free sulfate between
each Sample and its corresponding Control is the relative level of
sulfate released, as shown in Table 3 below.
9 TABLE 3 Relative Level of Sulfate Released (ppm) Example Extract
Nu Mu Iota Kappa 1 supernatant 101.0 15.3 6.4 6.7 (0-30% acetone) 2
pellet 48.1 7.1 2.4 3.2 (0-30% acetone) 3 supernatant 72.8 16.5 4.3
6.2 (30-60% acetone) 4 pellet 40.6 8.7 2.6 2.0 (30-60% acetone)
[0223] As shown in Table 3, Chondrus extract is active on the
nu-carrageenan. The first supernatant (0-30% acetone saturation)
and the second supernatant (30-60% supernatant) also display some
activity on the mu-carrageenan but the activity on this substrate
is relatively low as compared with the activity on the
nu-carrageenan.
EXAMPLES 5-38
[0224] As shown in the following Examples, in order to determine
the optimal conditions for the C. crispus sulfohydrolase, a partial
characterization of a fractionated C. crispus extract was performed
which included activity as a function of pH, temperature, enzyme
and substrate concentration. From these Examples, it appears that
the desulfatation of the nu-carrageenan should be performed for a
minimum of 6 to 15 hours at 48.degree. C. using a final substrate
concentration of 0.7% (w/v). These Examples also show that Chondrus
extract is characterized by a pH optimum around 7, using Tris-HCl
(50 mM, pH 7.1+10 mM 2-Mercaptoethanol) as buffer.
[0225] Unless otherwise stated, the nu-carrageenan listed in Table
1 was dissolved in Tris-HCl 50 mM (pH 7.1) buffer containing 10 mM
2-mercaptoethanol and dialyzed for 24 hr against 6.times.2 liters
of the buffer, using a Spectra/por membrane MWCO 6-8000 or 3500,
available from Spectrum Laboratories, Inc., Rancho Dominguez,
Calif., USA, in the same buffer. The substrate was used in the
reaction mixture at a final concentration of 0.6% (w/v).
[0226] To obtain sulfohydrolase extracts, C. crispus, which was
powdered in the manner described in Examples 1-4, was fractionated
with cold acetone at -20.degree. C. from 0 to 30% or from 30 to 60%
of acetone saturation.
[0227] The fractions were dialyzed for 16 hours in 4.times.2.5
liters of 50 mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol buffer.
The resulting C. crispus extract from 0-30% acetone saturation had
a protein concentration of about 170 .mu.g/ml, as measured by using
the Bio-Rad protein assay, available from Bio-Rad Laboratories
GmbH, Munich, Germany, with bovine serum albumin used as
standard.
EXAMPLES 5-13
[0228] These Examples are directed to determining the activity of
C. crispus extract on nu-carrageenan as a function of pH.
[0229] In these Examples, 300 mg of the nu-carrageenan were
dialyzed for 24 hr against 6.times.2 liters of MilliQ water,
available from Millipore Corporation, Bedford, Mass., USA, using a
Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum
Laboratories, Inc., Rancho Dominguez, Calif., USA. The final
concentration of carrageenan after dialysis was 1.1% (w/v).
[0230] Sample and Control reaction mixtures were prepared as shown
in Table 4 below.
10 TABLE 4 Component Control Sample Nu-carrageenan (1.1% (w/v) in
water) 500 .mu.l 500 .mu.l Buffer (100 mM Tris, Pipes, or Mes, as
250 .mu.l 250 .mu.l discussed below) 50 mM Tris-HCl pH 7.1 250
.mu.l 0 .mu.l Chondrus extract (in 50 mM Tris-HCl 0 .mu.l 250 .mu.l
pH 7.1) (0 to 30% acetone saturation)
[0231] Depending upon the pH required, as shown in Table 5 below,
the buffers used in the reaction mixtures were the following:
[0232] pH 5.5 to 6.5: 100 mM MES (2-(N-morpholino)ethanesulfonic
acid)+10 mM 2-mercaptoethanol;
[0233] pH 6.5 and 7.0: 100 mM Pipes (piperazine-N,N'
(2-ethanesulfonic acid)+10 mM 2-mercaptoethanol; and
[0234] pH 7.0 to 9.0: 100 mM Tris-HCl+10 mM 2-mercaptoethanol.
[0235] The pH of the buffer was varied by adding HCl in the case of
Tris-HCl or by adding NaOH in the cases of MES and Pipes.
[0236] The reaction mixtures were incubated for 26 hr at 29.degree.
C. After incubation, the amount of sulfate released was measured in
accordance with the free sulfate assay method described in the
specification. By comparing the amount of free sulfate in the
Samples and their corresponding Controls, the relative amount of
free sulfate, shown in Table 5 below, was calculated.
11 TABLE 5 Relative Level of Sulfate Released (ppm) % Relative
Example pH MES Pipes Tris-HCl Activity 5 5.5 2.7 6.6 6 6 20.0 48.7
7 6.5 31.2 75.9 8 6.5 29.8 72.5 9 7 32.6 79.3 10 7 41.1 100 11 8
32.1 78.1 12 8.5 23.0 56.0 13 9 23.5 57.2
[0237] Table 5 shows that the pH optimum of the C. crispus extract
was about 7.
EXAMPLES 14-21
[0238] These examples are directed to determining the activity of
C. crispus extract on nu-carrageenan as a function of quantity of
protein, including sulfohydrolase, in the extract.
[0239] Sample and Control reaction mixtures were prepared in the
proportions shown in Table 6 below.
12TABLE 6 Chondrus extract Nu-carrageenan (1.25% (w/v) (170
.mu.g/ml of 50 mM Tris-HCl in 50 mM Tris-HCl (pH 7.1) + 10 mM
protein) (0 to 30% (pH 7.1) + 10 mM Ex. 2-mercaptoethanol) acetone
saturation) 2-mercaptoethanol Control 500 .mu.l 0 .mu.l 500 .mu.l
14 500 .mu.l 50 .mu.l 450 .mu.l 15 500 .mu.l 100 .mu.l 400 .mu.l 16
500 .mu.l 150 .mu.l 350 .mu.l 17 500 .mu.l 200 .mu.l 300 .mu.l 18
500 .mu.l 250 .mu.l 250 .mu.l 19 500 .mu.l 300 .mu.l 200 .mu.l 20
500 .mu.l 400 .mu.l 100 .mu.l 21 500 .mu.l 500 .mu.l 0 .mu.l
[0240] The above Control and Sample mixtures having different
concentrations of sulfohydrolase extract were incubated for 30 hr
at 30.degree. C. at a pH of 7.1. After incubation, the amount of
sulfate released was measured in accordance with the free sulfate
assay method described in the specification. By comparing the
amount of free sulfate in the Samples and their corresponding
Controls, the relative amount of free sulfate, shown in Table 7
below, was calculated.
13TABLE 7 Relative Level of Sulfate Amount of protein Released in
30 hours Example (.mu.g) (ppm) 14 8.5 14.1 15 17 24.5 16 25.5 34.2
17 34 40.2 18 42.5 54.6 19 51 53.1 20 68 74.7 21 85 65.2
[0241] Table 7 shows, as expected, that increasing the amount of
enzyme preparation increases the amount of sulfate released.
EXAMPLES 22-32
[0242] These examples are directed to determining the activity of
C. crispus extract as a function of nu-carrageenan
concentration.
[0243] Control and Sample reaction mixtures were made in the same
manner as described in Table 1 above, except that the volume of the
reaction mixtures was reduced to 500 .mu.l, i.e., the volume of all
components was reduced by 50%, and except that the 30-60% acetone
saturation Chondrus extract was used.
[0244] In order to determine the optimal concentration of nu
carrageenan to use during the desulfatation reaction, 12 different
concentrations of nu carrageenan, ranging from 0 to 2.5% (w/v)
(final concentrations) were tested, as shown in Table 8 below.
[0245] After incubation for 15 hours at 37.degree. C., the amount
of sulfate released was measured in accordance with the free
sulfate assay method described in the specification. By comparing
the amount of free sulfate in the Samples and their corresponding
Controls, the relative amount of free sulfate, shown in Table 8
below, was calculated.
14TABLE 8 Relative Level of Nu-carrageenan in reaction mixture
Sulfate Released % relative Ex. (final % (w/v)) (ppm) activity 22
0.37 40.0 81.3 23 0.54 47.3 96.0 24 0.7 49.2 100 25 0.85 43.8 88.9
26 1.03 44.6 90.5 27 1.12 43.0 87.3 28 1.27 19.0 38.6 29 1.45 9.0
18.3 30 1.76 39.8 80.8 31 2.07 29.1 59.1 32 2.49 33.1 67.2
[0246] Table 8 shows that 0.7% (w/v) of nu-carrageenan (final
concentration in the reaction mixture) is the optimal concentration
of substrate to use for the desulfatation reaction by the nu
sulfohydrolase.
EXAMPLES 33-38
[0247] These examples are directed to determining the activity of
C. crispus extract on nu-carrageenan as a function of
temperature.
[0248] Control and Sample reaction mixtures were made in the same
manner as described in Examples 1-4 above, except that the 30-60%
acetone saturation Chondrus extract was used.
[0249] Reaction mixtures were incubated at 0, 13, 20, 30, 37 and
48.degree. C. at a pH of 7.1. In order to measure the amount of
free sulfate released as a function of time, small aliquots (about
0.2 ml) of reaction mixture were taken after 0, 2, 8, 26, 46 and 72
hr of incubation at the above mentioned temperatures.
15 TABLE 9 Relative Level of Sulfate Released (ppm) Temperature 46
72 Ex. (.degree. C.) 0 hours 2 hours 8 hours 26 hours hours hours
33 0 0.2 1.8 2.0 0 7.2 11.6 34 13 0 2.7 9.0 15.2 29.2 27.0 35 20
0.2 4.9 13.6 26.1 44.7 47.5 36 30 0.25 7.1 13.2 39.7 61.8 51.6 37
37 0.3 9.0 22.9 44.6 77.8 58.6 38 48 0.3 14.6 34.8 -- -- --
[0250] Table 9 shows that the amount of sulfate released from the
nu-carrageenan increases with the temperature. In particular,
48.degree. C. seems to be the optimal temperature for the crude
extract. In fact, an incubation of 8 hours at 48.degree. C.
releases as much free sulfate as a 40 hour incubation at room
temperature.
EXAMPLES 39-46
[0251] These examples are directed to studying the specificity of
pure, based on total protein content, C. crispus sulfohydrolase II
on different types of carrageenans.
[0252] Pure, based on total protein content, C. crispus
sulfohydrolase II was isolated by using the protocol described in
the specification. In particular, the fractions of pure C. crispus
sulfohydrolase II elute from the DEAE chromatography column with a
concentration of NaCl between 650 and 850 mM.
[0253] The specificity of the pure sulfohydrolase II was tested on
different types of carrageenans. In particular, Control and Sample
reaction mixtures as shown in Table 10 below, were made by using
the carrageenans shown in Table 11. Thus, the pure sulfohydrolase
II was tested on a nu enriched iota-carrageenan (shown as
preparation method "E" in Table 11). The pure sulfohydrolase II was
also tested on enzyme resistant fractions of nu enriched iota- and
iota-carrageenan incubated with iotase, as described below (shown
as preparation method "ER" in Table 11). The pure sulfohydrolase II
was further tested on neutral extracted iota- and
kappa-carrageenan, which was prepared as described below (shown as
preparation method "N" in Table 11). Still further, the pure
sulfohydrolase II was tested on the nu enriched iota- and mu
enriched kappa-carrageenan which were made as described below
(shown as preparation method "C" in Table 11).
[0254] The preparation method "E" of Table 11 for preparing nu
enriched iota-carrageenan was as follows. Fifteen (15) kg of
Eucheuma denticulatum was washed 3 times in 80% (w/v) of isopropyl
alcohol, dried in a drying chamber at 60.degree. C. until constant
weight and milled to 0.5 mm particle diameter. The milled seaweed
was extracted at 50.degree. C. for 4 hours in 1000 liter of 0.125 M
KCl/K.sub.2CO.sub.3 (9.15 kg KCl and 0.17 kg). During the
extraction, the pH was measured to ensure that it was between 8 and
9. If the pH was lower than 8, an appropriate amount of
K.sub.2CO.sub.3 was added.
[0255] The extract liquid was filtered on kiselgur-filter, and the
extract juice was washed in UF (ultrafiltration) with tap water
until the conductivity was at 2 mS/cm (<2000 .mu.S/cm) or lower
and stable. Then, the juice was concentrated to about 65-70 liter
total. The pH was adjusted with 0.1 M NaOH to approximately 8.5. In
this regard, 1-10 deciliter of the 0.1 M NaOH was added with a
pipette while stirring and closely monitoring the pH. A
precipitation test was conducted in 100% (w/v) isopropyl
alcohol--and if precipitate is formed, all is precipitated. If
precipitation is poor, the juice was concentrated by evaporation
under vacuum and pH and temperature are closely followed. The juice
must not exceed 9.2 in pH. In some cases, it was necessary to let
the carrageenan precipitate overnight. The precipitated carrageenan
was washed a couple of times in 100% (w/v) isopropyl alcohol. All
steps were conducted at room temperature. The carrageenan was
freeze-dried. NMR results indicated that the precursor level was
enriched to about 32% of total weight.
[0256] The iota-carrageenan was prepared in the same manner as the
iota-carrageenan shown of Table 1 above.
[0257] The preparation of enzyme resistant fractions (method "ER"
of Table 11) of the nu enriched iota- and iota-carrageenans was
carried out as follows. The starting material was either the nu
enriched iota-carrageenan was obtained via method "E" described
above, or the iota-carrageenan listed in Table 1.
[0258] With either of these starting materials, one gram of the
carrageenan to be made enzyme resistant was dissolved in 50 mM
tris-HCl (pH 7.0)+100 mM NaCl and 5 mM CaCl.sub.2. The resulting
digestion mixture was incubated for 4 hours at 37.degree. C. by
additions of 0.2 U of iota carrageenase, which had been purified in
accordance with POTIN et al., "Purification and Characterization of
a New K-Carrageenase from a Marine Cytophaga-like Bacterium", Eur.
J. Biochem., 201, pp. 241-247 (1991), the disclosure of which is
herein incorporated by reference in its entirety, per mg of
carrageenan with 2 hours intervals. One unit of enzyme activity (U)
is defined as the amount of enzyme which produces an increase of
0.1 absorbance unit at 237 nm per minute in the reducing sugar
assay in accordance with KIDBY et al., "A Convenient Ferricyanide
Estimation of Reducing Sugars in the Nanomole Range", Analytical
Biochemistry, 55, pp. 321-325 (1973), the disclosure of which is
herein incorporated by reference in its entirety. The first enzyme
resistant fraction (ERF1) was obtained after precipitation with 2
volumes of ethanol 50% (v/v). After centrifugation for 20 min at
10,000.times.g and 4.degree. C., the pellet was collected and
considered as ERF1. Two (2) volumes of 95% (v/v) ethanol were then
added to the supernatant. After centrifugation under the same
conditions as before, the pellet was collected and named ERF2. ERF1
and ERF2 were separately redissolved in water and dialyzed
5.times.2 liters of 20 mM (NH.sub.4)HCO.sub.3, using a Spectra/por
membrane MWCO 6-8000 or 3500, available from Spectrum Laboratories,
Inc., Rancho Dominguez, Calif., USA. The ERF1 and ERF2 were then
freeze-dried.
[0259] The neutral extraction method (i.e., method "N" of Table 11
below) was as follows. Extraction of 200 g of seaweed, as listed in
Table 11, was conducted in 10 liters of tap water for 35 minutes at
110.degree. C., followed by filtration, concentration, and
precipitation in isopropyl alcohol, as described in THERKELSEN,
"Carrageenan", Industrial Gums: Polysaccharides and their
Derivatives, 3rd ed., pp. 145-180, (1993), the disclosure of which
is herein incorporated by reference in its entirety.
[0260] The preparation method "C" was in accordance with
preparation method "E", described above, except that the starting
material was Cottonii (=Kappaphycus alvarezii).
[0261] Referring to Table 11, the reaction mixtures were incubated
for 14 hours at a pH of 7.1 at 48.degree. C. The amount of sulfate
released was measured in accordance with the free sulfate assay
method described in the specification. By comparing the amount of
free sulfate in the Samples and their corresponding Controls, the
relative amount of free sulfate, shown in Table 11 below, was
calculated.
16TABLE 10 Component Control Sample Carrageenan, as listed in Table
5 (1.4% (w/v) in 200 .mu.l 200 .mu.l MilliQ water) Pure
sulfohydrolase II boiled for 10 minutes 200 .mu.l 0 .mu.l Pure
sulfohydrolase II 0 .mu.l 200 .mu.l
[0262]
17TABLE 11 Relative Level of Sulfate Preparation Carrageenan
Released Ex. Carrageenan Method Source (ppm) 39 Nu enriched iota E
E. spinosum 87.7 40 ERF1 ER E. spinosum 4.9 41 ERF2 ER E. spinosum
77.3 42 Neutral extracted iota N E. spinosum 43.8 43 Nu enriched
iota C G. skottsbergii 9.0 44 Neutral extracted iota N G.
skottsbergii 7.4 45 Mu enriched kappa C K. alvarezii 1.4 46 Neutral
extracted kappa N K. alvarezii 1.6
[0263] Table 11 shows, as expected from the screening study, that
the C. crispus sulfohydrolase II is not active on kappa-carrageenan
or on its precursor, i.e., mu-carrageenan. The C. crispus
sulfohydrolase II is only active on the neutral extracted
iota-carrageenan from Spinosum or its nu-enriched counterparts.
Surprisingly, ERF1 of this nu-carrageenan is not a good substrate
for sulfohydrolase II. While not wishing to be bound by theory, the
ERF1 is believed to contain relatively high molecular weight
nu-carrageenan. In contrast, sulfohydrolase II is active on ERF2.
While not wishing to be bound by theory, the ERF2 is believed to
contain relatively medium or small molecular weight nu-carrageenan.
The observed activity on ERF2 and the observed inactivity on ERF1
is not completely understood.
EXAMPLES 47-53
[0264] These examples are directed to studying the influence of
temperature on the activity/stability of pure, based on protein
content, C. crispus sulfohydrolase II on different types of
carrageenans.
[0265] Pure, based on protein content, C. crispus sulfohydrolase II
was isolated by using the protocol described in the specification.
In particular, fractions 59 to 61 from DEAE sepharose
chromatography were pooled and used as the source of the
sulfohydrolase II.
[0266] One volume of the pool of fractions 59 to 61 from the DEAE
sepharose chromatography was incubated with one volume of
nu-carrageenan, made in accordance with the procedures of Examples
5-12, 1.4% (w/v) in 50 mM tris-HCl+10 mM 2-mercaptoethanol (pH
7.1). Reference mixtures were prepared the same way except that the
enzyme (pure sulfohydrolase II) was boiled for 10 minutes under
atmospheric pressure prior to incubation with the
nu-carrageenan.
[0267] As shown in Table 12, reaction mixtures as well as reference
mixtures were incubated at different temperatures, from 0.degree.
C. to 60.degree. C. Aliquots were taken after 3, 8, 15, 24, 32, and
40 hours of incubation at the above-mentioned temperatures. Results
are expressed as the relative amount of sulfate released, measured
by using the assay described in the specification, as a function of
the period of incubation.
18 TABLE 12 Temp. Relative Level of Sulfate Release at Time (hours)
Ex. (.degree. C.) 3 8 15 24 32 40 47 0 0 0 1.7 2.1 3.9 3.2 48 12 0
0 6.0 6.1 10.3 12.9 49 20 2.4 4.7 13.6 14.0 25.3 30.5 50 30 4.7 --
21.4 29.0 32.3 27.0 51 37 9.0 15.7 34.3 41.2 53.5 35.4 52 48 --
26.6 55.7 73.0 97.2 97.7 53 60 15.2 16.3 14.6 33.3 35.3 --
[0268] As seen from Table 12, the sulfohydrolase appears to have
maximum activity around 48.degree. C. Above that temperature, there
is a loss of activity due to the denaturation of the enzyme.
EXAMPLES 54-95
[0269] These Examples are directed to characterizing the
sulfohydrolase activity of extracts from Kappaphycus alvarezii
(Cottonii).
[0270] Cottonii was provided alive by Genu Philippines, Cebu,
Philippines. The Cottonii was frozen in liquid nitrogen upon
arrival and kept at -80.degree. C. until further use. Prior to
extraction the Cottonii was ground manually to a fine powder in a
mortar, using liquid nitrogen which was poured with a baker into
the mortar to keep the Cottonii frozen during the grinding.
EXAMPLES 54-57
[0271] These Examples are directed to treating various carrageenans
with various Cottonii sulfohydrolase extracts to screen for
activity. Extracts of Cottonii were prepared as described in
Examples 1-4 for C. crispus. In this case, however, an additional
step of fractionation was performed as the supernatant was also
brought from 60 to 85% saturation in cold acetone. The amount of
acetone to add was calculated as described in Examples 1-4. After
each step of fractionation, centrifugation was performed as
described in Examples 1-4. The pellets were then redissolved in 50
mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol, before being dialyzed
overnight at 4.degree. C. against 3.times.3 liters of the above
mentioned buffer. The volume of samples after dialysis was about
1.5 ml.
[0272] Control and Sample reaction mixtures were made in accordance
with Examples 1-4, except that the extract was from Cottonii as
opposed to C. crispus. Thus, as shown in Table 13 below, in some
cases, raw extract from Cottonii was fractionated with acetone.
[0273] As shown in Table 13, incubation of the carrageenan was
performed for 40 hr at 25.degree. C. with various extracts. The
amount of released sulfate was measured using the assay method
described in the specification.
19 TABLE 13 Sulfate released in Cottonii various substrates (ppm)
Example Extract Nu Mu Iota Kappa 54 Raw 0 18.9 3.5 7.2 55 0-30%
acetone (pellet) 0 85.9 2.2 24.1 56 30-60% acetone (pellet) 58.9
37.1 0 1.6 57 60-85% acetone (pellet) 0 1 0 17
[0274] As shown in Table 13, even when it is not fractionated, the
extract of Cottonii is active toward the mu-carrageenan. The same
extract, fractionated from 0 to 30% or from 30 to 60% acetone shows
a higher activity toward the same substrate. One of the extracts
also shows activity toward nu-carrageenan.
EXAMPLES 58-75
[0275] These examples are directed to determining the activity of
the Cottonii sulfohydrolase as a function of mu-carrageenan
concentration.
[0276] A 30-60% acetone fraction (pellet) of Cottonii extract was
dialyzed overnight at 4.degree. C. against 3.times.3 liters of 50
mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol by using a MWCO 6-8000
or 3500 dialysis membrane available from Spectrum Laboratories,
Inc., Rancho Dominguez, Calif., USA.
[0277] Control and Sample reaction mixtures having the components
listed in Table 14 were prepared. To prepare these reaction
mixtures, two stock solutions were prepared. Stock 1 was 1.25%
(w/v) of mu-carrageenan and Stock 2 was 5% (w/v) mu-carrageenan,
both in 50 mM tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol. In Table
14, the buffer is 50 mM tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol.
The concentration of protein in the extracts was estimated to be
about 105 .mu.g/ml, using the Bio-Rad protein assay, available from
Bio-Rad Laboratories GmbH, Munich, Germany, with bovine serum
albumin used as standard.
20TABLE 14 Final Concentration Extract Stock 1 Stock 2 Buffer of Mu
Ex. (.mu.l) (.mu.l) (.mu.l) (.mu.l) (% (w/v)) Control 125 0 0 375 0
58 125 5 0 370 0.012 59 125 10 0 365 0.025 60 125 20 0 355 0.05 61
125 50 0 325 0.125 62 125 75 0 300 0.187 63 125 100 0 275 0.25 64
125 150 0 225 0.375 65 125 200 0 175 0.5 66 125 250 0 125 0.625 67
250 0 74 676 0.37 68 250 0 110 640 0.55 69 250 0 140 610 0.7 70 205
0 170 580 0.85 71 250 0 200 550 1 72 250 0 220 530 1.1 73 250 0 300
450 1.5 74 250 0 350 400 1.75 75 250 0 400 350 2
[0278] The Control and Sample reaction mixtures were incubated for
40 hours at 25.degree. C. at a pH of 7.1 with different
concentrations of mu-carrageenan as shown in Table 15, below. The
relative amount of sulfate released was measured using the assay
method described in the specification.
21TABLE 15 Final Concentration Relative Level of of Mu Sulfate
Released Example (% (w/v)) (ppm) 58 0.012 3.8 59 0.025 5.1 60 0.05
5.8 61 0.125 11.9 62 0.187 17.9 63 0.25 19.7 64 0.37 22.2 65 0.375
23.2 66 0.5 31.2 67 0.5 26.7 68 0.625 32.8 69 0.7 32.6 70 0.85 36.9
71 1 29.2 72 1.1 29.6 73 1.5 66.1 74 1.75 65.4 75 2 55.9
[0279] As seen from Table 15, around 1.75% (w/v) mu-carrageenan was
the optimal concentration of substrate to use for the desulfatation
reaction by the Cottonii extract. In fact at this concentration,
the amount of sulfate released was about twice as much as the
amount of sulfate released for mu-carrageenan concentrations of 0.5
to 1% (w/v).
EXAMPLES 76-81
[0280] These Examples are directed to determining the activity of
the Cottonii sulfohydrolase on mu-carrageenan as a function of
temperature.
[0281] A 0-30% acetone fraction (pellet) of Cottonii extract was
dialyzed overnight at 4.degree. C. against 3.times.3 liters of 50
mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol by using a MWCO 6-8000
or 3500 dialysis membrane available from Spectrum Laboratories,
Inc., Rancho Dominguez, Calif., USA.
[0282] Control and Sample reaction mixtures were made in the same
manner as Examples 54-57. Thus, the amount of the Cottonii extract
was 250 .mu.l. Additionally, the amount of mu-carrageenan solution
(1.25% (w/v) in 50 mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol) in
the Sample reaction mixtures was 500 .mu.l.
[0283] The incubations were conducted at a pH of 7.1 at the
different temperatures listed in Table 16, below. Aliquots of the
reaction mixture were removed at the different times listed in
Table 16, below, and the relative amount of sulfate released was
measured by using the method described in the specification.
22 TABLE 16 Relative Amount of Sulfate Released (ppm) Temperature
48 70 Example (.degree. C.) 3 hours 9 hours 27 hours hours hours 76
4 2.4 2.6 25.5 15.9 42.1 77 12 10.2 6.4 29.5 38.8 79.4 78 20 11
18.4 53.5 59.4 79.8 79 30 17.8 34.8 -- 61.7 90.4 80 37 24 49.7 74.4
73.2 90.4 81 48 11.9 61.8 81.2 77.2 105
[0284] Table 16 shows that although the extract is active at low
temperature (4.degree. C.), the mu sulfohydrolase extract from
Cottonii displayed a maximum of activity at 48.degree. C. In this
regard, it should be noted that 48.degree. C. was the maximum
temperature tested such that the extract may have higher activity
at even higher temperatures. In any case, at 48.degree. C.,
significant sulfate was released after 9 hours of incubation of the
enzyme with the mu-carrageenan.
EXAMPLES 82-95
[0285] These examples are directed to determining the activity of
the Cottonii sulfohydrolase on mu-carrageenan as a function of
pH.
[0286] The Cottonii extract used in these Examples was estimated to
have a concentration of proteins of about 105 .mu.g/ml, using the
Bio-Rad protein assay, available from Bio-Rad Laboratories GmbH,
Munich, Germany, with bovine serum albumin used as standard. Sample
and Control reaction mixtures were prepared as shown in Table 17
below.
23 TABLE 17 Component Control Sample Mu-carrageenan (1.2% (w/v) in
water) 250 .mu.l 250 .mu.l Buffer (100 mM Tris, Pipes, Mes, or 125
.mu.l 125 .mu.l Bis-Tris, as discussed below) 50 mM Tris-HCl pH 7.1
+ 10 mM 2- 125 .mu.l 0 .mu.l mercaptoethanol Cottonii extract (in
50 mM Tris-HCl pH 0 .mu.l 125 .mu.l 7.1 + 10 mM
2-mercaptoethanol)
[0287] In these Examples, the pH was varied generally in accordance
with the method of Examples 5-13 which are directed determining the
activity of the C. crispus sulfohydrolase. In addition to the
buffers described for C. crispus, in order to determine the pH
optimum, bis-tris buffer (100 mM bis tris+2-mercaptoethanol 10 mM)
was used (from pH 8.0 to 9.5). The bis-tris being
(bis[2-Hydroxyethyl]iminotris[hydroxymethyl]methane;2--
bis[2-Hydroxyethyl]amino-2-[hydroxymethyl]-1,3-propanediol),
available from Sigma Chemical, St. Louis, Mo., USA.
[0288] The reaction mixtures were incubated for 18 hr at 48.degree.
C. After incubation, the amount of sulfate released was measured in
accordance with the free sulfate assay method described in the
specification. By comparing the amount of free sulfate in the
Samples and the Control, the relative amount of free sulfate, shown
in Table 18 below, was calculated.
24 TABLE 18 Sulfate Released (ppm) Example pH MES Pipes Tris-HCl
Bis-Tris 82 5.5 1.94 83 6 19.8 84 6.5 49.8 85 6.5 58.0 86 7 58.1 87
7 47.4 88 7.5 54.4 89 8.2 56.5 90 8.2 73.2 91 8.5 69.0 92 8.5 71.6
93 9 85.6 94 9 18.8 95 9.5 41.0
[0289] Table 18 shows that the pH optimum of the mu-sulfohydrolase
is between 8.2 and 9.0, depending on the buffer used in the
reaction mixture. Thus, the optimal pH for the Cottonii extract is
more alkaline than the optimal pH for Chondrus crispus extract.
EXAMPLES 96-104 AND COMPARATIVE EXAMPLE 1
[0290] These Examples and Comparative Example are directed to
determining the activity of the Cottonii sulfohydrolase on
mu-carrageenan as a function of quantity of proteins.
[0291] The concentration of proteins in the Cottonii extract was
estimated to be 105 .mu.g/ml for the Examples and Comparative
Example. Reaction mixtures in which the amount of protein was
varied were made in accordance with Table 19 below. The reaction
mixtures were incubated for 18 hours at 48.degree. C. at a pH of
7.1.
[0292] In Table 19, the buffer was 50 mM Tris-HCl pH 7.1+10 mM
2-mercaptoethanol. The amount of sulfate released, as measured in
accordance with the method described in the specification, is shown
in Table 19 below.
25TABLE 19 1.2% (w/v) Quantity Cottonii Mu-carrageenan of Sulfate
Extract Buffer in water Protein Released Example (.mu.l) (.mu.l)
(.mu.l) (.mu.g) (ppm) Comp. 1 0 250 250 0 1.9 96 5 245 250 0.52 5.7
97 10 240 250 1.05 7.2 98 20 230 250 2.10 10.4 99 50 200 250 5.20
17.9 100 75 175 250 7.90 26.2 101 100 150 250 10.50 30.8 102 150
100 250 15.70 50.2 103 200 50 250 21.00 66.8 104 250 0 250 26.20
84.5
[0293] Table 19 shows that the mu sulfohydrolase is an active
enzyme since as little as 5 .mu.g of total proteins was enough to
release a significant amount of sulfate.
EXAMPLE 105
[0294] This Example is directed to isolation and partial
purification of the mu-sulfohydrolase enzyme from Cottonii.
[0295] Fifty (50) g of fresh Cottonii provided by Genu Philippines,
Cebu, Philippines were frozen in liquid nitrogen and ground using a
Grinomix GM 200 miller, available from Retsch BV, Germany. The
powder was then allowed to thaw for about 60 hours at 4.degree. C.
in 1.5 volumes, i.e., 75 ml, of 50 mM Tris-HCl (pH 9.5)+10 mM
2-mercaptoethanol+500 mM KCl. The extract was centrifuged at
4.degree. C. at 24,000.times.g for 90 minutes.
[0296] The supernatant was then tested for sulfohydrolase activity
with 1.5% (w/v) nu- or mu-carrageenan in 50 mM Tris-HCl pH 7.1+10
mM 2-mercaptoethanol. Incubation with the Cottonii extract was
performed for 20 hours at 48.degree. C. The incubation mixture
contained 250 .mu.l 50 mM Tris-HCl+10 mM 2-mercaptoethanol at pH
7.1, 500 .mu.l of extract, and 500 .mu.l of substrate (1.5% (w/v)
in Tris-HCl (50 mM)+10 mM 2-mercaptoethanol at pH 7.1. The amount
of sulfate released was measured as described in the
specification.
[0297] Sulfate release was highest with the iota-precursor (80 ppm
after 20 hours) and lowest for the kappa-precursor (10-30 ppm after
20 hours). In particular, 11 ppm of free sulfate were released when
the substrate was the mu-carrageenan described in Table 1, whereas
29 ppm of free sulfate were released when the substrate was the
mu-carrageenan made in accordance with the "C" method described in
Examples 39-46.
[0298] To further purify the sulfohydrolase, the extract was
brought to 30% saturation of ammonium sulfate by adding 16.4 g of
ammonium sulfate/100 ml of sample. After 16 hours at 4.degree. C.,
the sample was then centrifuged at 24,000.times.g for 90 minutes at
4.degree. C. The supernatant (40 ml) was loaded at a flow rate of 3
ml/min on a Phenyl Sepharose 6 fast flow (high sub) column,
available from Amersham Pharmacia Biotech AB, Uppsala, Sweden,
previously equilibrated in 50 mM Tris-HCl (pH 8.7)+10 mM
2-mercaptoethanol+500 mM KCl+30% of saturation of ammonium sulfate.
The column was washed with this buffer until the absorbance at 280
nm was negligible. Then, the proteins were eluted using a
decreasing gradient in ammonium sulfate, as described below and in
Table 20:
26 Buffer A: 50 mM Tris-HCl (pH 8.7) + 10 mM 2-mercaptoethanol +
500 mM KCl + 30% of saturation of ammonium sulfate Buffer B: 50 mM
Tris-HCl (pH 8.7) + 10 mM 2-mercaptoethanol + 500 mM KCl Fraction
Size: 5 ml Flow Rate: 3 ml/min
[0299]
27TABLE 20 Time (min) % of Buffer A % of Buffer B 0 100 0 120 0
100
EXAMPLES 106-109 AND COMPARATIVE EXAMPLE 2
[0300] These Examples are directed to purification of
sulfohydrolase I and II from C. crispus.
[0301] Both sulfohydrolase I and II from Chondrus crispus were
purified to homogeneity. The extraction of the enzymes was started
with two batches of 0.6-0.7 kg of fresh seaweed, followed by two
runs on a Phenyl Sepharose column. Active fractions (in terms of
sulfate released) were pooled, dialyzed for 36 hours against
5.times.10 liters of 50 mM Tris-HCl buffer (pH 7.1) containing 10
mM 2-mercaptoethanol (buffer A), using a Spectra/por membrane MWCO
6-8000 or 3500, available from Spectrum Laboratories, Inc., Rancho
Dominguez, Calif., USA. The dialyzed fractions were then loaded on
to a DEAE Sepharose column, available from Amersham Pharmacia
Biotech AB, Uppsala, Sweden, giving the pure sulfohydrolase II.
Half of one fraction, which contains sulfohydrolase I as well as
many other proteins, was loaded to a HiTrap heparin column,
available from Amersham Pharmacia Biotech AB, Uppsala, Sweden,
giving the pure sulfohydrolase I.
[0302] The concentration of the pure sulfohydrolase I and II in the
fractions was very low and could not be determined using Bio-Rad
Protein assay available from Bio-Rad Laboratories GmbH, Munich,
Germany, with bovine serum albumin used as standard, as the amount
of pure sulfohydrolase I and II present in solution is below the
detection limit.
[0303] Reactions mixtures as shown in Table 21 below were incubated
for 14 hours at 48.degree. C. In these reaction mixtures, different
amounts of almost pure (about 80% (w/w) based on the total amount
of proteins) sulfohydrolase II having a concentration estimated to
be 30 .mu.g/ml using the Bio-Rad Protein assay, available from
Bio-Rad Laboratories, Munich, Germany, was incubated with
iota-precursor, obtained in accordance with method "E" described
above in Examples 39-46, giving a gel. In contrast, when the enzyme
was boiled at 100.degree. C. for 10 minutes prior to incubation,
incubation under the same conditions did not result in a gel. After
incubation, all the reaction mixtures were boiled for 10 minutes at
100.degree. C. in order to stop the reaction.
[0304] Comparative Example 2 serves as a control and was prepared
without addition of sulfohydrolase in the reaction mixture.
28TABLE 21 Nu-carrageenan Sulfohydrolase II, 30 .mu.g/ml Water
(1.4% (w/v) in water) Examples (.mu.l) (.mu.l) (.mu.l) Comp. 2 0
1500 1500 106 100 1400 1500 107 250 1250 1500 108 500 1000 1500 109
1000 500 1500
[0305] The gel strength and complex viscosity of the incubated
samples were measured as shown in Table 22. The measurements in
Table 22 are based on a temperature sweep in which the temperature
was decreased from 80 to 10.degree. C. and then increased from 10
to 80.degree. C. in sequence. A deformation is applied to the
sample at various times during the sweep and the resulting force is
measured. The testing was conducted on a Bohlin VOR Rheometer,
having a cup-bob system (C14), available from Bohlin Reologi AB,
Sjobo, Sweden. The torque element was 0.24 gcm, with a frequency of
0.5 Hz, and an amplitude of 10+80%.
[0306] The force is converted into an elastic and a viscous modulus
(G' and G", respectively) and a complex viscosity (.rho.*). Gel T.
is the temperature where G' crosses G" and is higher than G", when
running a sweep where the temperature is decreased. Melt T. is the
temperature where G" is higher than G' when running a sweep where
the temperature is increased. The gel strength is defined as G' gel
or G' melt and is G' obtained at 10.degree. C. The complex
viscosity (.eta.*) is the viscosity at 10.degree. C.
29TABLE 22 Gel Melt G' gel G' melt .eta.* gel .eta.* melt T. T. at
10.degree. C. at 10.degree. C. at 10.degree. C. at 10.degree. C.
Ex. (.degree. C.) (.degree. C.) (Pa) (Pa) (Pa .multidot. s) (Pa
.multidot. s) Comp. 2 -- -- -- -- 0.0041 0.0346 106 -- -- 4 0.124
0.097 0.135 107 25.3 35.5 38 46 11.65 17.38 108 51.4 58.4 76 69
24.54 21.868 109 64.4 68.1 39 39 12.19 12.16
[0307] Table 22 shows that the gel strength and the complex
viscosity increase with increasing enzyme concentration and appears
to reach a maximum at approximately 5 .mu.g/ml, after which the gel
strength and complex viscosity decrease and become like an alkali
treated iota-carrageenan. A certain amount of sulfohydrolase is
needed to make a measurable gel, about 1 .mu.g/ml. Low amounts (2.5
.mu.g/ml) of enzyme created a soft gel whereas higher amounts (5
and 10 .mu.g/ml) gave a stronger gel.
EXAMPLE 110 AND COMPARATIVE EXAMPLES 3-5
[0308] These Examples are directed to incubations of iota- and
nu-carrageenan with partially purified C. crispus extracts.
[0309] The iota-carrageenan and nu-carrageenan in Table 24 were
both extracted from Eucheuma denticulatum (Spinosum), as described
below.
[0310] Comparative Example 3 serves as a control and involves
iota-carrageenan, which is a commercial extract, and which has been
treated with calcium hydroxide to fully remove 6-O-sulfate groups
and serves as a control.
[0311] The nu-carrageenan for Comparative Examples 4-5 and the
Examples was extracted in the following manner. Twenty-five (25)
grams of freeze-dried and milled Spinosum dispersed in 2.5 liters
of 0.125 M KCl. The extraction was at room temperature for 24
hours. Filtration was conducted with Frisenette paperfilters
614-200 mm, available from A. G. Frisenette & s.o slashed.nner
APS, Ebeltoft, Denmark. As a result of clogging, the filters needed
to be replaced and a total of three filters was used. The liquid
was stored in refrigerator (5.degree. C.) overnight. The liquid was
then evaporated on Roto-Evaporator RE 120, available from
Laboratoriums Technik AG, Flawil, Switzerland, at 2-3 rpm to 50%
volume with waterbath at 40.degree. C. The pH was adjusted to
approximately 8.0 by adding 6 drops of 1 M NaOH. The carrageenan
was precipitated using 100% (v/v) isopropyl alcohol (IPA),
available from BP Chemicals Ltd., UK and freeze-dried. The
nu-carrageenan was manually broken into pieces having a maximum
size of 1 cm.sup.2 and dispersed in distilled water at a level of
1% (w/v) of nu-carrageenan. The nu-carrageenan was dialyzed against
tap water, using a Spectra/por membrane MWCO 6-8000 or 3500,
available from Spectrum Laboratories, Inc., Rancho Dominguez,
Calif., USA, until the conductivity in the water was 1.5 .mu.S/cm.
The pH of the nu-carrageenan solution was adjusted to 9.3, and then
the solution was precipitated and freeze-dried. The freeze-dried
composition was milled in a mortar. This extract had both enriched
nu-content and a high molecular weight.
[0312] Results are also shown for nu-carrageenan incubated with a
partially purified sulfohydrolase-containing fraction from Chondrus
crispus. The purification protocol was generally the same as that
shown in the specification, i.e., ammonium sulfate fractionation,
phenyl sepharose chromatography, except that Poros HQ
chromatography was conducted instead of DEAE Sepharose
chromatography. The Poros HQ (4.6.times.100 mm) chromatography
column being available from PerSeptive Biosystems, Foster City,
Calif., USA. The Poros HQ chromatography involved the same buffers
as used in the DEAE Sepharose chromatography, but elution was
performed as follows and as shown in Table 23:
30 Buffer A: 50 mM Tris-HCl + 10 mM 2- mercaptoethanol pH 7.1
Buffer B: 50 mM Tris-HCl + 10 mM 2- mercaptoethanol + 1 M NaCl pH
7.1 Flow Rate: 2 ml/min Fraction Size: 2 ml
[0313]
31TABLE 23 Time (min) % of Buffer A % of Buffer B 0 100 0 60 0
100
[0314] Fractions 28 to 37, which contain sulfohydrolase activity,
were pooled and used as the enzyme fraction as described below.
[0315] The nu-carrageenan was incubated in a mixture containing:
1.5 ml of 1.4% (w/v, in 50 mM Tris-HCl (pH 7.1)+10 mM
2-mercaptoethanol) of the nu-carrageenan and 1.5 ml of enzyme
fraction in 50 mM Tris-HCl (pH 7.1)/10 mM 2-mercaptoethanol.
[0316] The reference mixture of Comparative Example 5 was made
using a fraction from the phenyl sepharose chromatography that did
not display sulfohydrolase activity, i.e., did not release sulfate
from the nu-carrageenan. Comparative Example 5 is directed to
determining whether gelation was the result of interaction with
proteins or whether gelation was the result of interaction with
sulfohydrolase.
[0317] After 20 hours incubation at 48.degree. C., carrageenan was
removed from an aliquot (about 0.2 ml) of the reaction mixture by
centrifugation at 3320 g for 1 hr at 30.degree. C. in a Microcon-10
unit, available from Amicon Bioseparations, Millipore Corporation,
Bedford, Mass., USA, in order to measure the amount of free sulfate
released as described in the specification. In contrast, the other
measurements (i.e., galactose, 3,6 AG, and viscosity measurements)
were directly performed on the reaction mixtures.
[0318] The main structural characteristics of nu-carrageenan
extracted from E. denticulatum (Spinosum), and of the same
nu-carrageenan incubated with some protein or with a partially
purified enzyme fraction containing both sulfohydrolases from C.
crispus, are summarized in Table 24.
[0319] The viscometric measurements of Table 24 were carried out on
the reaction mixture described above with the exception that after
incubation at 48.degree. C., the viscosity of the reaction mixture
was directly measured using a programmable Brookfield rheometer
model DV III, available from Brookfield Engineering Laboratories,
Stoughton, Mass., USA, thermostated at 48.degree. C. Measurements
were performed with a CP52 spindle for 10 minutes using a shear
rate of 120 rpm.
[0320] In Table 24, sulfohydrolase activity was assayed by
measuring the amount of sulfate released upon incubation of the
enzyme extract on the nu carrageenan. The amount of free sulfate
present in the filtrate was measured in accordance with the method
described in the specification.
32TABLE 24 3,6- Brookfield Galactose AnhydroGal Free Sulfate
Viscosity Ex. Content (mol %) (mol %) (% w/w) (cP) Comp. 3 iota- 50
50 0.6 Too high carrageenan (gel) Comp. 4 nu-carrageenan 68 32 NA
<5 Comp. 5 nu-carrageenan + 71 29 0.4 <5 protein 110
nu-carrageenan + 59 41 2.3 40 partially purified sulfohydrolase I
and II
[0321] Table 24 shows that in the presence of sulfohydrolases the
sulfate is released and that the 3,6 AG level increases which leads
to a gel being formed.
EXAMPLES 111-116 AND COMPARATIVE EXAMPLES 6-11
[0322] These Examples are directed to incubating nu-carrageenan
with separate fractions of sulfohydrolase I and II from C. crispus
to examine the effect on viscosity.
[0323] To obtain the sulfohydrolase I and II, the purification
procedure generally involved following the protocol described in
the specification. The only difference here being that the starting
material was 2 kg.
[0324] Fractions 21 and 40 were obtained from the same purification
experiment, whereas fractions 23 and 48 were obtained from a
separate run using the same procedure. Normally, when using 0.6-0.7
kg of seaweed, the sulfohydrolase I elutes first after DEAE
Sepharose chromatography and the sulfohydrolase II elutes last, as
was the case with fractions 23 and 48. However, in the purification
with a batch of 2 kg of seaweed, the order of fractions was once
reversed, such that sulfohydrolase II was in fraction 21 and
sulfohydrolase I was in fraction 40.
[0325] In Table 25, "21+nu", "23+nu", "40+nu", and "48+nu" indicate
a mixture of 1 ml of fraction 21, 23, 40, and 48, respectively (in
50 mM Tris-HCl+10 mM 2-mercaptoethanol pH 7.1), 2 ml of MilliQ
water, and 3 ml of 1.4% (w/v, in MilliQ water) nu-carrageenan
obtained in accordance with method "E" of Examples 39-46, which was
incubated for 15 hours at 48.degree. C., and then inactivated at
100.degree. C. for 15 minutes. Controls involved mixtures having
the same treatment, except boiled enzyme, i.e., enzyme "@100" as
shown in Table 25, was used. "40+nu+21" and "23+nu+48" indicate a
mixture of 1 ml of fraction 40 and 23, respectively (in 50 mM
Tris-HCl+10 mM 2-mercaptoethanol pH 7.1), 2 ml of MilliQ water, and
3 ml of 1.4% (w/v, in MilliQ water) nu-carrageenan obtained in
accordance with method "E" of Examples 39-46, which was incubated
for 15 hours at 48.degree. C., then inactivated at 100.degree. C.
for 15 minutes, then to which is added 1 ml of fraction 21 and 48,
respectively (in 50 mM Tris-HCl+10 mM 2-mercaptoethanol pH 7.1),
and then incubated for 6 hours at 48.degree. C.
[0326] Results are summarized in Table 25 below.
33TABLE 25 Approx. Sulfate Viscosity Increase Fraction MW Release T
Mol % Ex. Number (kDa) Y/N ppm Y/N cP (.degree. C.) Iota 111 21 +
nu 35 Y 139 Y 12 40 92 (sulfo II) Comp. 6 21@100 + nu 35 7 N 0.0 40
77 (sulfo II) 112 23 + nu 62 Y 194 N 0.3 55 87 (sulfo I) Comp. 7
23@100 + nu 62 18 N 0.0 55 77 (sulfo I) 113 40 + nu 62 Y 69 N 0.0
40 83 (sulfo I) Comp. 8 40@100 + nu 62 16 N 0.0 40 76 (sulfo I) 114
48 + nu 35 Y 93 Y 5.6 55 83 (sulfo II) Comp. 9 48@100 + nu 35 12 Y
0.8 55 76 (sulfo II) 115 40 + nu + 21 62/35 Y 105 Y 29 40 95 (sulfo
I&II) 116 23 + nu + 48 62/35 Y ? Y 12 55 89 (sulfo
I&II)
[0327] Table 25 shows that after incubation of nu-carrageenan with
the enzymes, sulfohydrolase I did not increase viscosity of the
samples whereas sulfohydrolase II did increase viscosity.
[0328] Comparing Examples 113 and 115 shows that incubating the
iota-precursor, i.e., nu-carrageenan, with first the random enzyme
(sulfohydrolase I) followed by inactivation and thereafter
incubation with the processive enzyme (sulfohydrolase II), results
in a higher viscosity when compared to using the processive enzyme
alone.
[0329] The enzymatic modified samples were analyzed by NMR to
determine the mode of action of both enzymes. The data indicates
that both enzymes are sulfohydrolases, releasing sulfate from the
C-6 position of the iota-precursor (nu) and subsequently inducing
formation of the 3,6-anhydrobridge.
[0330] The gel strength and complex viscosity of the incubated
samples was measured as shown in Table 28. In Table 28, the sample
for Comparative Example 10 was made by dispersing 0.7% (w/v) of the
iota-carrageenan listed in Table 1 in 20 ml of ion exchanged water
containing 2 ml of 0.5% (w/v) of potassium chloride and 2 ml of
0.25% (w/v) of calcium chloride. The solution was heated to
80.degree. C. and evaporated to 5 ml. The sample for Comparative
Example 11 was nu-carrageenan without enzyme.
[0331] The measurements in Table 28 are based on the methods
described in Tables 26 and 27. Table 26 shows the method for
determining the gelling profile, with a start temperature of
80.degree. C. Table 27 shows the method for determining the melting
profile, with a start temperature of 10.degree. C. Unless otherwise
noted in Tables 26-27, measurements were conducted using a Bohlin
VOR Rheometer, as described in Examples 106-109 and Comparative
Example 2.
34TABLE 26 Sample Ramp/ T Time Int. Freq. Amp. Mode Hold (.degree.
C.) (s) (s) (Hz) (%) oscillation ramp 61 1140 20 0.5 80 oscillation
ramp 10 3060 20 0.5 10
[0332]
35TABLE 27 Sample Ramp/ T Time Int. Freq. Amp. Mode Hold (.degree.
C.) (s) (s) (Hz) (%) oscillation ramp 58 2280 20 0.5 10 oscillation
ramp 80 1320 20 0.5 80
[0333]
36TABLE 28 .eta.* at .eta.* at Gel T. Melt T. 40.degree. C.
10.degree. C. G' Ex. component (.degree. C.) (.degree. C.) (cP)
(cP) (Pa) Comp. 10 iota 60.8 65.3 8372 12287 38.4 111 21 + nu 37.1
39.2 42 15120 47.3 113 40 + nu 22.5 28.8 4.3 6557 20.6 115 40 + nu
+ 21 49.4 56.8 3979 13560 42.4 Comp. 11 nu -- -- -- -- --
[0334] As shown in Table 28, the enzyme preparations were able to
influence the gelling and melting temperature of the polymer. The
viscosity of the enzymatic modified polymer was lower than the
traditional iota carrageenan. The difference in viscosity and
gelling point might be caused by ionic and concentration effects
and can be optimized for the enzymatic prepared samples.
[0335] While the invention has been described in connection with
certain preferred embodiments so that aspects thereof may be more
fully understood and appreciated, it is not intended to limit the
invention to these particular embodiments. On the contrary, it is
intended to cover all alternatives, modifications and equivalents
as may be included within the scope of the invention as defined by
the appended claims.
Sequence CWU 1
1
23 1 25 PRT Chondrus crispus 1 Lys Glu Gly Cys Glu Thr Ala Ile Val
Gly Ala Gly Ile Gly Gly Ala 1 5 10 15 Tyr Ser Ala Phe Arg Leu Ala
Ser Pro 20 25 2 14 PRT Chondrus crispus 2 Gly Leu Ile Gly Leu Asn
Asn Leu Val Thr Pro Met Glu Lys 1 5 10 3 17 PRT Chondrus crispus 3
Met Ser Gly Val Glu Gln Val Tyr Phe Asp Asp Leu His Ala Gln Ile 1 5
10 15 Lys 4 17 PRT Chondrus crispus 4 Glu Ile Arg Asp Arg Val Glu
Glu Leu Glu Lys Glu Glu Asp Tyr Ala 1 5 10 15 Lys 5 11 PRT Chondrus
crispus 5 Gly Arg Thr Asn Val Tyr Glu Leu Tyr Ala Lys 1 5 10 6 11
PRT Chondrus crispus 6 Trp Val Val Asn Ile Val Ile Asn Gly Val Arg
1 5 10 7 11 PRT Chondrus crispus 7 Gln Val Leu Glu Leu Glu Phe Thr
Val Val Arg 1 5 10 8 8 PRT Chondrus crispus 8 Leu Ser Pro Leu Thr
Phe Gln Arg 1 5 9 16 PRT Chondrus crispus UNSURE (9)..(9) any amino
acid 9 Ile Asn Asp Asn Leu Val Tyr Gln Xaa Gly Asn Leu Pro Ala Gly
Lys 1 5 10 15 10 13 PRT Chondrus crispus 10 Asn Tyr Asn Ile Gln Asn
Thr Asp Gly Ser Val Phe Arg 1 5 10 11 6 PRT Chondrus crispus 11 Met
Thr Val Glu Phe Gln 1 5 12 17 PRT Chondrus crispus 12 Ala Ala Arg
Gly Ala Arg Gly Gly Arg Thr Gly Tyr Gly Ala Arg Ala 1 5 10 15 Cys
13 20 PRT Chondrus crispus 13 Gly Thr Arg Thr Thr Tyr Thr Gly Asp
Ala Thr Arg Thr Thr Arg Thr 1 5 10 15 Ala Gly Thr Thr 20 14 20 PRT
Chondrus crispus 14 Ala Ala Asn Ala Cys Asn Tyr Trp Asn Cys Cys Arg
Thr Cys Ile Gly 1 5 10 15 Thr Gly Thr Thr 20 15 17 DNA Chondrus
crispus 15 attaaccctc actaaag 17 16 17 DNA Chondrus crispus 16
aatacgactc actatag 17 17 2020 DNA Chondrus crispus 17 gacagccctc
cccaacatgg ggctcaccta cgttcttttg tctgttcttg tcttacaagc 60
aacccacgca cttgccaaag agggatgcga gaccgctata gttggagccg gaattggtgg
120 cgcttattct gccttccgtt tggcaagccc atcagtctgt atctttgaag
ccaaccgtcg 180 cccaggaggg cgcatcttga ccgttcggga tccaagcgcc
tcgtttctga acttcactat 240 tgaccttggt gcgtatcgct accatcgtgc
acaccatcgt cttgtccgcc tcgttgctga 300 agacctactc aaccttcctg
tggcttgcta tacggacttg ctcaacaacc gaaaagattg 360 tccggacgcg
acgattcgtc tcttttcaac tcgagggaac gtgcttggag ctcttggagg 420
ccggattgct caagacttaa taaagaagta cggaccgttc ctgccatatg tgattcagag
480 gagctttcgg tggggacaag gaaagccctt gaaggaaaga cggacaatgt
ctgggttgct 540 aatcgggcca aactcagtaa tcaaagagat ccgagatcgc
gttgaggagc ttgagaaaga 600 ggaagactat gcgaaagcga tgcagattgc
ggacgaaatc attgcggcaa tgcaagatgg 660 ctcgtacaga ggcatcccgt
attcagagat cagtttgatg caagtcgcga tccgtgaagg 720 ctttacgaca
gaggagatgc aactggaaac ggacttctca tttctcagca gtgtggaaag 780
gcggcagacg ctagaataca acgggcagct ttcaatcagg caaatggcac tagaaaaggg
840 acttataggg cttaacaatc tagtgacgcc gatggagaag cggcgtggag
tgctacgtag 900 agcgggcatg atcacgctcg tggacggcct gcttgagcgg
gcgatgaagg gcggtgtgca 960 ggtacaatat gggaagaagg tcgtgagaat
tacgcgcacc ggaaatgaga agaggcctat 1020 aagactcaag ttcgaggacg
gcgggatggt agaagtgaag aacgtgattc tcaacattgg 1080 caagccgggg
ctgattgctc ttgggctgga ctcggagccg atgatgagca ccaaggagcc 1140
tttccggcga gcggtcgagc gaaactttgt gctgagctta tccaagacgt attgtttctg
1200 ggaagacgcg tggtggttga caaagttggg gcaacgggat gggcgtattc
aggttccttc 1260 agattcgatg cagtcaatgc gataccacga tggacacgtc
gtgtgcaagg acgagagaag 1320 gcttaaaagt tgccgtggcg gattgctcgc
gtcgtactcg gggggcgatc agatggggct 1380 tggggctgct ttgcatgcgc
acgtgcataa tgctaaaccg tacacaccgc tgacaagcag 1440 tgacaacgta
gtgaaattga ttccagggaa gatgtctgga gtagagcaag tatactttga 1500
tgacctgcac gcacaaatca agcgtgtgca caagaggtcg gtggagcgga aggggcttga
1560 cgtggacaag gtgatttcca agccggctat gtgcctattt gcggattggc
gggaggtggg 1620 tactcatgcg gccatggggc cgggaaaagg aagaacgaat
gtgtacgagt tgtacgctaa 1680 accggttagt gatttgagaa tcgcactggt
aaacgaagcg tggagtgggg accaagggtg 1740 ggcggagggg agcttgagaa
gcgcagagcg ggcgctgttc caccaattcg gaatggagaa 1800 gccagagtgg
atggataagg agtatcaccg gtcggtgatt gagaggtaca accaggggtg 1860
atttcggcgc gggcatacag tatgcgtgtc gttcgtgaag ttgtagcaaa gggctaatcc
1920 ttccacctgt cgtctcggcg caaagaataa aagctcgaag attagtggtg
acgttagaag 1980 taggcataca gaaccaaaaa aaaaaaaaaa aaaaaaaaaa 2020 18
1399 DNA Chondrus crispus Unsure (18)..(18) any nucleotide 18
aaatttaaag gaattttnaa aaaattttaa agcttaaatt tgccatttgc cattaaggtg
60 ggcaattgtt ggaaagcgat cggttgggcc tttttgtttt tacgcaagct
ggaaaagggg 120 attgtgctcc aaggcgatta agttgggtaa cgcaagggtt
ttcccagtca gacgttgtaa 180 aacgacggcc agtgaattgt aatacgactc
actatagggc gaattgggta ccgggccccc 240 cctcgagttt tttttttttt
ttttttttca gaggcgaaag gtgtcgttta ctgaaatctc 300 aagtacggga
gcaactccgc ccatctctac tcatcgttca ctacaagttg cagacactat 360
ttcacgctca tcacgaggta agggaaacgg tactcgatac ctttactcac tcctcttaga
420 cagtgaactc gagctcaaga acctgcctgc cgtccacgtt cgagcggccc
aacctgcgtt 480 caaccctgac tccgttgatc acaatgttga ctacccacct
tgtgcggtta ttgaagcagt 540 tgttgaaatg gaacagcttg acagtgtact
tccctggaac catgttcgtg aacgacacag 600 cttccttgcc ggccggcaag
ttgccgcact ggtacaccaa gttgtcattg atacgtcctc 660 cctgggagcc
cggcggccca tcaacggaaa ggtcgaaatc atcggacgaa tcccacgcca 720
cgtccaccac gatcggccgc acaaccgtct ggaatgagac gcagtccctg gcgtcggacg
780 tcgcgatcgt gcggaacacg gacccatcgg tgttctgaat gttgtagttg
cggatcggaa 840 gacggatgca gaggtcgtcg aggaagtcct cctggttacc
cacagggcta cggcgggaga 900 cacccctgag ttcgcgctgg aacgtgagag
gggaaagacg gaagtagttt ggcgggaacg 960 ggtggctcag tccgtttggc
ctgtacctgt tgatgggggt ggtgcgtgag cccgattcaa 1020 cgagggcaac
gccagttttg cggatgctgc cgagcgtccg gccgcccggg aagttacggc 1080
agacggatgc ctgcagcccg ggggatccac tagttctaga gcggccgcca ccgcggtgga
1140 gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
tcatggtcat 1200 agctgttttc tgtgtgaaat tggtatccgc tcacaattca
cacaacatac cagccccgaa 1260 acataaatgt taaagcctgg gtggctaatg
agtgagctta cttacattaa ttggnttggc 1320 nntacttgcc cctttttaaa
tcggggaaac ctttcngccn actntnttaa anaatcggcc 1380 aaccccccgg
gganagggg 1399 19 1387 DNA Chondrus crispus 19 tttttgccca
ttgcccatca aggtgggcaa tttttggaaa ggcgatcggg tggggccttt 60
tggtaattac gcaagctgca aaaggggaat gtgctgcaag ccgattaagt tgggtaacgc
120 cagggttttc ccagtcagaa cgttgtaaac gcgggcagtg aattgtaata
cgactcacta 180 tagggcgaat tgggtaccgg gccccccctc gagttttttt
tttttttttt tttttttttt 240 tttttttttt tttggagatg aaaggtgtcg
tttactgata ccaagtacgg cgcaactccg 300 cccatctcta ctcatcgtca
ctacaggttg caaacactat tccacgacca tgacgattta 360 agggaacgct
acttgggacc ttccactcat tcctcttaga cggtgaactc gagctcgaga 420
accctcctgc cgtccacatt cgaccgcccc aacctgcgtt gaattcttac cccgttgacc
480 acaacgttca ccacccactt cgtacgctta ttgaagcagt tgttgaagtg
gaacagcgtg 540 accgtgtact tccctcgaac catgttttcg aaggacacag
cttccttgcc ggccggagag 600 tcgccgcacg cggccaccaa gttgtcattg
atacggcctc ctacagatcc agggggacca 660 acaacggaaa ggtcgaagtc
atcggacgaa tcccacgcca cgtccaccac gatcggccgc 720 acaaccgtcc
ggaatgaaag gcagtccctg gcgtccgacg tcgagatcgt ccggaacaca 780
gtcccatccg tgttctgaat gttgtagttg cggatcggaa gacggatgca gaggtcgtcg
840 aggaactcct cctggttacc catggcgcca cgacgagcga cgcccttgcg
gtcgcgctgg 900 aatgtcagag gcgtcagacg gaagtagttt ctcgggaacg
cgggtctcag tccattaggc 960 cggtacctgt tgatcggcgt ggtgcctgag
cctgattcaa tgagcgccgc gccagtggtg 1020 cgaacgttgc cgagcgttct
cccgccgggg aagttacggc agatgaatgc tgagcgggag 1080 tagtctggcc
cgcggagaag gatcttcgca cccttgggac gcatggagcc atcagcgcgg 1140
cagaagcgga accggcatga gcggccgcat tgcgcgttcg ctgtgaaggc gatagcggcc
1200 aggagaagga cgaacaggaa tgaagcgttc attgttggta tgtgaagact
gccgcaccga 1260 aatgaaggct gctggtgccg aattcctgca gcccggggga
tccactagtt ctagagcggc 1320 cgccaccgcg gtggagctcc agcttttgtt
ccctttagtg agggttaatt tcgagcttgg 1380 cgtaatg 1387 20 1460 DNA
Chondrus crispus 20 atctcgaaat taaccctcac taaagggaac aaaagctgga
gctccaccgc ggtggcggcc 60 gctctagaac tagtggatcc cccgggctgc
aggaattcgg caccagcagc cttcatttcg 120 gtgcggcagt cttcacatac
caacaatgaa cgcttcattc ctgttcgtcc ttctcctggc 180 cgctatcgcc
ttcacagcga acgcccaatg cggccgctca tgccggttcc gcttctgccg 240
cgctgatggc tccatgcgtc ccaagggtgc gaaggtcctt ctccgcgggc cagactactc
300 ccgctcagca ttcatctgcc gtaacttccc tggcgggaga acgctcggca
acgttcgcac 360 cactggcgcg gcgctcattg aatcaggctc aggcaccacg
ccgatcaaca ggtaccggcc 420 caatggactg agacccgcgt tcccgagaaa
ctacttccgt ctgacgcctc tgacattcca 480 gcgcgaccgc aagggcgtcg
ctcgtcgtgg cgccatgggt aaccaggagg agttcctcga 540 cgacctctgc
atccgtcttc cgatccgcaa ctacaacatt cagaacaagg atgggtctgt 600
gttccggacg atctcgacgt cggacgccag ggactgcctt tcattccgga cggttgtgcg
660 gccgatcgtg gtggacgtgg cgtgggattc gtccgatgac ttcgaccttt
ccgttgttgg 720 tccccctgga tctgtaggag gccgtatcaa tgacaacttg
gtggccgcgt gcggcgactc 780 tccggccggc aaggaagctg tgtccttcga
aaacatggtt cgagggaagt acacggtcac 840 gctgttccac ttcaacaact
gcttcaataa gcgtacgaag tgggtggtga acgttgtggt 900 caacggggta
aggattcaac gcaggttggg gcggtcgaat gtggacggca ggagggttct 960
cgagctcgag ttcaccgtct aagaggaatg agtggaaggt cccaagtagc gttcccttac
1020 atcgtcatgg tcgtggaata gtgtttgcaa cctgtagtga cgatgagtag
agatgggcgg 1080 agttgcgccg cacttggtat cagtaaacga cacctttcat
ctctgttcga aaaaaaaaaa 1140 aaaaaaaact cgaggggggg cccggtaccc
aattcgccct atagtgagtc gaattacaat 1200 tcactggccg cgttttacaa
cgtcgtgact gggaaaaccc tgcgttaccc aacttaatcg 1260 ctttgaagac
aatccctttt tccaacttgg ggtaaaaacc aaaagggccc gcaccattcg 1320
cctttccaaa aatttgccca cccttaatgg caaaatggca aattttaagc cttaaatttt
1380 ttttaaaaat tcccgttaaa tttttttaaa ataacttatt tttttaacca
ataggcccaa 1440 atcgggaaaa tcccttttaa 1460 21 1068 DNA Chondrus
crispus 21 gaattcggca cgagcagcct tcatttcggt gcggcagtct tcacatacca
acaatgaacg 60 cttcattcct gttcgtcctt ctcctggccg ctatcgcctt
cacagcgaac gcccaatgcg 120 gccgctcatg ccggttccgc ttctgccgcg
ctgatggctc catgcgtccc aagggtgcga 180 agatccttct ccgcgggcca
gactactccc gctcagcatt catctgccgt aacttccccg 240 gcgggagaac
gctcggcaac gttcgcacca ctggcgcggc gctcattgaa tcaggctcag 300
gcaccacgcc gatcaacagg taccggccca atggactgag acccgcgttc ccgagaaact
360 tcttccgtct gacgcctctg acattccagc gcgaccgcaa gggcgtcgct
cgtcgtggcg 420 ccatgggtaa ccaggaggag ttcctcgacg acctctgcat
ccgtcttccg atccgcaact 480 acaacattca gaacaaggat gggtctgtgt
tccggacgat ctcgacgtcg gacgccaggg 540 actgcctttc attccggacg
gttgtgcggc cgatcgtggt ggacgtggcg tgggattcgt 600 ccgatgactt
cgacctttcc gttgttggtc cccctggatc tgtaggaggc cgtatcaatg 660
acaacttggt ggccgcgtgc ggcgactctc cggccggcaa ggaagctgtg tccttcgaaa
720 acatggttcg agggaagtac acggtcacgc tgttccactt caacaactgc
ttcaataagc 780 gtacgaagtg ggtggtgaac gttgtggtca acggggtaag
gattcaacgc aggttggggc 840 ggtcgaatgt ggacggcagg agggttctcg
agctcgagtt caccgtctaa gaggaatgag 900 tggaaggtcc caagtagcgt
tcccttacat cgtcatggtc gtggaatagt gtttgcaacc 960 tgtagtgacg
atgagtagag atgggcggag ttgcgccgca cttggtatca gtaaacgaca 1020
cctttcatct ctgttcgaaa aaaaaaaaaa aaaaaaaaaa aactcgag 1068 22 614
PRT Chondrus crispus 22 Met Gly Leu Thr Tyr Val Leu Leu Ser Val Leu
Val Leu Gln Ala Thr 1 5 10 15 His Ala Leu Ala Lys Glu Gly Cys Glu
Thr Ala Ile Val Gly Ala Gly 20 25 30 Ile Gly Gly Ala Tyr Ser Ala
Phe Arg Leu Ala Ser Pro Ser Val Cys 35 40 45 Ile Phe Glu Ala Asn
Arg Arg Pro Gly Gly Arg Ile Leu Thr Val Arg 50 55 60 Asp Pro Ser
Ala Ser Phe Leu Asn Phe Thr Ile Asp Leu Gly Ala Tyr 65 70 75 80 Arg
Tyr His Arg Ala His His Arg Leu Val Arg Leu Val Ala Glu Asp 85 90
95 Leu Leu Asn Leu Pro Val Ala Cys Tyr Thr Asp Leu Leu Asn Asn Arg
100 105 110 Lys Asp Cys Pro Asp Ala Thr Ile Arg Leu Phe Ser Thr Arg
Gly Asn 115 120 125 Val Leu Gly Ala Leu Gly Gly Arg Ile Ala Gln Asp
Leu Ile Lys Lys 130 135 140 Tyr Gly Pro Phe Leu Pro Tyr Val Ile Gln
Arg Ser Phe Arg Trp Gly 145 150 155 160 Gln Gly Lys Pro Leu Lys Glu
Arg Arg Thr Met Ser Gly Leu Leu Ile 165 170 175 Gly Pro Asn Ser Val
Ile Lys Glu Ile Arg Asp Arg Val Glu Glu Leu 180 185 190 Glu Lys Glu
Glu Asp Tyr Ala Lys Ala Met Gln Ile Ala Asp Glu Ile 195 200 205 Ile
Ala Ala Met Gln Asp Gly Ser Tyr Arg Gly Ile Pro Tyr Ser Glu 210 215
220 Ile Ser Leu Met Gln Val Ala Ile Arg Glu Gly Phe Thr Thr Glu Glu
225 230 235 240 Met Gln Leu Glu Thr Asp Phe Ser Phe Leu Ser Ser Val
Glu Arg Arg 245 250 255 Gln Thr Leu Glu Tyr Asn Gly Gln Leu Ser Ile
Arg Gln Met Ala Leu 260 265 270 Glu Lys Gly Leu Ile Gly Leu Asn Asn
Leu Val Thr Pro Met Glu Lys 275 280 285 Arg Arg Gly Val Leu Arg Arg
Ala Gly Met Ile Thr Leu Val Asp Gly 290 295 300 Leu Leu Glu Arg Ala
Met Lys Gly Gly Val Gln Val Gln Tyr Gly Lys 305 310 315 320 Lys Val
Val Arg Ile Thr Arg Thr Gly Asn Glu Lys Arg Pro Ile Arg 325 330 335
Leu Lys Phe Glu Asp Gly Gly Met Val Glu Val Lys Asn Val Ile Leu 340
345 350 Asn Ile Gly Lys Pro Gly Leu Ile Ala Leu Gly Leu Asp Ser Glu
Pro 355 360 365 Met Met Ser Thr Lys Glu Pro Phe Arg Arg Ala Val Glu
Arg Asn Phe 370 375 380 Val Leu Ser Leu Ser Lys Thr Tyr Cys Phe Trp
Glu Asp Ala Trp Trp 385 390 395 400 Leu Thr Lys Leu Gly Gln Arg Asp
Gly Arg Ile Gln Val Pro Ser Asp 405 410 415 Ser Met Gln Ser Met Arg
Tyr His Asp Gly His Val Val Cys Lys Asp 420 425 430 Glu Arg Arg Leu
Lys Ser Cys Arg Gly Gly Leu Leu Ala Ser Tyr Ser 435 440 445 Gly Gly
Asp Gln Met Gly Leu Gly Ala Ala Leu His Ala His Val His 450 455 460
Asn Ala Lys Pro Tyr Thr Pro Leu Thr Ser Ser Asp Asn Val Val Lys 465
470 475 480 Leu Ile Pro Gly Lys Met Ser Gly Val Glu Gln Val Tyr Phe
Asp Asp 485 490 495 Leu His Ala Gln Ile Lys Arg Val His Lys Arg Ser
Val Glu Arg Lys 500 505 510 Gly Leu Asp Val Asp Lys Val Ile Ser Lys
Pro Ala Met Cys Leu Phe 515 520 525 Ala Asp Trp Arg Glu Val Gly Thr
His Ala Ala Met Gly Pro Gly Lys 530 535 540 Gly Arg Thr Asn Val Tyr
Glu Leu Tyr Ala Lys Pro Val Ser Asp Leu 545 550 555 560 Arg Ile Ala
Leu Val Asn Glu Ala Trp Ser Gly Asp Gln Gly Trp Ala 565 570 575 Glu
Gly Ser Leu Arg Ser Ala Glu Arg Ala Leu Phe His Gln Phe Gly 580 585
590 Met Glu Lys Pro Glu Trp Met Asp Lys Glu Tyr His Arg Ser Val Ile
595 600 605 Glu Arg Tyr Asn Gln Gly 610 23 278 PRT Chondrus crispus
23 Met Asn Ala Ser Phe Leu Phe Val Leu Asn Leu Ala Ala Ile Ala Phe
1 5 10 15 Thr Ala Asn Ala Gln Cys Gly Arg Ser Cys Arg Phe Arg Phe
Cys Arg 20 25 30 Ala Asp Gly Ser Met Arg Pro Lys Gly Ala Lys Ile
Leu Leu Arg Gly 35 40 45 Pro Asp Tyr Ser Arg Ser Ala Phe Ile Cys
Arg Asn Phe Pro Gly Gly 50 55 60 Arg Thr Leu Gly Asn Val Arg Thr
Thr Gly Ala Ala Leu Ile Glu Ser 65 70 75 80 Gly Ser Gly Thr Thr Pro
Ile Asn Arg Tyr Arg Pro Asn Gly Leu Arg 85 90 95 Pro Ala Phe Pro
Arg Asn Phe Phe Arg Leu Thr Pro Leu Thr Phe Gln 100 105 110 Arg Asp
Arg Lys Gly Val Ala Arg Arg Gly Ala Asn Gly Asn Gln Glu 115 120 125
Glu Phe Leu Asp Asp Leu Cys Ile Arg Leu Pro Ile Arg Asn Tyr Asn 130
135 140 Ile Gln Asn Lys Asp Gly Ser Val Phe Arg Thr Ile Ser Thr Ser
Asp 145 150 155 160 Ala Arg Asp Cys Leu Ser Phe Arg Thr Val Val Arg
Pro Ile Val Val 165 170 175 Asp Val Ala Trp Asp Ser Ser Asp Asp Phe
Asp Leu Ser Val Val Gly 180 185 190 Pro Pro Gly Ser Val Gly Gly Arg
Ile Asn Asp Asn Leu Val Ala Ala 195 200 205 Cys Gly Asp Ser Pro Ala
Gly Lys Glu Ala Val Ser Phe Glu Asn Met 210 215 220 Val Arg Gly Lys
Tyr Thr Val Thr Leu Phe His Phe Asn Asn Cys Phe 225 230 235 240 Asn
Lys Arg Thr Lys Trp Val Val Asn Val Val Val Asn Gly Val Arg 245 250
255 Ile Gln Arg Arg Leu Gly Arg Ser Asn Val Asp Gly Arg Arg Val Leu
260 265 270 Glu Leu Glu Phe Thr Val 275
* * * * *
References