U.S. patent application number 10/448139 was filed with the patent office on 2003-11-06 for use of alpha-1,4-glucan lyase for preparation of 1,5-d-anhydrofructose.
This patent application is currently assigned to DANISCO A/S. Invention is credited to Bojko, Maja, Bojsen, Kirsten, Christensen, Tove, Kragh, Karsten, Marcussen, Jan, Nielsen, John, Yu, Shukun.
Application Number | 20030207409 10/448139 |
Document ID | / |
Family ID | 27547208 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207409 |
Kind Code |
A1 |
Yu, Shukun ; et al. |
November 6, 2003 |
Use of alpha-1,4-glucan lyase for preparation of
1,5-D-anhydrofructose
Abstract
A method of preparing the sugar 1,5-D-anhydrofructose is
described. The method comprises treating an .alpha.-1,4-glucan with
an .alpha.-1,4-glucan lyase wherein the enzyme is used in
substantially pure form. In a preferred embodiment, if the glucan
contains links other than and in addition to the .alpha.-1,4-links,
the .alpha.-1,4-glucan lyase is used in conjunction with a suitable
reagent that can break the other links.
Inventors: |
Yu, Shukun; (Malmo, SE)
; Bojsen, Kirsten; (Allerod, DK) ; Kragh,
Karsten; (Viby J, DK) ; Bojko, Maja;
(Gentofte, DK) ; Nielsen, John; (Copenhagen S,
DK) ; Marcussen, Jan; (Copenhagen K, DK) ;
Christensen, Tove; (Allerod, DK) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
DANISCO A/S
|
Family ID: |
27547208 |
Appl. No.: |
10/448139 |
Filed: |
May 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10448139 |
May 30, 2003 |
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09280197 |
Mar 29, 1999 |
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09280197 |
Mar 29, 1999 |
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08633719 |
Jul 8, 1996 |
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08633719 |
Jul 8, 1996 |
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PCT/EP94/03397 |
Oct 15, 1994 |
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Current U.S.
Class: |
435/105 ;
435/232 |
Current CPC
Class: |
C12N 9/00 20130101; C12N
9/88 20130101; C12P 19/02 20130101 |
Class at
Publication: |
435/105 ;
435/232 |
International
Class: |
C12P 019/02; C12N
009/88 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 1993 |
GB |
9321304.9 |
Oct 15, 1993 |
GB |
9321305.6 |
Oct 15, 1993 |
GB |
9321301.5 |
Oct 15, 1993 |
GB |
9321302.3 |
Oct 15, 1993 |
GB |
9321303.1 |
Claims
1. A method of preparing the sugar 1,5-D-anhydrofructose comprising
treating an .alpha.-1,4-glucan with the enzyme .alpha.-1,4-glucan
lyase characterised in that enzyme is used in substantially pure
form.
2. A method according to claim 1 wherein if the glucan contains
links other than and in addition to the .alpha.-1,4-link the
.alpha.-1,4-glucan lyase is used in conjunction with a suitable
reagent that can break the other links.
3. A method according to claim 2 wherein the glucan is starch and a
hydrolase, preferably a glucanohydrolase, is used in conjunction
with the .alpha.-1,4-glucan lyase.
4. A method according to claim 2 or claim 3 wherein the hydrolase
is at least one of pullanase or isoamylase.
5. A method according to any preceding claim wherein the
.alpha.-1,4-glucan lyase is bound to a support or, more preferably,
is in a dissolved form.
6. A method according to any preceding claim wherein the enzyme is
isolated from either a fungus, preferably Morchella costata or
Morchella vulgaris, or from a fungally infected algae, preferably
Gracilariopsis lemaneiformis or from algae alone, preferably
Gracilariopsis lemaneiformis.
7. A method according to claim 6 wherein the enzyme is isolated
and/or further purified from the fungus or from the fungally
infected algae or from algae alone using a gel that is not degraded
by the enzyme.
8. A method according to claim 7 wherein the gel is based on
dextrin or derivatives thereof, preferably the gel is a
cyclodextrin--more preferably beta-cyclodextrin.
9. A method according to any of the preceding claims wherein the
enzyme comprises the amino acid sequence SEQ. ID. No. 1. or the
amino acid sequence SEQ. ID. No. 2 or the amino acid sequence SEQ.
ID. No. 5. or the amino acid sequence SEQ. ID. No. 6, or any
variant thereof.
10. A method according to any preceding claim wherein the enzyme is
obtained from the expression of a nucleotide sequence coding for
the enzyme.
11. A method according to claim 10 wherein the nucleotide sequence
is a DNA sequence.
12. A method according to claim 11 wherein the DNA sequence
comprises a sequence that is the same as, or is complementary to,
or has substantial homology with, or contains any suitable codon
substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4
or SEQ. ID. No. 7 or SEQ. ID. No. 8.
13. The method according to claim 3 or any claim dependent thereon
wherein the starch is used in high concentration--such as up to
about 25% solution.
14. The method according to any one of the preceding claims wherein
the substrate is treated with the enzyme in the presence of a
buffer.
15. The method according to any one of claims 1 to 13 wherein the
substrate is treated with the enzyme in the presence of at least
substantially pure water.
16. The method according to any one of the preceding claims wherein
the substrate is treated with the enzyme in the absence of a
co-factor.
17. The method according to any one of the preceding claims wherein
the enzyme is used in combination with amylopectin or dextrin.
18. A method of preparing the sugar 1,5-D-anhydrofructose
comprising treating an .alpha.-1,4-glucan with the enzyme
.alpha.-1,4-glucan lyase characterised in that enzyme comprises the
amino acid sequence SEQ. ID. No. 1. or the amino acid sequence SEQ.
ID. No. 2 or the amino acid sequence SEQ. ID. No. 5. or the amino
acid sequence SEQ. ED. No. 6, or any variant thereof.
19. The sugar 1,5-D-anhydrofructose when prepared by the method of
the present invention.
20. The use of a reagent that can increase the hydrophobicity of
the reaction medium to increase the stability and activity of the
GL enzyme.
21. Use of AF as an anti-oxidant.
22. Use of AF as a sweetener.
Description
[0001] The present invention relates to the use of an enzyme, in
particular .alpha.-1,4-glucan lyase ("GL"), to prepare
1,5-D-anhydrofructose ("AF") from substrates based on
.alpha.-1,4-glucan.
[0002] The present invention also relates to the use of a sugar, in
particular 1,5-D-anhydrofructose ("AF"), as an anti-oxidant, in
particular as an anti-oxidant for food stuffs and beverages.
[0003] The present invention relates to the use of
1,5-D-anhydrofructose ("AF") as a sweetener, in particular as a
sweetener for foodstuffs and beverages, preferably human foodstuffs
and beverages.
[0004] FR-A-2617502 and Baute et al in Phytochemistry [1988] vol.
27 No. 11 pp3401-3403 report on the production of AF in Morchella
vulgaris by an apparent enzymatic reaction. The yield of production
of AF is quite low. Despite a reference to a possible enzymatic
reaction, neither of these two documents presents any amino acid
sequence data for any enzyme let alone any nucleotide sequence
information. These documents say that AF can be a precursor for the
preparation of the antibiotic pyrone microthecin.
[0005] Yu et al in Biochimica et Biophysica Acta [1993] vol 1156
pp313-320 report on the preparation of GL from red seaweed and its
use to degrade .alpha.-1,4-glucan to produce AF. The yield of
production of AF is quite low. Despite a reference to the enzyme GL
this document does not present any amino acid sequence data for
that enzyme let alone any nucleotide sequence information coding
for the same. This document also suggests that the source of GL is
just algal.
[0006] A typical .alpha.-1,4-glucan based substrate is starch.
Today, starches have found wide uses in industry mainly because
they are cheap raw materials.
[0007] Starch degrading enzymes can be grouped into various
categories. The starch hydrolases produce glucose or
glucose-oligomers. A second group of starch degrading enzymes are
phosphorylases that produce glucose-1-phosphate from starch in the
presence of inorganic phosphate.
[0008] AF has also been chemically synthesised--see the work of
Lichtenthaler in Tetrahedron Letters Vol 21 pp 1429-1432. However,
this chemical synthesis involves a large number of steps and does
not yield large quantities of AF.
[0009] The chemical synthetic route for producing AF is therefore
very expensive.
[0010] There is therefore a need for a process that can prepare AF
in a cheap and easy manner and also in a way that enables large
quantities of AF to be made.
[0011] Furthermore, anti-oxidants are typically used to prevent
oxygen having any deleterious effect on a substance such as a
foodstuff. Two commonly used anti-oxidants are GRINDOX 142 and
GRINDOX 1029. These anti-oxidants contain many components and are
quite expensive to make.
[0012] There is therefore a need to have a simpler and cheaper form
of anti-oxidant.
[0013] Furthermore, sweeteners are often used in the preparation of
foodstuffs and beverages. However, many sweeteners are expensive
and complex to prepare.
[0014] There is therefore a need to have a simpler and cheaper form
of sweetener.
[0015] According to the present invention there is provided a
method of preparing the sugar 1,5-D-anhydrofructose comprising
treating an .alpha.-1,4-glucan with the enzyme .alpha.-1,4-glucan
lyase characterised in that enzyme is used in substantially pure
form.
[0016] Preferably if the glucan contains links other than and in
addition to the .alpha.-1,4-links the .alpha.-1,4-glucan lyase is
used in conjunction with a suitable reagent that can break the
other links--such as a hydrolase--preferably glucanohydrolase.
[0017] Preferably the glucan is starch or a starch fraction
prepared chemically or enzymatically. If prepared enzymatically the
reaction can be performed before the addition of the
.alpha.-1,4-glucan lyase or the reactions can be performed
simultaneously. The suitable reagent can be an auxiliary enzyme.
Preferred auxiliary enzymes are alpha- or beta-amylases. Preferably
a debranching enzyme is used. More preferably the auxiliary enzyme
is at least one of pullanase or isoamylase.
[0018] Preferably the .alpha.-1,4glucan lyase either is bound to a
support or, more preferably, is in a dissolved form.
[0019] Preferably the enzyme is isolated from either a fungus,
preferably Morchella costata or Morchella vulgaris, or from a
fungally infected algae, preferably Gracilariopsis lemaneiformis,
or from algae lone, preferably Gracilariopsis lemaneiformis.
[0020] Preferably the enzyme is isolated and/or further purified
from the fungus or from the fungally infected algae or algae alone
using a gel that is not degraded by the enzyme.
[0021] Preferably the gel is based on dextrin or derivatives
thereof.
[0022] Preferably the gel is a cyclodextrin--more preferably
beta-cyclodextrin.
[0023] Preferably the enzyme comprises the amino acid sequence SEQ.
ID. No. 1. or the amino acid sequence SEQ. ID. No. 2 or the amino
acid sequence SEQ. ID. No. 5 or the amino acid SEQ. I.D. No. 6, or
any variant thereof.
[0024] In an alternative preferable embodiment, the enzyme
comprises any one of the amino acid sequences shown in SEQ. ID.
Nos. 9-11, or any variant thereof.
[0025] The term "any variant thereof" means any substitution of,
variation of, modification of, replacement of, deletion of or
addition of an amino acid from or to the sequence providing the
resultant enzyme has lyase activity.
[0026] Preferably the enzyme is used in combination with
amylopectin or dextrin.
[0027] Preferably, the enzyme is obtained from the expression of a
nucleotide sequence coding for the enzyme.
[0028] Preferably the nucleotide sequence is a DNA sequence.
[0029] Preferably the DNA sequence comprises a sequence that is the
same as, or is complementary to, or has substantial homology with,
or contains any suitable codon substitutions for any of those of,
SEQ. ID. No. 3 or SEQ. ID. No. 4 or SEQ. ID. No. 7 or SEQ. ID. No.
8.
[0030] In an alternative preferable embodiment, the DNA sequence
comprises any one of the sequences that are the same as, or are
complementary to, or have substantial homology with, or contain any
suitable codon substitutions as shown as SEQ. ID. Nos. 12-14.
[0031] The expression "substantial homology" covers homology with
respect to structure and/or nucleotide components and/or biological
activity.
[0032] The expression "contains any suitable codon substitutions"
covers any codon replacement or substitution with another codon
coding for the same amino acid or any addition or removal thereof
providing the resultant enzyme has lyase activity.
[0033] In other words, the present invention also covers a modified
DNA sequence in which at least one nucleotide has been deleted,
substituted or modified or in which at least one additional
nucleotide has been inserted so as to encode a polypeptide having
the activity of a glucan lyase, preferably having an increased
lyase activity.
[0034] Preferably the starch is used in high concentration--such as
up to about 25% solution.
[0035] Preferably the substrate is treated with the enzyme in the
presence of a buffer.
[0036] More preferably the substrate is treated with the enzyme in
the presence of substantially pure water.
[0037] Preferably the substrate is treated with the enzyme in the
absence of a co-factor.
[0038] According to the present invention there is also provided a
method of preparing the sugar 1,5-D-anhydrofructose comprising
treating an .alpha.-1,4-glucan with the enzyme .alpha.-1,4-glucan
lyase characterised in that enzyme comprises the amino acid
sequence SEQ. ID. No. 1. or the amino acid sequence SEQ. ID. No. 2
or the amino acid sequence SEQ. ID. No. 5. or the amino acid
sequence SEQ. ID. No. 6, or any one of the amino acid sequences
SEQ. I.D. Nos. 9-11, or any variant thereof.
[0039] According to the present invention there is also provided
the sugar 1,5-D-anhydrofructose when prepared by the method of the
present invention.
[0040] AF prepared by the present method was confirmed and
characterised by .sup.13C NMR.
[0041] One of key advantages of the present method is that the
sugar 1,5-D-anhydrofructose can be prepared in much larger
quantities than before and by a method that is relatively easier
and cheaper than the known processes. For example the sugar can now
be prepared in amounts of for example greater than 100 g--such as
500 g--compared to the prior art methods when only much smaller
amounts were and could be produced--such as micro gram amounts.
[0042] Typical reactions that can be catalyzed by GL can be
summarised as follows:
[0043] 1). Amylopectin.fwdarw.AF+limit dextrin
[0044] 2). Amylose.fwdarw.AF+limit dextrin
[0045] 3). Dextrin.fwdarw.AF+glucose
[0046] In reaction 1), the ratio of the two products depend on the
structure of amylopectin or the distribution of
.alpha.-1,6-glucosidic linkages in the amylopectin molecules.
[0047] In reaction 2) and 3), the ratio of the products depends on
the degree of polymerisation (DP) number of the substrate. In
reaction 3 the ratio between AF and glucose depends upon the DP.
For example if the dextrin contains 10 glucose units the ratio
AF:glucose would be 9:1.
[0048] Another advantage of the present invention is that glucans
that contain links other than .alpha.-1,4-links can be
substantially degraded--whereas before only partial degradation was
achieved. The substantial degradation of the 1,5-D-anhydrofructose
precursor is one of the factors leading to the increased yields of
1,5-D-anhydrofructose.
[0049] Other advantages are AF is a naturally occurring substance
and therefore it has a potential for human purposes. For example,
it can be converted to the antibiotic microthecin by AF dehydrase.
Antibiotics are known for their uses in food bio-preservation,
which is an important area in food technology. However, to date,
the preparation of AF and also microthecin has had a number of
disadvantages. For example, only small quantities could be
produced. Also, the process was costly.
[0050] The present invention overcomes these problems by providing
a larger production of and much cheaper production of AF and so
also other products such as microthecin. In this regard, it is
possible to prepare gram to kilogram amounts of AF.
[0051] A further advanatge is that the lyase is stable for at least
one year at 4.degree. C. and can be lyophilized without loss of
activity.
[0052] Another advantage is that the lyase produces AF directly
from starches and does not need the presence of any co-factors.
[0053] Another advantage is that the enzyme can be used in pure
water. This result is very surprising.
[0054] Based on the simple properties of the present lyase, one can
expect that the production cost of AF will be comparable to that of
glucose. This is especially advantageous that the present lyase
does not necessarily require the presence of any co-factors which
are generally very expensive.
[0055] In general .alpha.-1,4-glucans can be used as substrate for
the enzyme.
[0056] As a preferred substrate, starch is used.
[0057] In a preferred process, soluble or gelatinized starch or
starch hydrolysate are used. The starch hydrolysates can be
prepared either chemically or enzymatically.
[0058] If an enzyme is used for the partial starch degradation the
enzyme can either be added before the addition of the lyase or any
other additional starch degrading reagent (such as the enzyme
glucanohydrolase) which may be added simultaneously.
[0059] The lyase will convert the glucan to AF. The enzyme will
attach the substrate from the non reducing end and leave only the
reducing sugar unconverted. The residual glucose can be removed by
known methods some of which have been described here.
[0060] Using the reaction described here pure AF can be produced
and also in large amounts.
[0061] In one embodiment, the .alpha.-1,4-glucan lyase is purified
from the fungally infected algae--such as Gracilariopsis
lemaneiformis--by affinity chromatography on .beta.-cyclodextrin
Sepharose, ion exchange chromatography on Mono Q HR 5/5 and gel
filtration on Superose 12 columns. The purified enzyme produces
1,5-anhydro-D-fructose. from .alpha.-1,4-glucans.
[0062] The fungal lyase isolated from fungal infected
Gracilariopsis lemaneiformis is characterized as having a pH
optimum at 3.5-7.5 when amylopectin is used, a temperature optimum
at 50.degree. C. and a pI of 3.9.
[0063] In another embodiment, the .alpha.-1,4-glucan lyase is
purified from the fungus Morchella costata by affinity
chromatography on .beta.-cyclodextrin Sepharose, ion exchange
chromatography on Mono Q BR 5/5 and gel filtration on Superose 12
columns. The purified enzyme produces 1,5-anhydro-D-fructose from
.alpha.-1,4-glucans.
[0064] The fungal lyase shows a pI around 5.4 as determined by
isoelectric focusing on gels with pH gradient of 3 to 9. The
molecular weight determined by SDS-PAGE on 8-25% gradient gels was
110 kDa. The enzyme exhibited a pH optimum in the range pH 5-7. The
temperature optimum was found to be between 30-45.degree. C.
[0065] In another embodiment, the .alpha.-,1,4-glucan lyase is
purified from the fungus Morchella vulgaris by affinity
chromatography on .beta.-cyclodextrin Sepharose, ion exchange
chromatography on Mono Q HR 5/5 and gel filtration on Superose 12
columns. The purified enzyme produces 1,5-anhydro-D-fructose from
.alpha.-1,4-glucans.
[0066] In another embodiment, the .alpha.-1,4-glucan lyase is
purified from algae--such as Gracilariopsis lemaneiformis--by
affinity chromatography on .beta.-cyclodextrin Sepharose, ion
exchange chromatography on Mono Q HR 5/5 and gel filtration on
Superose 12 columns. The purified enzyme produces
1,5-anhydro-D-fructose from .alpha.-1,4-glucans.
[0067] Typical pH and temperature optima for the lyase catalyzed
reaction for some of the GL enzymes according to the present
invention are as follows:
1 Optimal Optimal GL sources Optimal pH pH range temperature M.
costata 6.5 5.5-7.5 37 C; 40 C.sup.a M. vulgaris 6.4 5.9-7.6 43 C;
48 C.sup.a Fungal infected 3.8 3.7-4.1 40 C; 45 C.sup.a
Gracilariopsis lemaneformis .sup.aParameters determined using
glycogen as substrate; other parameters determined using
amylopectin as substrate.
[0068] The enzymes of the present invention convert amylose and
amylopectin to 1,5-anhydrofructose.
[0069] Among the maltosaccharides tested, we found that the lyase
showed low activity towards maltose, and lower activity to
maltotriose and maltoheptaose with the highest activity to
maltotetraose and maltopentaose. The enzyme showed no substrate
inhibition up to a concentration 10 mg ml.sup.-1 among these
maltosaccharides.
[0070] The enzymes from each of the preferred sources has been
sequenced and the amino acid sequences are presented later. Also
presented later are the DNA sequences coding for the enzymes.
[0071] The present invention therefore describes a new starch
degrading enzyme--namely a new .alpha.-1,4-glucan lyase. This is an
enzyme that has been purified and characterized for the first
time.
[0072] As mentioned above, the present invention also relates to
some specific uses of AF.
[0073] In particular, the present invention relates to the use of
1,5-D-anhydrofructose ("AF"), as an anti-oxidant, in particular as
an anti-oxidant for food stuffs and beverages.
[0074] Therefore according to the present invention there is
provided the use of 1,5-D-anhydrofructose (AF) as an
antioxidant.
[0075] Preferably AF is or is used in an edible substance.
[0076] Preferably AF is used in or as a foodstuff or beverage.
[0077] Preferably, AF is used in combination with another
anti-oxidant.
[0078] Preferably the AF is prepared by the method according to the
present invention.
[0079] The main advantages of using AF as an anti-oxidant are that
it is a natural product, it is non-metabolisable, it is easy to
manufacture, it is water-soluble, and it is generally
non-toxic.
[0080] In a preferred embodiment the present invention therefore
relates to the enzymatic preparation of pure AF which can be used
as an attractive water soluble antioxidant for food and non-food
purposes. In the application examples are given for the use of AF
as an antioxidant in food formulations.
[0081] In the accompanying examples it is seen that AF is
comparable with known high quality commercial available food
antioxidants.
[0082] Non-food examples include use in polymer chemistry as oxygen
scavengers during the synthesis of polymers. Also, AF could be used
for the synthesis of bio-degradable plastic.
[0083] Experiments have shown that AF can be an efficient reducing
agent (antioxidant), as it can easily reduce 3,5-dinitrosalicylic
acid to 3-amino-5-nitrosalicylic acid.
[0084] AF is a naturally occurring substance and therefore it has a
tremendous potential for use as an acceptable antioxidant. AF can
also be converted into the antibiotic microthecin by AF dehydrase.
Antibiotics are known for their uses in food biopreservation, an
important area in food biotechnology.
[0085] In another aspect, the present invention also relates to the
use of 1,5-D-anhydrofructose as a sweetener, in particular as a
sweetener for foodstuffs and beverages, preferably human foodstuffs
and beverages.
[0086] Thus according to this aspect of the present invention there
is provided the use of 1,5-D-anhydrofructose as a sweetener.
[0087] Preferably the AF is used as or in a human foodstuff or
beverage.
[0088] The AF may be used in any desired amount such as a 5%
soution or 100mg/kg to 500 mg/kg.
[0089] The advantages of using AF as a sweetener are that it is a
natural product, it is generally non-toxic, it is water soluble, it
is non-metabolisable and it is easy to manufacture.
[0090] The present invention therefore also relates to a novel
application of AF as a sweetener.
[0091] Preferably the AF is prepared by the method according to the
present invention.
[0092] Further aspects of the present invention include:
[0093] a method of preparing the enzyme .alpha.-1,4-glucan lyase
(GL) Comprising isolating the enzyme from a fungally infected
algae, fungus or algae alone;
[0094] an enzyme comprising the amino acid sequence SEQ. ID. No. 1.
or SEQ. ID. No. 2 or SEQ. ID. No. 5. or SEQ. ID. No. 6, or any
variant thereof;
[0095] an enzyme comprising the amino acid sequence SEQ. ID. No. 9.
or SEQ. ID. No. 10 or SEQ. ID. No. 11, or any variant thereof;
[0096] a nucleotide sequence coding for the enzyme
.alpha.-1,4-glucan lyase, preferably wherein the sequence is not in
its natural environment (i.e. it does not form part of the natural
genome of a cellular organism capable of expressing the enzyme,
preferably wherein the nucleotide sequence is a DNA sequence;
[0097] a nucleotide sequence wherein the DNA sequence comprises at
least a sequence that is the same as, or is complementary to, or
has substantial homology with, or contains any suitable codon
substitutions for any of those of, SEQ. ID. No. 3 or SEQ. ID. No. 4
or SEQ. ID. No. 7 or SEQ. ID. No. 8, preferably wherein the
sequence is in isolated form;
[0098] a nucleotide sequence wherein the DNA sequence comprises at
least a sequence that is the same as, or is complementary to, or
has substantial homology with, or contains any suitable codon
substitutions for any of those of, SEQ. ID. No. 12 or SEQ. ID. No.
13 or SEQ. ID. No. 14, preferably wherein the sequence is in
isolated form; and
[0099] the use of beta-cyclodextrin to purify an enzyme, preferably
GL.
[0100] Other preferred embodiments of the present invention include
any one of the following: A transformed host organism having the
capability of producing AF as a consequence of the introduction of
a DNA sequence as herein described; such a transformed host
organism which is a microorganism--preferably wherein the host
organism is selected from the group consisting of bacteria, moulds,
fungi and yeast; preferably the host organism is selected from the
group consisting of Saccharomyces, Kluyveromyces, Aspergillus,
Trichoderma Hansenula, Pichia, Bacillus Streptomyces, Eschericia
such as Aspergillus oryzae, Saccharomyces cerevisiae, bacillus
sublilis, Bacillus amyloliquefascien, Eschericia coli. A method for
preparing the sugar 1,5-D-anhydrofructose comprising the use of a
transformed host organism expressing a nucleotide sequence encoding
the enzyme .alpha.-1,4-glucan lyase, preferably wherein the
nucleotide sequence is a DNA sequence, preferably wherein the DNA
sequence is one of the sequences hereinbefore described; A vector
incorporating a nucleotide sequence as hereinbefore described,
preferably wherein the vector is a replication vector, preferably
wherein the vector is an expression vector containing the
nucleotide sequence downstream from a promoter sequence, preferably
the vector includes a marker (such as a resistance marker);
Cellular organisms, or cell line, transformed with such a vector; A
method of producing the product .alpha.-1,4-glucan lyase or any
nucleotide sequence or part thereof coding for same, which
comprises culturing such an organism (or cells from a cell line)
transfected with such a vector and recovering the product.
[0101] In particular, in the expression systems, the enzyme should
preferably be secreted to ease its purification. To do so the DNA
encoding the mature enzyme is fused to a signal sequence, a
promoter and a terminator from the chosen host.
[0102] For expression in Aspergillus niger the gpdA (from the
Glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus
nidulans) promoter and signal sequence is fused to the 5' end of
the DNA encoding the mature lyase. The terminator sequence from the
A. niger trpC gene is placed 3' to the gene (Punt, P. J. et al
1991-(1991): J. Biotech. 17, 19-34). This construction is inserted
into a vector containing a replication origin and selection origin
for E. coli and a selection marker for A. niger. Examples of
selection markers for A. niger are the amdS gene, the argB gene,
the pyrG gene, the hygB gene, the BmIR gene which all have been
used for selection of transformants. This plasmid can be
transformed into A. niger and the mature lyase can be recovered
from the culture medium of the transformants. Eventually the
construction could be transformed into a protease deficient strain
to reduce the proteolytic degradation of the lyase in the culture
medium (Archer D. B. et al 1992--Biotechnol. Lett. 14,
357-362).
[0103] Instead of Aspergillus niger as host, other industrial
important microorganisms for which good expression systems are
known could be used such as: Aspergillus oryzae, Aspergillus sp.,
Trichoderma sp., Saccharomyces cerevisiae, Kluyveromyces sp.,
Hansenula sp., Pichia sp., Bacillus subtilis, B. amyloliquefaciens,
Bacillus sp., Streptomyces sp. or E. coli.
[0104] The following samples were deposited in accordance with the
Budapest Treaty at the recognised depositary The National
Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23
St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on
Jun. 20, 1994:
[0105] E. Coli containing plasmid pGL1 (NCIMB 40652)--[ref.
DH5alpha-pGL1]; and
[0106] E. Coli containing plasmid pGL2 (NCIMB 40653)--[ref.
DH5alpha-pGL2].
[0107] The following sample was accepted as a deposit in accordance
with the Budapest Treaty at the recognised depositary The Culture
Collection of Algae and Protozoa (CCAP) at Dunstaffnage Marine
Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom, PA34
4AD on Oct. 11, 1994:
[0108] Fungally infected Gracilariopsis lemaneiformis (CCAP
1373/1)--[ref. GLQ-1 (Qingdao)].
[0109] Thus highly preferred embodiments of the present invention
include a GL enzyme obtainable from the expression of the GL coding
sequences present in plasmids that are the subject of either
deposit NCIMB 40652 or deposit NCIMB 40653; and a GL enzyme
obtainable from the fungally infected algae that is the subject of
deposit CCAP 1373/1.
[0110] The following samples were deposited in accordance with the
Budapest Treaty at the recognised depositary The National
Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23
St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on
Oct. 3, 1994:
[0111] E. Coli containing plasmid pMC (NCIMB 40687)--[ref.
DH5alpha-pMC];
[0112] E. Coli containing plasmid pMV1 (NCIMB 40688)--(ref.
DH5alpha-pMV1]; and.
[0113] E. Coli containing plasmid pMV2 (NCIMB 40689)--[ref.
DH5alpha-pMV2].
[0114] Plasmid pMC is a pBluescript II KS containing a 4.1 kb
fragment isolated from a genomic library constructed from Morchella
costata. The fragment contains a gene coding for .alpha.-1,4-glucan
lyase.
[0115] Plasmid pMV1 is a pBluescript II KS containing a 2.45 kb
fragment isolated from a genomic library constructed from Morchella
vulgaris. The fragment contains the 5' end of a gene coding for
.alpha.-1,4-glucan lyase.
[0116] Plasmid MV2 is a pPUC19 containing a 3.1 kb fragment
isolated from a genomic library constructed from Morchella
vulgaris. The fragment contains the 3' end of a gene coding for
.alpha.-1,4-glucan lyase.
[0117] In the following discussions, MC represents Morchella
costata and MV represents Morchella vulgaris.
[0118] As mentioned, the GL coding sequence from Morchella vulgaris
was contained in two plasmids. With reference to FIG. 15 pMV1
contains the nucleotides from position 454 to position 2902; and
pMV2 contains the nucleotides downstream from (and including)
position 2897. With reference to FIGS. 12 and 13, to ligate the
coding sequences one can digest pMV2 with restriction enzymes EcoRI
and BamHI and then insert the relevant fragment into pMV1 digested
with restriction enzymes EcoRI and BamHI.
[0119] Thus highly preferred embodiments of the present invention
include a GL enzyme obtainable from the expression of the GL coding
sequences present in plasmids that are the subject of either
deposit NCIMB 40687 or deposit NCIMB 40688 and deposit NCIMB
40689.
[0120] The following sample was also accepted as a deposit in
accordance with the Budapest Treaty at the recognised depositary
The Culture Collection of Algae and Protozoa (CCAP) at Dunstaffnage
Marine Laboratory PO Box 3, Oban, Argyll, Scotland, United Kingdom,
PA34 4AD on Oct. 11, 1994:
[0121] Fungally infected Gracilariopsis lemaneiformis (CCAP
1373/2)--[ref. GLSC-1 (California)].
[0122] Thus a highly preferred embodiment of the present invention
includes a GL enzyme obtainable from the algae that is the subject
of deposit CCAP 1373/2.
[0123] The present invention will now be described only by way of
example.
[0124] In the following Examples reference is made to the
accompanying figures in which:
[0125] FIG. 1 shows stained fungally infected algae;
[0126] FIG. 2 shows stained fungally infected algae;
[0127] FIG. 3 shows sections of fungal hypha;
[0128] FIG. 4 shows sections of fungally infected algae;
[0129] FIG. 5 shows a section of fungally infected algae;
[0130] FIG. 6 shows a plasmid map of pGL1;
[0131] FIG. 7 shows a plasmid map of pGL2;
[0132] FIG. 8 shows the amino acid sequence represented as SEQ.
I.D. No. 3 showing positions of the peptide fragments that were
sequenced;
[0133] FIG. 9 shows the alignment of SEQ. I.D. No. 1 with SEQ. I.D.
No. 2;
[0134] FIG. 10 is a microphotograph;
[0135] FIG. 11 shows a plasmid map of pMC;
[0136] FIG. 12 shows a plasmid map of pMV1;
[0137] FIG. 13 shows a plasmid map of pMV2;
[0138] FIG. 14 shows the GL coding sequence and part of the 5' and
3' non-translated regions for genomic DNA obtained from Morchella
costata;
[0139] FIG. 15 shows the GL coding sequence and part of the 5' and
3' non-translated regions for genomic DNA obtained from Morchella
vulgaris;
[0140] FIG. 16 shows a comparison of the GL coding sequences and
non-translated regions from Morchella costata and Morchella
vulgaris;
[0141] FIG. 17 shows the amino acid sequence represented as SEQ.
I.D. No. 5 showing positions of the peptide fragments that were
sequenced;
[0142] FIG. 18 shows the amino acid sequence represented as SEQ.
I.D. No. 6 showing positions of the peptide fragments that were
sequenced;
[0143] FIG. 19 shows a graph of oxygen consumption with and without
the presence of AF; and
[0144] FIG. 20 shows a TLC plate.
[0145] In more detail, FIG. 1 shows Calcoflour White stainings
revealing fungi in upper part and lower part of Gracilariopsis
lemaneiformis (108.times. and 294.times.).
[0146] FIG. 2 shows PAS/Anilinblue Black staining of Gracilariopsis
lemaneiformis with fungi. The fungi have a significant higher
content of carbohydrates.
[0147] FIG. 3 shows a micrograph showing longitudinal and grazing
sections of two thin-walled fungal hypha (f) growing between thick
walls (w) of algal cells. Note thylacoid membranes in the algal
chloroplast (arrows).
[0148] FIG. 4 shows the antisense detections with clone 2 probe
(upper row) appear to be restricted to the fungi illustrated by
Calcoflour White staining of the succeeding section (lower row)
(46.times. and 108.times.).
[0149] FIG. 5 shows intense antisense detections with clone 2 probe
are found over the fungi in Gracilariopsis lemaneiformis
(294.times.).
[0150] FIG. 6 shows a map of plasmid pGL1--which is a pBluescript
II KS containing a 3.8 kb fragment isolated from a genomic library
constructed from fungal infected Gracilariopsis lemaneiformis. The
fragment contains a gene coding for alpha-1,4-glucan lyase.
[0151] FIG. 7 shows a map of plasmid pGL2--which is a pBluescript
II SK containing a 3.6 kb fragment isolated from a genomic library
constructed from fungal infected Gracilariopsis lemaneiformis. The
fragment contains a gene coding for alpha-1,4-glucan lyase.
[0152] FIG. 9 shows the alignment of SEQ. I.D. No. 1 (GL1) with
SEQ. I.D. No. 2 (GL2). The total number of residues for GL1 is
1088; and the total number of residues for GL2 is 1091. In making
the comparison, a structure-genetic matrix was used (Open gap cost:
10; Unit gap cost: 2). In FIG. 9 the character to show that two
aligned residues are identical is `:`; and the character to show
that two aligned residues are similar is `.`. Amino acids said to
be `similar` are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W. Overall
there is an identity of 845 amino acids (i.e. 77.67%); a similarity
of 60 amino acids (5.51%). The number of gaps inserted in GL1 are 3
and the number of gaps inserted in GL2 are 2.
[0153] FIG. 10 is a microphotograph of a fungal hypha (f) growing
between the algal walls (w). Note grains of floridean starch (s)
and thylakoids (arrows) in the algal cell.
[0154] In FIG. 14, the total number of bases is 4726--and the DNA
sequence composition is: 1336 A; 1070 C; 1051 G; 1269 T. The ATG
start codon is shown in bold. The introns are underlined. The stop
codon is shown in italics.
[0155] In FIG. 15, the total number of bases is 4670--and the DNA
sequence composition is: 1253 A; 1072 C; 1080 G; 1265 T. The ATG
start codon is shown in bold. The introns are underlined. The stop
codon is shown in italics.
[0156] In FIG. 16, the two aligned sequences are those obtained
from MC (total number of residues: 1066) and MV (total number of
residues: 1070). The comparison matrix used was a structure-genetic
matrix (Open gap cost: 10; Unit gap cost: 2). In this Figure, the
character to show that two aligned residues are identical is `:`.
The character to show that two aligned residues are similar is `.`.
The amino acids said to be `similar` are: A,S,T; D,E; N,Q; R,K;
I,L,M,V; F,Y,W. Overall there is: Identity: 920 (86.30%);
Similarity: 51 (4.78%). The number of gaps inserted in MC is 1 and
the number of gaps inserted in MV is 1.
[0157] In the attached sequence listings: SEQ. I.D. No. 5 is the
amino-acid sequence for GL obtained from Morchella costata; SEQ.
I.D. No. 6 is the amino-acid sequence for GL obtained from
Morchella vulgaris; SEQ. I.D. No. 7 is the nucleotide coding
sequence for GL obtained from Morchella costata; and SEQ. I.D. No.
8 is the nucleotide coding sequence for GL obtained from Morchella
vulgaris.
[0158] In SEQ. I.D. No. 5 the total number of residues is 1066. The
GL enzyme has an amino acid composition of:
2 46 Ala 13 Cys 25 His 18 Met 73 Thr 50 Arg 37 Gln 54 Ile 43 Phe 23
Trp 56 Asn 55 Glu 70 Leu 56 Pro 71 Tyr 75 Asp 89 Gly 71 Lys 63 Ser
78 Val
[0159] In SEQ. I.D. No. 6 the total number of residues is 1070. The
GL enzyme has an amino acid composition of:
3 51 Ala 13 Cys 22 His 17 Met 71 Thr 50 Arg 40 Gln 57 Ile 45 Phe 24
Trp 62 Asn 58 Glu 74 Leu 62 Pro 69 Tyr 74 Asp 87 Gly 61 Lys 55 Ser
78 Val
[0160] Experiments
[0161] 1 The Soluble Enzyme System:
[0162] 1.1. Effect of pH on the Stability and Activity of the Lyase
Isolated from Fugal Infected Gracilariopsis lemaneiformis.
[0163] Two buffer systems, namely HOAc and NaOAc and sodium
citrate--citric acid in a concentration of 5 mM--were tested at
37.degree. C. The pH range tested was from pH 3 to pH 5.2. The
lyase showed maximum activity in a pH range between 3.6 to 4.2. At
pH 3, the stability and activity of the enzyme decreased by about
90%. At pH 5.2, the activity decreased by about 64 %. However, the
enzyme was considerably more stable at this pH than at pH 3, as the
AF yield obtained at pH 5.2 was 75% of the AF yield obtained at pH
3.8. Slightly higher AF yield was obtained in the HOAc and NaOAc
buffer than in citrate buffer. This is not due to any differential
effect of the two buffers (final conc. is 125 .mu.M in the AF assay
mixture) in the AF assay method.
[0164] 1.2. Effect of Temperature on the Activity and Stability of
the Lyase.
[0165] This experiment was conducted at optimal pH range. At
25.degree. C. the production of AF was linear up to at least 9
days. This indicates that no loss of activity and stability of the
lyase occurred within 9 days. With increasing temperature, the
stability of the enzyme decreased.
[0166] The half life of the enzyme activity at the following
temperature was:
4 30.degree. C. 5 days 37.degree. C. 2.5 days 40.degree. C. less
than 1 day 50.degree. C. less than 1 day
[0167] 1.3. Effect of Substrate Concentration on the Stability of
the Lyase and AF Yield.
[0168] It was observed that amylopectin and dextrins have a
stabilizing effect on the lyase while the smallest substrate
maltose does not. This was verified for both the soluble enzyme
system and the immobilized enzyme system.
[0169] AF yield increases with the increase in amylopectin
concentration up to 25%. In the case of dextrin, the AF yield
decreases when the concentration exceeds 30% (30%, 40% and 50% were
tested).
[0170] 1.4 Activation and Inactivation of Lyase
[0171] No metal ions are found necessary for the activity and the
enzyme catalysed reaction can surprisingly proceed in pure water.
The fact that the addition of EDTA in the reaction mixture up to 20
mM had little effect on the activity clearly demonstrates that
metal ions are not essential for the activity of the lyase enzyme
according to the present invention.
[0172] This means that in the AF purification step, the ion
exchange chromatography step that takes away salts from the
reaction system can be omitted, if water is used as reaction
medium. However, inclusion of NaCl in the reaction mixture in a
concentration of 0.85% (0.145 M) can increase the AF yield up to
1-fold.
[0173] 1.5. Substrate Specificity
[0174] Upon cooling solubilized starch will tend to form rigid gels
when the starch concentration becomes to high. Therefore it is an
advantage to utilize partly degraded starch as substrate for the
1,4-glucan lyase.
[0175] The specificity of .alpha.-1,4-glucan lyase isolated from M.
costata for different oligosaccharides was tested. The
oligosaccharides were maltose (G2), maltotriose (G3), maltotetraose
(G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7).
The oligosaccharides were dissolved in H.sub.2O at a concentration
of 8 mg/ml. The enzyme assay contained 150 .mu.l substrate
G2/G3/G4/G5/G6/G7, 120 .mu.l 0.1M MES pH 6.3 and 30 .mu.l purified
enzyme. The reaction mixture was incubated for 60 min at 30.degree.
C. Afterwards the reaction was stopped by boiling for 3 min and 900
.mu.l absolute ethanol was added for precipitation. After
centrifugation at 20.000.times. g for 5 min at 4.degree. C. the
supernatant was transferred to a new eppendorf tube and
lyophilized.
[0176] The freeze-dried samples were dissolved in 1000 .mu.l
H.sub.2O and were filtrated through a 0.22 .mu.m Millipore filter
before 25 .mu.l of the sample was loaded on the Dionex HPLC.
[0177] 1.7 HPLC
[0178] Analytical Procedures.
[0179] Analyses were performed on a Dionex 4500 i chromatography
system consisting of a GPM-2 pump and a PED detector which was used
in pulse-amperometric detection mode.
[0180] The anion exchange columns were a CarboPac PA-100
(4.times.250 mm) and a CarboPac PA-100 guard column (3.times.25 mm)
from Dionex.
[0181] The eluent were 200 mM sodium hydroxide (A), 500 mM sodium
acetate (B) and 18 M ohm de-ionized water (C). The pump was
programmed in 2 different ways, method no. 1 and method no. 2:
[0182] Method No. 1:
5 Time, min 0.0 3.0 3.1 26.9 29.0 % A 10 10 50 50 10 % B 0 0 0 32 0
% C 90 90 50 18 90
[0183] Method No. 2:
6 Time, min. 0.0 30 % A 10 10 % B 0 0 % C 90 90
[0184] Standards:
[0185] Glucose, maltose, maltotriose, maltotetraose, maltopentaose,
maltohexaose and maltoheptaose (all from Sigma) and
1,5-anhydrofructose were used as standards. All compounds were
dissolved in 18 M ohm de-ionized water which was filtered through a
0.22 .mu.m Millipore filter before use.
[0186] 1.7 Results:
[0187] The analyses show that the purified enzyme which was
isolated from M. costata indeed was able to use
maltooligosaccharides as substrate 1 for 1,5-anhydrofructose
formation.
[0188] When maltose was used as substrate, almost no
1,5-anhydrofructose was formed but when the other
maltooligosaccharides (G3-G7) were used, high amounts of this
compound were produced.
[0189] It is clear that higher amounts of 1,5-anhydrofructose were
obtained when a longer maltooligosaccharide was used.
[0190] This observation corresponds perfectly well with the theory
of the lyase forming 1,5-anhydrofructose from the non-reducing end
of the substrate, leaving only the terminal glucose molecule
unchanged.
[0191] 1.8 Formation of AF
[0192] .alpha.-1,4-glucan lyase from M. costata hydrolyses starch
to the end-product 1,5-anhydrofructose. The end-product was shown
by HPLC, method 2. The enzyme assay contained 500 .mu.l amylopectin
(20 mg/ml, dissolved in H.sub.2O), 400 .mu.l 0.1 M MES pH 6.3 and
100 .mu.l purified enzyme. The reaction mixture was incubated at
30.degree. C. and the reaction was stopped by boiling after 30 or
120 min incubation. High-molecular oligosaccharides were
precipitated by addition of 3 vol abs. ethanol and the sample was
centrifuged and freeze-dried as described above. The samples were
dissolved in 125 .mu.l H.sub.2O and 25 .mu.l were applied on the
HPLC column.
[0193] The HPLC elution profile clearly shows that
.alpha.-1,4-glucan lyase from M. costata produces
1,5-anhydrofructose by hydrolysis of starch. Equal amounts of
1,5-anhydrofructose were found after 30 and 120 min. incubation
which indicate that the enzyme activity is not inhibited by the
endproduct 1,5-anhydrofructose.
[0194] .sup.13C No spectra (water) of AF prepared in this way shows
that it adopts one major form giving rise to the following signals:
.delta.93.5 (quart, C-2), 81.5 (CH, C-5), 77.7 (CH, C-3), 72.6
(CH.sub.2, C-1), 69,8 (CH, C4), 62.0 (CH.sub.2, C-6). Assignments
are based on H--H C--H and C--H 2D correlation spectra.
[0195] 1.6. The Cooperative Effect of Lyase with Pullulanase and
Isoamylase.
[0196] As it can be seen from Table 1, the inclusion of pullulanase
in the reaction mixture will obviously increase the AF yield by
about 15-23 %, depending on whether soluble starch or amylopectin
is used as substrate.
7TABLE The cooperation of pullulanase and lyase in the production
of AF. Substrate Lyase Pullulanase AF Yield (%) Glc Yield (%)
Solubl. + - 51 0 Starch - + 0 0.37 + + 66.0 3.9 Amylo- + - 48.0 0
pectin - + 0 0.33 + + 71.3 3.7 +, enzyme added, - enzyme
omitted.
[0197] The reaction mixture contained 0.3 ml 2% potato amylopectin
(Sigma) in water or 0.3 ml 2% soluble starch (Merck), 2 .mu.l lyase
and 0.36 units pullulanase (BM) as indicated.
[0198] The reaction was carried out at 30.degree. C. for 1 day. At
the end of the reaction, samples were taken for AF and Glc
analysis.
[0199] In the case of isoamylase, the advantage is that the optimal
pH of the lyase overlaps with that of Pseudomonas isoamylase (pH
3.0-4.5). The problem, however, is that isoamylase will produce an
excess amount of long chain amylose that precipitates from the
solution, and therefore is no longer suitable as a substrate for
the lyase. It can be expected that the cooperation of the lyase
with isoamylase will be efficient, if the chain of amylose is not
too long.
[0200] 2. The Immobilized Enzyme System
[0201] Immobilization of the lyase was achieved by using
succinimide-activated Sepharose (Affigel 15 gel, Bio-Rad) and
glutaradehye-activated Silica gel (BM). The recovery of lyase
activity after immobilization on Affigel 15 gel was between 40% to
50%. There may be some lyase that is still active after
immobilization, but is inaccessible to the substrate because of the
steric hindrance, especially in the case of macromolecules like
starches. Immobilized enzymes used in the industry usually have an
activity recovery of around 50%.
[0202] The most interesting thing of the Affigel 15 gel immobilized
lyase is that its stability has been greatly improved at pH 5.5.
When the column was operated at this pH, the stability was at least
16 days long. The pH shift in the stability is very important
considering the optimal pH of pullulanase which is around pH 5.5.
This is the prerequisite for the lyase and pullulanase to cooperate
efficiently in the same reactor with the same physico-chemical
environment. The soluble lyase has an optimal pH between 3.6 and
4.2, and at this pH range pullulanase shows little or no
activity.
[0203] With the silica gel immobilized lyase, the activity recovery
is very high, around 80-100%. However, the silica gel immobilized
enzyme was not stable when the column was operated neither at pH
3.8 nor pH 5.5. It is possible that some lyase was adsorbed on the
surface of the silica gel beads and was slowly released from the
silica gel after each washing of the column. It may therefore be
the adsorbed lyase that contributes to the high recovery rate and
the decrease in column activity.
[0204] 3. Purification of AF
[0205] 3.1. The Lyase-Amylopectin/Soluble Starch System
[0206] In this system, the reaction system contained AF, limit
dextrin, the lyase, and buffer salts at the end of the reaction. AF
was separated from the macromolecules limit dextrin and the lyase)
by ethanol (final conc. 50 %) precipitation. Unprecipitated
low-molecular-weight amylopectin was separated by ultrafiltration
using Amicon YM3 membranes (cut-off 3,000). Ethanol was removed by
evaporation at 40.degree. C. in a rotary evaporator. Buffer salts
were removed from AF by mixed ion exchangers. Purified solid AF was
obtained by freeze-drying.
[0207] 3.2. The Lyase-Pullulanase/Amylopectin/Soluble Starch
System.
[0208] In this system the final products are AF and glucose. If at
least a substantially pure sample of AF is to be prepared, the
by-product glucose must be removed. This can be achieved by
enzymatic methods. First the glucose is converted into gluconic
acid and hydrogen peroxide by glucose oxidase.
[0209] Catalase is needed to dispel H.sub.2O.sub.2 formed.
H.sub.2O.sub.2 will oxidize AF into two new compounds which are at
present of unknown structure. The other impurities in the AF
preparation are the oxidation products of AF. It was observed that
AF can slowly be oxidized by air-level of oxygen, especially at
high temperature, high AF concentration and long time of
exposure.
[0210] Gluconic acid was removed together with the buffer salts by
ion exchange chromatography.
[0211] In this system, the low-molecular-weight amylopectin
molecules may alternatively be hydrolysed by amyloglucosidase
instead of using ultrafiltration.
[0212] 3.3. The Purity Checking of AF.
[0213] The purity of the AF preparations were confirmed by TLC,
Dionex and NMR.
[0214] 3.4 Analysis of the Antioxidative Activity of Anhydro
Fructose.
[0215] Electrochemical Oxygen Consumption:
[0216] Method.
[0217] The activity of AF was investigated in a methyl linoleate
emulsion as described by Jorgensen and Skibsted (Z. Lebensm.
Unters. Forsch. (1993) 196: 423-429) with minor modifications: To
5.00 ml of a 1.33 mM methyl linoleate emulsion in 5.0 mM aqueous
phosphate buffer with pH=5.8 and 0.2 w/w % Tween 20 as emulsifier
was added AF in the following concentrations: 0, 15, 146 and 680
.mu.M. The oxidation in the system was initiated by addition of 50
.mu.l 0.26 M metmyoglobin (MMb) final concentration 0.26 mM.
Immediately after initiating the reaction the sample was injected
to a thermostated (25.0.+-.0.1.degree. C.) 70 .mu.l closed cell,
effectively excluding diffusion of oxygen into the system. The
oxygen consumption was measured by a Clark electrode, which was
connected to a PC data collection program. The relative oxygen
concentration (%) was registered every 30s.
[0218] Results.
[0219] Curves corresponding to oxygen consumption for the different
samples are illustrated in FIG. 19. For samples without addition of
AF a relative decrease in oxygen concentration is seen immediately
after injection of the sample. For samples containing AF a
lag-phase is observed before the curve breaks off and the oxygen
concentration is reduced. After the lag-phase only a minor
reduction in the oxygen consumption rate is observed compared to
samples without AF added. A tendency for samples having the highest
amount of AF to have the longest lag-phase is observed. As well the
rate for oxygen consumption is lower for these samples, which is
seen by a smaller slope of the curves compared to the slope for the
references (0 .mu.M).
[0220] ESR Analysis
[0221] Method.
[0222] Hydroxyl radicals were generated by a Fenton reaction with
H.sub.2O.sub.2 (0.17 mM) and FeSO.sub.4 (4.8 .mu.M). The generated
radicals were trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO,
9.7 mM). AF was added in concentrations of 1.3 mM and 6.3 mM. A
water soluble extract of rosemary (Rosmaninus officinalis L.) was
analyzed in a concentration of 0.25 mg/ml (in grams equivalent to
1.26 mM AF). Measurements were carried out at room temperature
(20.+-.1.degree. C.) after 120 s and repeated for the same reaction
mixture after 300 s with the following spectrometer settings:
Center field 3475.60 G; sweep width 55 G; microwave power 20 mW;
modulation frequency 100 kHz; modulation amplitude 1.01 G; receiver
gain 1.00-10.sup.5; conversion time 81.92 ms time constant 163.84
ms and sweep time 83.89 s.
[0223] Results.
[0224] The generated hydroxyl radicals were trapped by DMPO. The
spin adduct gives rise to a characteristic 1:2:2:1 ESR spectrum.
The peak height of the spectrum is proportional to the quantitative
amount of generated spin adduct. Addition of both DMPO and AF will
set up a competition between the spin trap and AF. A reduction of
peak height will indicate a good scavenging activity of AF.
8TABLE Peak height of ESR-spectra. H.sub.2O.sub.2 = 0.17 mM and
Fe.sup.2+ = 4.8 .mu.M. Anhydro Rosemary Peak height Peak height
fructose [mM] extract [mg/ml] [120 s] [300 s] 0 0 2475 2780 1.3 0
2634 2545 6.3 0 1781 1900
[0225] At a concentration of 1.3 mM AF no scavenging activity of
hydroxyl radicals is seen, at 6.3 mM Af the peak height is reduced,
indicating that a part of the generated hydroxyl radicals is
scavenged by AF.
[0226] 4. Use of AF AS AN Anti-Oxidant
EXAMPLE 4.1
[0227] Use of AF as an Anti-Oxidant in a 50% Mayonnaise.
[0228] 50% mayonnaise is used for salads, open sandwiches, etc. in
both the catering and the retail trades. The low oil content of 50%
mayonnaise makes it suitable for low-calorie applications.
[0229] A typical mayonnaise composition is as follows:
9 Soya oil 50.0% Tarragon vinegar (10%) 4.0% Egg yolk 3.5% Sugar
3.0% Salt 1.0% Potassium sorbate 0.1% Water 35.2% MAYODAN 602 3.0%
Lemon flavouring 10251 0.2% MAYODAN 602 ensures a fine, stable oil
dispersion and the required viscosity, thereby providing 50%
mayonnaise with a long shelf life. Flavouring 10251 is a natural
lemon flavouring which provides mayonnaise with the fresh taste of
lemon.
[0230] Typically the mayonnaise is prepared by the following
method:
[0231] 1) Dry mix the MAYODAN 602, sugar and salt. Disperse in oil
in a ratio of 1 part powder to 2 parts oil.
[0232] 2) Add flavouring and potassium sorbate to the water and
pour into the Koruma mixer. Add 1).
[0233] 3) Add the egg yolk.
[0234] 4) Add the oil continuously in a vacuum.
[0235] 5) After 2/3 of the oil has been added (slowly), blend the
tarragon vinegar with the remaining 1/3 of the oil, and add.
[0236] The following data show that when AF is added to the
mayonnaise as an anti-oxidant the results are comparable to the
known food anti-oxididants GRINDOX 142 and GRINDOX 1029.
[0237] GRINDOX 142:
10 Ascorbyl palmitate 10% Propyl gallate 20% Citric acid 10% Food
grade emulsifier 60% Form at 25.degree. C. paste Colour grey to
pale brown Density 1.1 g/ml (All percentages are by weight)
[0238]
11 Ascorbyl palmitate 20% Natural tocopherols 20% Food grade
emulsifier 60% Form at 25.degree. C. paste Colour light brown
Density at 25.degree. C. 1,0 g/ml (All percentages are by
weight).
[0239] In the test procedure the anti-oxidants were added to the
mayonnaise to provide an anti-oxidant concentration in the order of
about 500 ppm. The mayonnaise was then placed in a bomb calorimeter
at temperature 80.degree. C. containing pure O.sub.2. An induction
period to the onset of substantial oxidation of the product is then
measured.
[0240] The results were as follows.
12 Samples: IP (hours) 1. Blank 28.0 2. +500 ppm GRINDOX 142 35.0
3. +500 ppm GRINDOX 1029 33.3 4. +550 ppm GRINDOX 1029 34.3 5. +500
ppm 1,5 anhydro-D-fructose 32.0 (IP hours = Induction Period)
[0241] These results show that AF is an excellent food anti-oxidant
and is comparable with the known foodstuffs anti-oxidants GRINDOX
142 or GRINDOX 1029.
EXAMPLE 4.2
[0242] Use of AF as an Anti-Oxidant in a Salad Dressing
[0243] Yogurt Salad Dressing with 50% Oil
[0244] Yogurt salad dressing with 50% oil is used for salads,
potatoes, raw vegetable salad, meat, fish and boiled
vegetables.
13 Composition Soya oil 50.0% Yogurt (plain) 39.0% Vinegar (10%)
3.5% Sugar 3.0% Egg yolk 2.0% Salt 1.0% Potassium sorbate 0.1%
MAYODAN 525 1.4% Acid masking flavouring 2072 0.02%
[0245] MAYODAN 525 provides unique emulsion stability, prevents
syneresis, ensures uniform oil dispersion and viscosity, improves
tolerance to production processes and ensures a long shelf
life.
[0246] Flavouring 2072 is a nature-identical, acid masking
flavouring reducing the acidulated taste of dressing without
affecting its pH value.
[0247] Process
[0248] 1. Dry mix MAYODAN 525, sugar and salt. Disperse in oil in a
ratio of 1 part powder to 2 parts oil.
[0249] 2. Fill flavouring, potassium sorbate and yogurt into the
Koruma mixer. Add 1).
[0250] 3. Add the egg yolk.
[0251] 4. Add the oil continuously in a vacuum.
[0252] 5. After 2/3 of the oil has been added (slowly), blend the
vinegar with the remaining 1/3 of the oil, and add.
[0253] 6. Add spices if required.
[0254] Test Results:
14 Sample: IP hours PF 1. Blank 37.2 1.00 2. 500 ppm
anhydrofructose 39.5 1.06 3. 800 ppm GRINDOX 1032 43.3 1.07 (IP -
Induction Period); (PF - Protection Period)
[0255] Protection Factor (PF):
[0256] For each temperature defined as
[0257] PF=IP of the oil with added antioxidant/IP of the same oil
without added antioxidant
[0258] Life Extension LE) %:
LE=(PF-1.0).times.100
[0259] 6. Preparations of .alpha.-1,4-Glucan Lyase
[0260] Introduction
[0261] With regard to a further embodiments of the present
invention the enzyme .alpha.-1,4-glucan lyase for use in preparing
the AF may be isolated from a fungally infected algae, preferably
fungally infected Gracilariopsis lemaneiformis, more preferably
fungally infected Gracilariopsis lemaneiformis from Qingdao
(China).
[0262] Alternatively the enzyme may be obtained from a fungus. For
example, the fungus can be any one of Discina perlata, Discina
parma, Gyromitra gigas, Gyromitra infula, Mitrophora hybrida,
Morchella conica, Morchella costata, Morchella elata, Morchella
hortensis, Morchella rotunda, Morchella vulgaris, Peziza badia,
Sarcosphaera eximia, Disciotis venosa, Gyromitra esculenta,
Helvella crispa, Helvella lacunosa, Leptopodia elastica, Verpa
digitaliformis, and other forms of Morchella. Preferably the fungus
is Morchella costata or Morchella vulgaris.
[0263] With regard to a further embodiment of the present invention
the enzyme .alpha.-1,4-glucan lyase for use in preparing the AF may
be isolated from algae alone, preferably Gracilariopsis
lemaneiformis, more preferably Gracilariopsis lemaneiformis from
Santa Cruz (Calif.).
[0264] The initial enzyme purification can be performed by the
method as described by Yu et al (ibid). However, preferably, the
initial enzyme purification includes an optimized procedure in
which a solid support is used that does not decompose under the
purification step. This gel support further has the advantage that
it is compatible with standard laboratory protein purification
equipment. The details of this optimized purification strategy are
given later on. The purification is terminated by known standard
techniques for protein purification.
[0265] The purity of the enzyme can be readily established using
complementary electrophoretic techniques.
[0266] A. Source=Fungally Infected Algae
[0267] The following sequence information was used to generate
primers for the PCR reactions mentioned below and to check the
amino acid sequence generated by the respective nucleotide
sequences.
[0268] Amino Acid Sequence Assembled from Peptides from Fungus
Infected Gracilariopsis lemaneiformis
15 Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Met Tyr Gln Asn Ala Ala
Phe Gly Lys Pro Ile Ile Lys Ala Ala Ser Met Tyr Asn Asn Asp Ser Asn
Val Arg Arg Ala Gln Asn Asp His Phe Leu Leu Gly Gly His Asp Gly Tyr
Arg Ile Leu Cys Ala Pro Val Val Trp Glu Asn Ser Thr Glu Arg Glu Leu
Tyr Leu Pro Val Leu Thr Gln Trp Tyr Lys Phe Gly Pro Asp Phe Asp Thr
Lys Pro Leu Glu Gly Ala
[0269] The Amino Acid Sequence (27-34) used to Generate primer A
and B (Met Tyr Asn Asn Asp Ser Asn Val)
16 Primer A ATG TA(TC) AA(CT) AA(CT) GA(CT) TC(GATC) AA(CT) GT 128
mix Primer B ATG TA(TC) AA(CT) AA(CT) GA(CT) AG(CT) AA(CT) GT 64
mix
[0270] The Amino Acid Sequence (45-50) used to Generate Primer C
(Gly Gly His Asp Gly Tyr)
17 Primer C TA (GATC)CC (GA)TC (GA)TG (GATC)CC (GATC)CC 256 mix
[The sequence corresponds to the complementary strand.]
[0271] The Amino Acid Sequence (74-79) used to Generate Primer E
(Gln Trp Tyr Lys Phe Gly)
18 Primer E GG(GATC) CC(GA) AA(CT) TT(GA) TAG CA(CT) TG 64 mix [The
sequence corresponds to the complementary strand.]
[0272] The Amino Acid Sequence (1-6) used to Generate Primer F1 and
F2 (Tyr Arg Trp Gln Glu Val)
19 Primer F1 TA(TC) CG(GATC) TGG CA(GA) GA(GA) GT 32 mix Primer F2
TA(TC) AG(GA) TGG CA(GA) GA(GA) GT 16 mix
[0273] The Sequence Obtained from the First PCR Amplification
(Clone 1)
20 ATGTACAACA ACGACTCGAA CGTTCGCAGG GCGCAGAACG ATCATTTCCT
TCTTGGCGGC CACGACGGTT A Met Tyr Asn Asn Asp Ser Asn Val Arg Arg Ala
Gln Asn Asp His Phe Leu Leu Gly Gly His Asp Gly
[0274] The sequence Obtained from the Second PCR Amplification
(Clone 1)
21 ATGTACAACA ACGACTCGAA CGTTCGCAGG GCGCAGAACG ATCATTTTCCT
TCTTGGTGGA CATGATGGAT ATCGCATTTCT GTGCGCGCCT GTTGTGTGGG AGAATTCGAC
CGAACGNGAA TTGTACTTGC CCGTGCTGAC CCAATGGTAC AAATTCGGCC C Met Tyr
Asn Asn Asp Ser Asn Val Arg Arg Ala Gln Asn Asp His Phe Leu Leu Gly
Gly His Asp Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Trp Glu Asn Ser
Thr Glu Arg Glu Leu Tyr Leu Pro Val Leu Thr Gln Trp Tyr Lys Phe Gly
Pro
[0275] The Sequence Obtained from the Third PCR Amplification
(Clone2)
22 TACAGGTGGC AGGAGGTGTT GTACACTGCT ATGTACCAGA ATGCGGCTTT
CGGGAAACCG ATTATCAAGG CAGCTTCCAT GTACGACAAC GAGAGAAACG TTCGCGGCGC
ACAGGATGAC CACTTCCTTC TCGGCGGACA CGATGGATAT CGTATTTTGT GTGCACCTGT
TGTGTGGGAG AATACAACCA GTCGCGATCT GTACTTGCCT GTGCTGACCA GTGGTACAAA
TTCGGCCC Tyr Arg Trp Gln Glu Val Leu Tyr Thr Ala Met Tyr Gln Asn
Ala Ala Phe Gly Lys Pro Ile Ile Lys Ala Ala Ser Met Tyr Asp Asn Asp
Arg Asn Val Arg Gly Ala Gln Asp Asp His Phe Leu Leu Gly Gly His Asp
Gly Tyr Arg Ile Leu Cys Ala Pro Val Val Trp Glu Asn Thr Thr Ser Arg
Asp Leu Tyr Leu Pro Val Leu Thr Lys Trp Tyr Lys Phe Gly
[0276] A.1. Cytological Investigations of Gracilariopsis
lemaneiformis
[0277] A.1.1.1 Detection of Fungal Infection in Gracilariopsis
lemaneiformis
[0278] Sections of Gracilariopsis lemaneiformis collected in China
were either hand cut or cut from paraffin embedded material.
Sectioned material was carefully investigated by light microscopy.
Fungal hyphae were clearly detected in Gracilariopsis
lemaneiformis.
[0279] The thalli of the Gracilariopsis lemaneiformis are composed
of cells appearing in a highly ordered and almost symmetric manner.
The tubular thallus of G. lemaneiformis is composed of large,
colourless central cells surrounded by elongated, slender,
ellyptical cells and small, round, red pigmented peripherial cells.
All algal cell types are characterized by thick cell walls. Most of
the fungal hyphae are found at the interphase between the central
layer of large cells and the peripherial layer. These cells can
clearly be distinguished from the algae cells as they are long and
cylindrical. The growth of the hyphae is observed as irregularities
between the highly ordered algae cells. The most frequent
orientation of the hypha is along the main axis of the algal
thallus. Side branches toward the central and periphery are
detected in some cases. The hypha can not be confused with the
endo/epiphytic 2nd generation of the algae.
[0280] Calcofluor White is known to stain chitin and cellulose
containing tissue. The reaction with chitin requires four
covalently linked terminal n-acetyl glucosamine residues. It is
generally accepted that cellulose is almost restricted to higher
plants although it might occur in trace amounts in some algae. It
is further known that chitin is absent in Gracilaria.
[0281] Calcofluor White was found to stain domains corresponding to
fungi hyfa cell walls in sectioned Gracilariopsis lemaneiformis
material.
[0282] The hypha appear clear white against a faint blue background
of Gracilaria tissue when observed under u.v. light--see FIG. 1.
Chitin is the major cell wall component in most fungi but absent in
Gracilaria. Based upon these observations we conclude that the
investigated algae is infected by a fungi. 40% of the lower parts
of the investigated Gracilariopsis lemaneiformis sections were
found to be infected with fungal hyphae. In the algae tips 25% of
the investigated Gracilariopsis lemaneiformis sections were found
to be infected.
[0283] Staining of sectioned Gracilariopsis lemaneiformis with
Periodic acid Schiff (PAS) and Aniline blue black revealed a
significantly higher content of carbohydrates within the fungal
cells as compared with the algae cells--see FIG. 2. Safranin O and
Malachit Green showed the same colour reaction of fungi cells as
found in higher plants infected with fungi.
[0284] An Acridin Orange reaction with sectioned Gracilariopsis
lemaneiformis showed clearly the irregularly growth of the
fungus.
[0285] A. 1.1.2 Electron Microscopy
[0286] Slides with 15 .mu.m thick sections, where the fungus was
detected with Calcofluor White were fixed in 2% OsO.sub.4, washed
in water and dehydrated in dimethoxypropane and absolute alcohol. A
drop of a 1:1 mixture of acetone and Spurr resin was placed over
each section on the glass slide, and after one hour replaced by a
drop of pure resin. A gelatin embedding capsule filled with resin
was placed face down over the section and left over night at
4.degree. C. After the polymerization at 55.degree. C. for 8 hrs,
the thick sections adhering to the resin blocks could can be
separated from the slide by immersion in liquid nitrogen.
[0287] Blocks were trimmed and 100 nm thick sections were cut using
a diamond knife on a microtome. The sections were stained in
aqueous uranyl acetate and in lead citrate. The sections were
examined in an electron microscope at 80 kV.
[0288] The investigation confirmed the ligth microscopical
observations and provided further evidence that the lyase
producing, chinese strain of G. lamneiformis is infected by a
fungal parasite or symbiont.
[0289] Fungal hyphae are build of tubular cells 50 to 100 .mu.m
long and only few microns in diameter. The cells are serially
arranged with septate walls between the adjacent cells. Ocasional
branches are also seen. The hyphae grow between the thick cell
walls of algal thallus without penetrating the wall or damaging the
cell. Such a symbiotic association, called mycophycobiosis, is
known to occur between some filamentous marine fungi and large
marine algae (Donk and Bruning, 1992--Ecology of aquatic fungi in
and on algae. In Reisser, W.(ed.): Algae and Symbioses: Plants,
Animals, Fungi, Viruses, Interactions Explored. Biopress
Ltd.,Bristol.)
[0290] Examining the microphotograph in FIG. 10, several
differences between algal and fungal cells can be noticed. In
contrast to several Am thick walls of the alga, the fungal walls
are only 100-200 nm thick. Plant typical organells as chloroplasts
with thyllacoid membranes as well as floridean starch grains can be
seen in algal cells, but not in the fungus.
[0291] Intercellular connections of red algae are characterized by
specific structures termed pit plugs, or pit connections The
structures are prominent, electron dense cores and they are
important features in algal taxonomy (Pueschel, C. M.: An expanded
survey of the ultrastructure of Red algal pit plugs. J. Phycol. 25,
625, (1989)). In our material, such connections were frequently
observed in the algal thallus, but never between the cells of the
fungus.
[0292] A. 1.2 In Situ Hybridization Experiments
[0293] In situ hybridization technique is based upon the principle
of hybridization of an antisense ribonucotide sequence to the mRNA.
The technique is used to visualize areas in microscopic sections
where said mRNA is present. In this particular case the technique
is used to localize the enzyme .alpha.-1,4-glucan lyase in sections
of Gracilariopsis lemaneiformis.
[0294] A. 1.2.1 Preparation of .sup.35S Labelled Probes for In Situ
Hybridization
[0295] A 238 bp PCR fragment from a third PCR amplification--called
clone 2 (see above)--was cloned into the pGEM-3Zf(+) Vector
(Promega). The transcription of the antisense RNA was driven by the
SP6 promotor, and the sense RNA by the T7 promotor. The
Ribonuclease protection assay kit (Ambion) was used with the
following modifications. The transcripts were run on a 6%
sequencing gel to remove the unincorporated nucleotide and eluted
with the elution buffer supplied with the T7RNA polymerase in vitro
Transcription Kit (Ambion). The antisense transcript contained 23
non-coding nucleotides while the sense contained 39. For
hybridization 10.sup.7 cpm/ml of the .sup.35S labelled probe was
used.
[0296] In situ hybridization was performed essentially as described
by Langedale et. al.(1988). The hybridization temperature was found
to be optimal at 45.degree. C. After washing at 45.degree. C. the
sections were covered with KodaK K-5 photographic emulsion and left
for 3 days at 5.degree. C. in dark (Ref: Langedale, J. A.,
Rothermel, B. A. and Nelson, T. (1988). Genes and development 2:
106-115. Cold Spring Harbour Laboratory).
[0297] The in situ hybridization experiments with riboprobes
against the mRNA of .alpha.-1,4-glucan lyase, show strong
hybridizations over and around the hypha of the fungus detected in
Gracilariopsis lemaneiformis--see FIGS. 4 and 5. This is considered
a strong indication that the .alpha.-1,4-glucan lyase is produced.
A weak random background reactions were detected in the algae
tissue of both Gracilariopsis lemaneiformis. This reaction was
observed both with the sense and the antisense probes. Intense
staining over the fungi hypha was only obtained with antisense
probes.
[0298] These results were obtained with standard hybridisation
conditions at 45.degree. C. in hybridization and washing steps. At
50.degree. C. no staining over the fungi was observed, whereas the
background staining remained the same. Raising the temperature to
55.degree. C. reduced the background staining with both sense and
antisense probes significantly and equally.
[0299] Based upon the cytological investigations using
complementary staining procedures it is concluded that
Gracilariopsis lemaneiformis is fungus infected. The infections are
most pronounced in the lower parts of the algal tissue.
[0300] In sectioned Gracilariopsis lemaneiformis material in situ
hybridization results clearly indicate that hybridization is
restricted to areas where fungal infections are found--see FIG. 4.
The results indicate that .alpha.-1,4-glucan lyase mRNA appears to
be restricted to fungus infected areas in Gracilariopsis
lemaneiformis. Based upon these observations we conclude that
.alpha.-1,4-glucan lyase activity is detected in fungally infected
Gracilariopsis lemaneiformis.
[0301] A.2. Enzyme Purification and Characterization
[0302] Purification of .alpha.-1,4-glucan lyase from fungal
infected Gracilariopsis lemaneiformis material was performed as
follows.
[0303] A.2. 1 Materials and Methods
[0304] The algae were harvested by filtration and washed with 0.9%
NaCl. The cells were broken by homogenization followed by
sonication on ice for 6.times.3 min in 50 mM citrate-NaOH pH 6.2
(Buffer A). Cell debris were removed by centrifugation at
25,000.times.g for 40 min. The supernatant obtained at this
procedure was regarded as cell-free extract and was used for
activity staining and Western blotting after separation on 8-25 %
gradient gels.
[0305] A.2.2 Separation by .beta.-Cyclodextrin Sepharose Gel
[0306] The cell-free extract was applied directly to a
.beta.-cyclodextrin Sepharose gel 4B column (2.6.times.18 cm) pre
equilibrated with Buffer A. The column was washed with 3 volumes of
Buffer A and 2 volumes of Buffer A containing 1 M NaCl.
.alpha.-1,4-glucan lyase was eluted with 2 % dextrins in Buffer A.
Active fractions were pooled and the buffer changed to 20 mM
Bis-tris propane-HCl (pH 7.0, Buffer B).
[0307] Active fractions were applied onto a Mono Q HR 5/5 column
pre-equilibrated with Buffer B. The fungal lyase was eluted with
Buffer B in a linear gradient of 0.3 M NaCl.
[0308] The lyase preparation obtained after -cyclodextrin Sepharose
chromatography was alternatively concentrated to 150 .mu.l and
applied on a Superose 12 column operated under FPLC conditions.
[0309] A.2.3 Assay for .alpha.-1,4-Glucan Lyase Activity and
Conditions for Determination of Substrate Specificity, pH and
Temperature Optimum
[0310] The reaction mixture for the assay of the .alpha.-1,4-glucan
lyase activity contained 10 mg ml.sup.-1 amylopectin and 25 mM
Mes-NaOH (pH 6.0). The reaction was carried out at 30.degree. C.
for 30 min and stopped by the addition of 3,5-dinitrosalicylic acid
reagent. Optical density at 550 nm was measured after standing at
room temperature for 10 min.
[0311] A.3. Amino Acid Sequencing of the .alpha.-1.4-Glucan Lyase
from Fungus Infected Gracilariopsis lemaneiformis
[0312] A.3.1 Amino Acid Sequencing of the Lyases
[0313] The lyases were digested with either endoproteinase Arg-C
from Clostridium histolyticum or endoproteinase Lys-C from
Lysobacter enzymogenes, both sequencing grade purchased from
Boehringer Mannheim, Germany. For digestion with endoproteinase
Arg-C, freeze dried lyase (0.1 mg) was dissolved in 50 .mu.M urea,
50 mM methylamine, 0.1 M Tris-HCl, pH 7.6. After overlay with
N.sub.2 and addition of 10 .mu.l of 50 mM DTT and 5 mM EDTA the
protein was denatured and reduced for 10 min at 50.degree. C. under
N.sub.2. Subsequently, 1 .mu.g of endoproteinase Arg-C in 10 .mu.l
of 50 mM Tris-HCl, pH 8.0 was added, N.sub.2 was overlayed and the
digestion was carried out for 6 h at 37.degree. C. For subsequent
cysteine derivatization, 12.5 .mu.l 100 mM iodoacetamide was added
and the solution was incubated for 15 min at RT in the dark under
N.sub.2.
[0314] For digestion with endoproteinase Lys-C, freeze dried lyase
(0.1 mg) was dissolved in 50 .mu.l of 8 M urea, 0.4 M
NH.sub.4HCO.sub.3, pH 8.4. After overlay with N.sub.2 and addition
of 5 .mu.l of 45 mM DTT, the protein was denatured and reduced for
15 min at 50.degree. C. under N.sub.2. After cooling to RT, 5 .mu.l
of 100 mM idoacetamide was added for the cysteines to be
derivatized for 15 min at RT in the dark under N.sub.2.
[0315] Subsequently, 90 .mu.l of water and 5 .mu.g of
endoproteinase Lys-C in 50 .mu.l of 50 mM tricine and 10 mM EDTA,
pH 8.0, was added and the digestion was carried out for 24 h at
37.degree. C. under N.sub.2.
[0316] The resulting peptides were separated by reversed phase HPLC
on a VYDAC C18 column (0.46.times.15 cm; 10 .mu.m; The Separations
Group; California) using solvent A: 0.1% TFA in water and solvent
B: 0.1% TFA in acetonitrile. Selected peptides were
rechromatographed on a Develosil C18 column (0.46.times.10 cm; 3
.mu.m; Dr. Ole Schou, Novo Nordisk, Denmark) using the same solvent
system prior to sequencing on an Applied Biosystems 476A sequencer
using pulsed-liquid fast cycles.
[0317] The amino acid sequence information from the enzyme derived
from fungus infected Gracilariopsis lemaneiformis is shown below,
in particular SEQ. ID. No. 1. and SEQ. ID. No. 2.
[0318] SEQ. I.D. No. 1 has:
[0319] Number of residues: 1088.
[0320] Amino acid composition (including the signal sequence)
23 61 Ala 15 Cys 19 His 34 Met 78 Thr 51 Arg 42 Gln 43 Ile 53 Phe
24 Trp 88 Asn 53 Glu 63 Leu 51 Pro 58 Tyr 79 Asp 100 Gly 37 Lys 62
Ser 77 Val
[0321] SEQ. I.D. No. 2 has:
[0322] Number of residues: 1091.
[0323] Amino acid composition (including the signal sequence)
24 58 Ala 16 Cys 14 His 34 Met 68 Thr 57 Arg 40 Gln 44 Ile 56 Phe
23 Trp 84 Asn 47 Glu 69 Leu 51 Pro 61 Tyr 81 Asp 102 Gly 50 Lys 60
Ser 76 Val
[0324] A.3.2 N-Terminal Analysis
[0325] Studies showed that the N-terminal sequence of native glucan
lyase 1 was blocked. Deblocking was achieved by treating glucan
lyase 1 blotted onto a PVDF membrane with anhydrous TFA for 30 min
at 40.degree. C. essentially as described by LeGendre et al. (1993)
[Purification of proteins and peptides by SDS-PAGE; In: Matsudaira,
P. (ed.) A practical guide to protein and peptide purification for
microsequencing, 2nd edition; Academic Press Inc., San Diego; pp.
74-101.]. The sequence obtained was TALSDKQTA, which matches the
sequence (sequence position from 51 to 59 of SEQ. I.D. No. 1)
derived from the clone for glucan lyase 1 and indicates
N-acetylthreonine as N-terminal residue of glucan lyase 1. Sequence
position 1 to 50 of SEQ. I.D. No. 1 represents a signal
sequence.
[0326] A.4. DNA Sequencing of Genes Coding for the
.alpha.-1.4-Glucan Lyase from Fungus Infected Gracilariopsis
lemaneiformis
[0327] A.4.1 Methods for Molecular Biology
[0328] DNA was isolated as described by Saunders (1993) with the
following modification: The polysaccharides were removed from the
DNA by ELUTTP-D (Schleicher & Schuell) purification instead of
gel purification. (Ref:Saunders, G. W. (1993). Gel purification of
red algal genomic DNA: An inexpensive and rapid method for the
isolation of PCR-friendly DNA. Journal of phycology 29(2): 251-254
and Schleicher & Schuell: ELUTIP-d. Rapid Method for
Purification and Concentration of DNA.)
[0329] A.4.2 PCR
[0330] The preparation of the relevant DNA molecule was done by use
of the Gene Amp DNA Amplification Kit (Perkin Elmer Cetus, USA) and
in accordance with the manufactures instructions except that the
Taq polymerase was added later (see PCR cycles) and the temperature
cycling was changed to the following:
25 PCR cycles: no of cycles C time (min.) 1 98 5 60 5 addition of
Taq polymerase and oil 35 94 1 47 2 72 3 1 72 20
[0331] A.4.3 Cloning of PCR Fragments
[0332] PCR fragments were cloned into pT7Blue (from Novagen)
following the instructions of the supplier.
[0333] A.4.4 DNA Sequencing
[0334] Double stranded DNA was sequenced essentially according to
the dideoxy method of Sanger et al. (1979) using the Auto Read
Sequencing Kit (Pharmacia) and the Pharmacia LKB A.L.F. DNA
sequencer. (Ref.: Sanger, F., Nicklen, S. and Coulson, A. R.(1979).
DNA sequencing with chain-determinating inhibitors. Proc. Natl.
Acad. Sci. USA 74: 5463-5467.)
[0335] The sequences are shown as SEQ. I.D. Nos. 1 and 2. In
brief:
[0336] SEQ. I.D. No. 3 has:
[0337] Total number of bases: 3267.
[0338] DNA sequence composition: 850 A; 761 C; 871 G; 785 T
[0339] SEQ. I.D. No. 4 has:
[0340] Total number of bases: 3276.
[0341] DNA sequence composition: 889 A; 702 C; 856 G; 829 T
[0342] A.4.5 Screening of the Library
[0343] Screening of the Lambda Zap library obtained from
Stratagene, was performed in accordance with the manufacturer's
instructions except that the prehybridization and hybridization was
performed in 2.times. SSC, 0.1% SDS, 10.times. Denhardt.times.s and
100 .mu.g/ml denatured salmon sperm DNA. To the hybridization
solution a 32P-labeled denatured probe was added. Hybridization was
performed over night at 55.degree. C. The filters were washed twice
in 2.times. SSC 0.1% SDS and twice in 1.times. SSC, 0.1% SDS.
[0344] A.4.6 Probe
[0345] The cloned PCR fragments were isolated from the pT7 blue
vector by digestion with appropriate restriction enzymes. The
fragments were separated from the vector by agarose gel
electrophoresis and the fragments were purified from the agarose by
Agarase (Boehringer Mannheim). As the fragments were only 90-240 bp
long the isolated fragments were exposed to a ligation reaction
before labelling with 32P-dCTP using either Prime-It random primer
Et (Stratagene) or Ready to Go DNA labelling kit (Pharmacia).
[0346] A.4.7 Results
[0347] A.4.7.1 Generation of PCR DNA Fragments Coding for
.alpha.-1,4-Glucan Lyase.
[0348] The amino acid sequences of three overlapping tryptic
peptides from .alpha.-1,4-glucan lyase were used to generate mixed
oligonucleotides, which could be used as PCR primers (see the
sequences given above).
[0349] In the first PCR amplification primers A/B (see above) were
used as upstream primers and primer C (see above) was used as
downstream primer. The size of the expected PCR product was 71 base
pairs.
[0350] In the second PCR amplification primers A/B were used as
upstream primers and E was used as downstream primer. The size of
the expected PCR product was 161 base pairs.
[0351] In the third PCR amplification primers F1 (see above) and F2
(see above) were used as upstream primers and E was used as
downstream primer. The size of the expected PCR product was 238
base pairs.
[0352] The PCR products were analysed on a 2% LMT agarose gel and
fragments of the expected sizes were cut out from the gel and
treated with Agarase (Boehringer Manheim) and cloned into the
pT7blue Vector (Novagen) and sequenced.
[0353] The cloned fragments from the first and second PCR
amplification coded for amino acids corresponding to the sequenced
peptides (see above). The clone from the third amplification (see
above) was only about 87% homologous to the sequenced peptides.
[0354] A.4.7.2 Screening of the Genomic Library with the Cloned PCR
Fragments.
[0355] Screening of the library with the above-mentioned clones
gave two clones. One clone contained the nucleotide sequence of SEQ
I.D. No. 4 (gene 2). The other clone contained some of the sequence
of SEQ I.D. No. 3 (from base pair 1065 downwards) (gene 1).
[0356] The 5' end of SEQ. I.D. No. 3 (i.e. from base pair 1064
upwards) was obtained by the RACE (rapid amplification of cDNA
ends) procedure (Michael, A. F., Michael, K. D. & Martin, G.
R.(1988). Proc. Natl. Acad. Sci. USA 85:8998-99002.) using the 5'
race system from Gibco BRL. Total RNA was isolated according to
Collinge et al. (Collinge, D. B., Milligan D. E:, Dow, J. M.,
Scofield, G. & Daniels, M. J.(1987). Plant Mol Biol 8:
405-414). The 5' race was done according to the protocol of the
manufacturer, using lug of total RNA. The PCR product from the
second amplification was cloned into pT7blue vector from Novagen
according to the protocol of the manufacturer. Three independent
PCR clones were sequenced to compensate for PCR errors.
[0357] An additional PCR was performed to supplement the clone just
described with XbaI and NdeI restriction sites immediately in front
of the ATG start codon using the following oligonucleotide as an
upstream primer:
26 GCTCTAGAGCATGTTTTCAACCCTTGCG
[0358] and a primer containing the complement sequence of bp
1573-1593 in sequence GL1 (i.e. SEQ. I.D. No. 3) was used as a
downstream primer.
[0359] The complete sequence for gene 1 (i.e. SEQ. I.D. No. 3) was
generated by cloning the 3' end of the gene as a BamHI-HindIII
fragment from the genomic clone into the pBluescript It KS+ vector
from Stratagene and additionally cloning the PCR generated 5' end
of the gene as a XbaI-BamIE fragment in front of the 3' end.
[0360] Gene 2 was cloned as a HindIII blunt ended fragment into the
EcoRV site of pBluescript II SK+ vector from Stratagene. A part of
the 3' untranslated sequence was removed by a Sacd digestion,
followed by religation. HindIII and HpaI restriction sites were
introduced immediately in front of the start ATG by digestion with
HindIII and NarI and religation in the presence of the following
annealed oligonucleotides
27 AGCTTGTTAACATGTATCCAACCCTCACCTTCGTGG
ACAATTGTACATAGGTTGGGAGTGGAAGCACCGC
[0361] No introns were found in the clones sequenced.
[0362] The clone 1 type (SEQ. ID. No. 3) can be aligned with all
ten peptide sequences (see FIG. 8) showing 100% identity. Alignment
of the two protein sequences encoded by the genes isolated from the
fungal infected algae Gracilariopsis lemaneiformis shows about 78%
identity, indicating that both genes are coding for a
.alpha.-1,4-glucan lyase.
[0363] A.5. Expression of the GL Gene in Micro-Organisms (e.g.
Analyses of Pichia Lyase Transformants and Aspergillus Lyase
Transformants)
[0364] The DNA sequence encoding the GL was introduced into
microorganisms to produce an enzyme with high specific activity and
in large quantities.
[0365] In this regard, gene 1 (i.e. SEQ. I.D. No. 3) was cloned as
a NotI-HindIII blunt ended (using the DNA blunting kit from
Amersham International) fragment into the Pichia expression vector
pHIL-D2 (containing the AOX1 promoter) digested with EcoRI and
blunt ended (using the DNA blunting kit from Amersham
International) for expression in Pichia pastoris (according to the
protocol stated in the Pichia Expression Kit supplied by
Invitrogen).
[0366] In another embodiment, the gene 1 (i.e. SEQ. I.D. No. 3) was
cloned as a NotI-HindIII blunt ended fragment (using the DNA
blunting kit from Amersham International) into the Aspergillus
expression vector pBARMTE1 (containing the methyl tryptophan
resistance promoter from Neuropera crassa) digested with SmaI for
expression in Aspergillus niger (Pall et al (1993) Fungal Genet
Newslett. vol 40 pages 59-62). The protoplasts were prepared
according to Daboussi et al (Curr Genet (1989) vol 15 pp 453-456)
using lysing enzymes Sigma L-2773 and the lyticase Sigma L-8012.
The transformation of the protoplasts was followed according to the
protocol stated by Buxton et al (Gene (1985) vol 37 pp 207-214)
except that for plating the transformed protoplasts the protocol
laid out in Punt et al (Methods in Enzymology (1992) vol 216 pp
447-457) was followed but with the use of 0.6% osmotic stabilised
top agarose.
[0367] The results showed that lyase activity was observed in the
transformed Pichia pastoris and Aspergillus niger.
[0368] A.5.1 General Methods
[0369] Preparation of Cell-Free Extracts.
[0370] The cells were harvested by centrifugation at 9000 rpm for 5
min and washed with 0.9% NaCl and resuspended in the breaking
buffer (50 mM K-phosphate, pH 7.5 containing 1 mM of EDTA, and 5%
glycerol). Cells were broken using glass beads and vortex
treatment. The breaking buffer contained 1 mM PMSF (protease
inhibitor). The lyase extract (supernatant) was obtained after
centrifugation at 9000 rpm for 5 min followed by centrifugation at
20,000.times. g for 5 min.
[0371] Assay of lyase activity by alkaline 3,5-dinitrosalicylic
acid reagent (DNS)
[0372] One volume of lyase extract was mixed with an equal volume
of 4% amylopectin solution. The reaction mixture was then incubated
at a controlled temperature and samples were removed at specified
intervals and analyzed for AF.
[0373] The lyase activity was also analyzed using a radioactive
method.
[0374] The reaction mixture contained 10 .mu.l .sup.14C-starch
solution (1 .mu.Ci; Sigma Chemicals Co.) and 10 .mu.l of the lyase
extract. The reaction mixture was left at 25.degree. C. overnight
and was then analyzed in the usual TLC system. The radioactive AF
produced was detected using an Instant Imager (Pachard Instrument
Co., Inc., Meriden, Conn.).
[0375] Electrophoresis and Western Blotting
[0376] SDS-PAGE was performed using 8-25 % gradient gels and the
PhastSystem (Pharmacia). Western blottings was also run on a
Semidry transfer unit of the PhastSystem.
[0377] Primary antibodies raised against the lyase purified from
the red seaweed collected at Qingdao (China) were used in a
dilution of 1:100. Pig antirabbit IgG conjugated to alkaline
phosphatase (Dako A/S, Glostrup, Denmark) were used as secondary
antibodies and used in a dilution of 1:1000.
[0378] Part I, Analysis of the Pichia Transformantscontaining the
Above Mentioned Construct
[0379] Results:
[0380] 1. Lyase activity was determined 5 days after induction
(according to the manual) and proved the activity to be
intracellular for all samples in the B series.
28 Samples 11 12 13 15 26 27 28 29 30 of B series: Specific 139 81
122 192 151 253 199 198 150 activity: *Specific activity is defined
as nmol AF released per min per mg protein in a reaction mixture
containing 2% (w/v) of glycogen, 1% (w/v) glycerol in 10 mM
potassium phosphate buffer (pH 7.5). The reaction temperature was
45.degree. C.; the reaction time was 60 min.
[0381] A time course of sample B27 is as follows. The data are also
presented in FIG. 1.
29 Time (min) 0 10 20 30 40 50 60 Spec. act. 0 18 54 90 147 179
253
[0382] Assay conditions were as above except that the time was
varied.
[0383] 2. Western-blotting analysis.
[0384] The CFE of all samples showed bands with a molecular weight
corresponding to the native lyase.
[0385] MC-Lyase expressed intracellularly in Pichia pastoris
30 Names of culture Specific activity* A18 10 A20 32 A21 8 A22 8
A24 6
[0386] Part II, The Aspergilus Transformants
[0387] Results
[0388] I. Lyase activity was determined after 5 days
incubation(minimal medium containing 0.2% casein enzymatic
hydrolysate analysis by the alkaline 3,5-dinitrosalicylic acid
reagent.
[0389] 1). Lyase activity analysis of the culture medium
[0390] Among 35 cultures grown with 0.2% amylopectin included in
the culture medium, AF was only detectable in two cultures. The
culture medium of 5.4+ and 5.9+ contained 0.13 g AF/liter and 0.44
g/liter, respectively. The result indicated that active lyase had
been secreted from the cells. Lyase activity was also measurable in
the cell-free extract.
[0391] 2). Lyase activity analysis in cell-free extracts
31 Name of the culture Specific activity* 5.4+ 51 5.9+ 148 5.13 99
5.15 25 5.19 37 *The specific activity was defined as nmol of AF
produced per min per mg protein at 25.degree. C. +indicates that
0.2% amylopectin was added.
[0392] The results show that Gene 1 of GL was expressed
intracellular in A. niger.
[0393] Experiments with transformed E. coli (using cloning vectors
pQE30 from the Qia express vector kit from Qiagen) showed
expression of enzyme that was recognised by anti-body to the enzyme
purified from fungally infected Gracilariopsis lemaneiformis.
[0394] B. Source=Fungus
[0395] B.1. Enzyme Purification and Characterization of the
.alpha.-1.4-Glucan Lyase from the Fungus Morchella costata
[0396] B.1.1 Materials and Methods
[0397] The fungus Morchella costata was obtained from American Type
Culture Collection (ATCC). The fungus was grown at 25.degree. C. on
a shaker using the culture medium recommended by ATCC. The mycelia
were harvested by filtration and washed with 0.9% NaCl.
[0398] The fungal cells were broken by homogenization followed by
sonication on ice for 6.times.3 min in 50 mM citrate-NaOH pH 6.2
(Buffer A). Cell debris were removed by centrifugation at
25,000.times. g for 40 min. The supernatant obtained at this
procedure was regarded as cell-free extract and was used for
activity staining and Western blotting after separation on 8-25%
gradient gels.
[0399] B. 1.2 Separation by .beta.-Cyclodextrin Sepharose Gel
[0400] The cell-free extract was applied directly to a
.beta.-cyclodextrin Sepharose gel 4B column (2.6.times.18 cm) pre
equilibrated with Buffer A. The column was washed with 3 volumes of
Buffer A and 2 volumes of Buffer A containing 1 M NaCl.
.alpha.-1,4-glucan lyase was eluted with 2% dextrins in Buffer A.
Active fractions were pooled and the buffer changed to 20 mM
Bis-tris propane-HCl (pH 7.0, Buffer B).
[0401] Active fractions were applied onto a Mono Q HR 5/5 column
pre-equilibrated with Buffer B. The fungal lyase was eluted with
Buffer B in a linear gradient of 0.3 M NaCl. The lyase preparation
obtained after .beta.-cyclodextrin Sepharose chromatography was
alternatively concentrated to 150 .mu.l and applied on a Superose
12 column operated under FPLC conditions.
[0402] B. 1.3 Assay for .alpha.-1,4-Glucan Lyase Activity and
Conditions for Determination of Substrate Specificity, pH and
Temperature Optimum
[0403] The reaction mixture for the assay of the .alpha.-1,4-glucan
lyase activity contained 10 mg ml.sup.-1 amylopectin and 25 mM
Mes-NaOH (pH 6.0).
[0404] The reaction was carried out at 30.degree. C. for 30 min and
stopped by the addition of 3,5-dinitrosalicylic acid reagent.
Optical density at 550 nm was measured after standing at room
temperature for 10 min. 10 mM EDTA was added to the assay mixture
when cell-free extracts were used.
[0405] The substrate amylopectin in the assay mixture may be
replaced with other substrates and the reaction temperature may
vary as specified in the text.
[0406] In the pH optimum investigations, the reaction mixture
contained amylopection or maltotetraose 10 mg ml.sup.-1 in a 40 mM
buffer. The buffers used were glycine-NaOH (pH 2.0-3.5), HoAc-NaoAc
(pH 3.5-5.5), Mes-NaOH (pH 5.5-6.7), Mops-NaOH (6.0-8.0) and
bicine-NaOH (7.69.0). The reactions were carried out at 30.degree.
C. for 30 min. The reaction conditions in the temperature optimum
investigations was the same as above except that the buffer
Mops-NaOH (pH 6.0) was used in all experiments. The reaction
temperature was varied as indicated in the text.
[0407] SDS-PAGE, Native-PAGE and isoelectrofocusing were performed
on PhastSystem (Pharmacia, Sweden) using 8-25% gradient gels and
gels with a pH gradient of 3-9, respectively. Following
electrophoresis, the gels were stained by silver staining according
to the procedures recommended by the manufacturer (Pharmacia). The
glycoproteins were stained by PAS adapted to the PhastSystem. For
activity staining, the electrophoresis was performed under native
conditions at 6.degree. C.
[0408] Following the electrophoresis, the gel was incubated in the
presence of 1% soluble starch at 30.degree. C. overnight. Activity
band of the fungal lyase was revealed by staining with I.sub.2/KI
solution.
[0409] B. 1.4 Results
[0410] B.1.4.1 Purification, Molecular Mass and Isoelectric Point
of the .alpha.-1,4-Glucan Lyase
[0411] The fungal lyase was found to adsorb on columns packed with
.beta.-cyclodextrin Sepharose, starches and Red Sepharose. Columns
packed with .beta.-cyclodextrin Sepharose 4B gel and starches were
used for purification purposes.
[0412] The lyase preparation obtained by this step contained only
minor contaminating proteins having a molecular mass higher than
the fungal lyase. The impurity was either removed by ion exchange
chromatography on Mono Q HR 5/5 or more efficiently by gel
filtration on Superose 12.
[0413] The purified enzyme appeared colourless and showed no
absorbance in the visible light region. The molecular mass was
determined to 110 kDa as estimated on SDS-PAGE.
[0414] The purified fungal lyase showed a isoelectric point of pI
5.4 determined by isoelectric focusing on gels with a pH gradient
of 3 to 9. In the native electrophoresis gels, the enzyme appeared
as one single band. This band showed starch-degrading activity as
detected by activity staining. Depending the age of the culture
from which the enzyme is extracted, the enzyme on the native and
isoelectric focusing gels showed either as a sharp band or a more
diffused band with the same migration rate and pI.
[0415] B. 1.4.2 The pH and Temperature Optimum of the Fungal Lyase
Catalayzed Reaction
[0416] The pH optimum pH range for the fungal lyase catalyzed
reaction was found to be between pH 5 and pH 7.
[0417] B. 1.4.3 Substrate Specificity
[0418] The purified fungal lyase degraded maltosaccharides from
maltose to maltoheptaose. However, the degradation rates varied.
The highest activity achieved was with maltotetraose (activity as
100%), followed by maltohexaose (97%), maltoheptaose (76%),
maltotriose (56%) and the lowest activity was observed with maltose
(2%).
[0419] Amylopectin, amylose and glycogen were also degraded by the
fungal lyase (% will be determined). the fungal lyase was an
exo-lyase, not a endolyase as it degraded p-nitrophenyl
.alpha.-D-maltoheptaose but failed to degrade reducing end blocked
p-nitrophenyl .alpha.-D-maltoheptaose.
[0420] B. 1.5 Morchella Vulgaris
[0421] The protocols for the enzyme purification and
charaterisation of alpha 1,4-glucal lyase obtained from Morchella
vulgaris were the same as those above for Morchella costata (with
similar results).
[0422] B.2. Amino Acid Sequencing of the .alpha.-1,4-Glucan Lyase
from Fungus
[0423] B.2.1 Amino Acid Sequencing of the Lyases
[0424] The lyases were digested with either endoproteinase Arg-C
from Clostridium histolyticum or endoproteinase Lys-C from
Lysobacter enzymogenes, both sequencing grade purchased from
Boehringer Mannheim, Germany. For digestion with endoproteinase
Arg-C, freezedried lyase (0.1 mg) was dissolved in 50 .mu.l 10 M
urea, 50 mM methylamine, 0.1 M Tris-HCl, pH 7.6. After overlay with
N.sub.2 and addition of 10 .mu.l of 50 mM DTT and 5 mM EDTA the
protein was denatured and reduced for 10 min at 50.degree. C. under
N.sub.2. Subsequently, 1 .mu.g of endoproteinase Arg-C in 10 .mu.l
of 50 mM Tris-HCl, pH 8.0 was added, N.sub.2 was overlayed and the
digestion was carried out for 6 h at 37.degree. C.
[0425] For subsequent-cysteine derivatization, 12.5 .mu.l 100 mM
iodoacetamide was added and the solution was incubated for 15 min
at RT in the dark under N.sub.2.
[0426] For digestion with endoproteinase Lys-C, freeze dried lyase
(0.1 mg) was dissolved in 50 ml of 8 M urea, 0.4 M
NH.sub.4HCO.sub.3, pH 8.4. After overlay with N.sub.2 and addition
of 5 .mu.l of 45 mM DTT, the protein was denatured and reduced for
15 min at 50.degree. C. under N.sub.2. After cooling to RT, 5 .mu.l
of 100 mM iodoacetamide was added for the cysteines to be
derivatize for 15. min at RT in the dark under N.sub.2.
Subsequently, 90 .mu.l of water and 5 .mu.g of endoproteinase Lys-C
in 50 .mu.l of 50 mM tricine and 10 mM EDTA, pH 8.0, was added and
the digestion was carried out for 24 h at 37.degree. C. under
N.sub.2.
[0427] The resulting peptides were separated by reversed phase HPLC
on a VYDAC C18 column (0.46.times.15 cm; 10 .mu.m; The Separations
Group; California) using solvent A: 0.1% TFA in water and solvent
B: 0.1% TFA in acetonitrile. Selected peptides were
rechromatographed on a Develosil C18 column (0.46.times.10 cm; 3
.mu.m; Dr. Ole Schou, Novo Nordisk, Denmark) using the same solvent
system prior to sequencing on an Applied Biosystems 476A sequencer
using pulsed-liquid fast cycles.
[0428] The amino acid sequence information from the enzyme derived
from the fungus Morchella costata is shown FIG. 17.
[0429] The amino acid sequence information from the enzyme derived
from the fungus Morchella vulgaris is shown FIG. 18.
[0430] B.3. DNA Sequencing of Genes Coding for the
.alpha.-1,4Glucan Lyase from Fungus
[0431] B.3.1 Methods for Molecular Biology
[0432] DNA was isolated as described by Dellaporte et al
(1983--Plant Mol Biol Rep vol 1 pp19-21).
[0433] B.3.2 PCR
[0434] The preparation of the relevant DNA molecule was done by use
of the Gene Amp DNA Amplification Kit (Perkin Elmer Cetus, USA) and
in accordance with the manufactures instructions except that the
Taq polymerase was added later (see PCR cycles) and the temperature
cycling was changed to the following:
32 PCR cycles: no of cycles C time (min.) 1 98 5 60 5 addition of
Taq polymerase and oil 35 94 1 47 2 72 3 1 72 20
[0435] B.3.3 Cloning of PCR Fragments
[0436] PCR fragments were cloned into pT7Blue (from Novagen)
following the instructions of the supplier.
[0437] B.3.4 DNA Sequencing
[0438] Double stranded DNA was sequenced essentially according to
the dideoxy method of Sanger et al. (1979) using the Auto Read
Sequencing Kit (Pharmacia) and the Pharmacia LKB A.L.F. DNA
sequencer. (Ref: Sanger, F., Nicklen, S. and Coulson, A. R.(1979).
DNA sequencing with chain-determinating inhibitors. Proc. Natl.
Acad. Sci. USA 74: 5463-5467.)
[0439] B.3.5 Screening of the Libraries
[0440] Screening of the Lambda Zap libraries obtained from
Stratagene, was performed in accordance with the manufacturer's
instructions except that the prehybridization and hybridization was
performed in 2.times. SSC, 0.1% SDS, 10.times. Denhardt's and 100
.mu.g/ml denatured salmon sperm DNA.
[0441] To the hybridization solution a 32P-labeled denatured probe
was added. Hybridization was performed over night at 55.degree. C.
The filters were washed twice in 2.times. SSC, 0.1% SDS and twice
in 1.times. SSC, 0.1% SDS.
[0442] B.3.6 Probe
[0443] The cloned PCR fragments were isolated from the pT7 blue
vector by digestion with appropriate restriction enzymes. The
fragments were seperated from the vector by agarose gel
electrophoresis and the fragments were purified from the agarose by
Agarase (Boehringer Mannheim). As the fragments were only 90-240 bp
long the isolated fragments were exposed to a ligation reaction
before labelling with 32P-dCTP using either Prime-It random primer
kit (Stratagene) or Ready to Go DNA labelling kit (Pharmacia).
[0444] B.3.7 Results
[0445] B.3.7. 1 Generation of PCR DNA Fragments Coding for
.alpha.-1,4-Glucan Lyase.
[0446] The amino acid sequences (shown below) of three overlapping
tryptic peptides from .alpha.-1,4-glucan lyase were used to
generate mixed oligonucleotides, which could be used as PCR primers
for amplification of DNA isolated from both MC and MV.
33 Lys Asn Leu His Pro Gln His Lys Met Leu Lys Asp Thr Val Leu Asp
Ile Val Lys Pro Gly His Gly Glu Tyr Val Gly Trp Gly Glu Met Gly Gly
Ile Gln Phe Met Lys Glu Pro Thr Phe Met Asn Tyr Phe Asn Phe Asp Asn
Met Gln Tyr Gln Gln Val Tyr Ala Gln Gly Ala Leu Asp Ser Arg Glu Pro
Leu Tyr His Ser Asp Pro Phe Tyr
[0447] In the first PCR amplification primers A1/A2 (see below)
were used as upstream primers and primers B1/B2 (see below) were
used as downstream primer.
34 Primer A1: CA(GA)CA(CT)AA(GA)ATGCT(GATC)AA(GA)GA(CT)A- C Primer
A2: CA(GA)CA(CT)AA(GA)ATGTT(GA)AA(GA)GA- (CT)AC Primer B1:
TA(GA)AA(GATC)GG(GA)TC(GA)CT(G- A)TG(GA)TA Primer B2:
TA(GA)AA(GATC)GG(GA)TC(GAT- C)GA(GA)TG(GA)TA
[0448] The PCR products were analysed on a 2% LMT agarose gel and
fragments of the expected sizes were cut out from the gel and
treated with Agarase (Boehringer Manheim) and cloned into the
pT7blue Vector (Novagen) and sequenced.
[0449] The cloned fragments from the PCR amplification coded for
amino acids corresponding to the sequenced peptides (see above) and
in each case in addition to two intron sequences. For MC the PCR
amplified DNA sequence corresponds to the sequence shown as from
position 1202 to position 1522 with reference to FIG. 14. For MV
the PCR amplified DNA sequence corresponds to the sequence shown as
from position 1218 to position 1535 with reference to FIG. 15.
[0450] B.3.7.2 Screening of the Genomic Libraries with the Cloned
PCR Fragments.
[0451] Screening of the libraries with the above-mentioned clone
gave two clones for each source. For MC the two clones were
combined to form the sequence shown in FIG. 14 (see below). For MV
the two clones could be combined to form the sequence shown in FIG.
15 in the manner described above.
[0452] An additional PCR was performed to supplement the MC clone
with PstI, PvuII, AscI and NcoI restriction sites immediately in
front of the ATG start codon using the following oligonucleotide as
an upstream primer:
35 AAACTGCAGCTGGCGCGCCATGGCAGGATTTTCTGAT
[0453] and a primer containing the complement sequence of bp
1297-1318 in FIG. 4 was used as a downstream primer.
[0454] The complete sequence for MC was generated by cloning the 5'
end of the gene as a BglII-EcoRI fragment from one of the genomic
clone (first clone) into the BamHI-EcoRI sites of pBluescript II
KS+ vector from Stratagene. The 3' end of the gene was then cloned
into the modified pBluescript II KS+vector by ligating an NspV
(blunt ended, using the DNA blunting kit from Amersham
International)-EcoRI fragment from the other genomic clone (second
clone) after the modified pBluescript II KS+ vector had been
digested with EcoRI and EcoRV. Then the intermediate part of the
gene was cloned in to the further modified pBluescript II KS+
vector as an EcoRI fragment from the first clone by ligating that
fragment into the further modified pBluescript II KS+ vector
digested with EcoRI.
[0455] B.4. Expression of the GL Gene in Micro-Organisms
[0456] The DNA sequence encoding the GL can be introduced into
microorganisms to produce the enzyme with high specific activity
and in large quantities.
[0457] In this regard, the MC gene (FIG. 14) was cloned as a
XbaI-XhoI blunt ended (using the DNA blunting kit from Amersham
International) fragment into the Pichia expression vector pHIL-D2
(containing the AOX1 promoter) digested with EcoRI and blunt ended
(using the DNA blunting kit from Amersham International) for
expression in Pichia pastoris (according to the protocol stated in
the Pichia Expression Kit supplied by Invitrogen).
[0458] In another embodiment, the MC gene 1 (same as FIG. 14 except
that it was modified by PCR to introduce restriction sites as
described above) was cloned as a PvuII-XhoI blunt ended fragment
(using the DNA blunting kit from Amersham International) into the
Aspergillus expression vector pBARMTE1 (containing the methyl
tryptophan resistance promoter from Neuropera crassa) digested with
SmaI for expression in Aspergillus niger (Pall et al (1993) Fungal
Genet Newslett. vol 40 pages 59-62). The protoplasts were prepared
according to Daboussi et al (Curr Genet (1989) vol 15 pp 453456)
using lysing enzymes Sigma L-2773 and the lyticase Sigma L-8012.
The transformation of the protoplasts was followed according to the
protocol stated by Buxton et al (Gene (1985) vol 37 pp 207-214)
except that for plating the transformed protoplasts the protocol
laid out in Punt et al (Methods in Enzymology (1992) vol 216 pp
447-457) was followed but with the use of 0.6% osmotic stabilised
top agarose.
[0459] The results showed that lyase activity was observed in the
transformed Pichia pastoris and Aspergillus niger.
[0460] Analyses of Pichia Lyase Transformants and Aspergillus Lyase
Transformants
[0461] General Methods
[0462] Preparation of Cell-Free Extracts.
[0463] The cells were harvested by centrifugation at 9000 rpm for 5
min and washed with 0.9% NaCl and resuspended in the breaking
buffer (50 mM K-phosphate, pH 7.5 containing 1 mM of EDTA, and 5%
glycerol). Cells were broken using glass beads and vortex
treatment. The breaking buffer contained 1 mM PMSF (protease
inhibitor). The lyase extract (supernatant) was obtained after
centrifugation at 9000 rpm for 5 min followed by centrifugation at
20,000.times. g for 5 min.
[0464] Assay of lyase activity by alkaline 3,5-dinitrosalicylic
acid reagent (DNS)
[0465] One volume of lyase extract was mixed with an equal volume
of 4% amylopectin solution. The reaction mixture was then incubated
at a controlled temperature and samples were removed at specified
intervals and analyzed for AF.
[0466] The lyase activity was also analyzed using a radioactive
method.
[0467] The reaction mixture contained 10 .mu.l .sup.4C-starch
solution (1 .mu.Ci; Sigma Chemicals Co.) and 10 .mu.l of the lyase
extract. The reaction mixture was left at 25.degree. C. overnight
and was then analyzed in the usual TLC system. The radioactive AF
produced was detected using an Instant Imager (Pachard Instrument
Co., Inc., Meriden, Conn.).
[0468] Electrophoresis and Western Blotting
[0469] SDS-PAGE was performed using 8-25% gradient gels and the
PhastSystem (Pharmacia). Western blottings was also run on a
Semidry transfer unit of the PhastSystem. Primary antibodies raised
against the lyase purified from the red seaweed collected at
Qingdao (China) were used in a dilution of 1:100. Pig antirabbit
IgG conjugated to alkaline phosphatase (Dako A/S, Glostrup,
Denmark) were used as secondary antibodies and used in a dilution
of 1:1000.
[0470] Part I, Analysis of the Pichia Transformantscontaining the
Above Mentioned Construct
[0471] MC-Lyase expressed intracellularly in Pichia pastoris
36 Names of culture Specific activity* A18 10 A20 32 A21 8 A22 8
A24 6 *The specific activity was defined as nmol of AF produced per
min per mg protein at 25.degree. C.
[0472] Part II, The Aspergilus Transformants
[0473] Results
[0474] I. Lyase activity was determined after 5 days
incubation(minimal medium containing 0.2% casein enzymatic
hydrolysate analysis by the alkaline 3,5-dinitrosalicylic acid
reagent
[0475] Lyase activity analysis in cell-free extracts
37 Name of the culture Specific activity* 8.13 11 8.16 538 8.19 37
*The specific activity was defined as nmol of AF produced per min
per mg protein at 25.degree. C.
[0476] The results show that the MC-lyase was expressed
intracellular in A. niger.
[0477] II. Lyase activity test by radioactive method
[0478] The cell-free extracts of the following cultures contained
.sup.14C labelled AF
[0479] 51+, 54+, 55+, 59+, 512, 513, 514, 515, 516, 518, 519.
[0480] The TLC of the degradation products of the
.alpha.-1,4-glucan lyase reaction using .sup.14C-starch as
substrate are shown in FIG. 20. The reaction mixture was applied on
the TLC. The lane number corresponds to the name of the culture: 1,
512; 2, 513; 3, 514; 4, 515; 5, 516; 6, 517; 7, 518; 8, 519; 9,
520. The fast moving spots are AF.
[0481] C. Source=Algae Alone
[0482] The protocols for the enzyme purification and
charaterisation of alpha 1,4-glucal lyase obtained from
Gracilarioposis lemaneiformis (as obtained from Santa Cruz) were
essentially the same as those described above for, for example,
Morchella costata (with similar results).
[0483] 1. Characterization of .alpha.-1,4-glucan lyase from the
parasite-free red seaweed Gracilariopsis lemaneiformis collected in
California.
[0484] The amino acid composition of the lyase is given in the
following table.
38 Amino acid residues mol % of each residue Asx 15.42 Thr 5.24 Ser
6.85 Glx 9.46 Pro 5.46 Gly 9.08 Ala 5.38 1/2Cys 1.57 Val 6.60 Met
2.90 Ile 3.66 Leu 6.00 Tyr 6.00 Phe 4.37 His 1.65 Lys 4.44 Arg 4.17
Trp 1.75 Total: 100.00
[0485] 2. Sequence Analysis
[0486] Comparison of the peptide sequences from the Californian
algae with the amino acid sequence from the fungally infected algae
from China showed a high degree of homology (78 to 80% identity
between the amino acid sequence generated from the PCR fragments
and the corresponding sequences in the GL obtained from the algae
from China) between the two protein sequences.
[0487] Three Oligonucleotides was generated from these two
sequences from the Californian algae to generate a PCR fragment of
app. 970 bp.
39 Primer 1: ATGAC(GATC)AA(CT)TA(CT)AA(CT)TA(CT)GA(CT)AA Primer 2:
(AG)TG(GATC)GGCATCAT(GATC)GC(GATC)GG(- GATC)AC Primer 3:
GTCAT(GA)TC(CT)TGCCA(GATC)AC(G- A)AA(GA)TC
[0488] Primer 1 was used as the upstream primer and primer 2 was
used as the downstream primer in the first PCR amplification. In
the second PCR amplification primer 1 was used as the upstream
primer and primer 3 was used as the downstream primer. A PCR
fragment of the expected size was generated and cloned into the
pT7blue vector from Novagen. Three independent plasmids containing
a PCR fragment were sequenced and it was seen that these three
cloned PCR fragments contained the codons for peptide sequences
originating from three different proteins. This indicates that
there are at least three different genes coding for
.alpha.-1,4-glucan lyase in the Californian algae.
[0489] 3. The substrate concentration at which half of the maximal
velocity rate was reached is 3.76 mg/ml for amylopectin and 3.37
mg/ml for glycogen.
[0490] 4. The degradation rates of the lyase on various substrates
are given below.
40 Substrate AF released (nmol) Maltose 657 Maltotriose 654
Maltotetraose 670 Maltopentaose 674 Maltohexaose 826 Maltoheptaose
865 Dextrin 20 775 Dextrin 15 775 Dextrin 10 844 Amylopectin 732
Glycogen 592
[0491] Reaction conditions: The reaction mixture contained 10 mM of
HOAc--NaOAc (pH 3.8). The substrate concentration was 10 mg/ml. The
final volume was 100 ul after the addition of lyase and water. The
reaction time was 40 min at 45.degree. C.
[0492] The lyase was not able to degrade pullulan, nigeran
tetrasaccharide, trehalose, isomaltose, glucose, .alpha.-, .beta.-
and r-cyclodextrins. The lyase degraded panose and nigerose though
at a slow rate.
[0493] 5. The temperature optimum for the lyase was 48.degree. C.
when amylopectin was used as substrate and 50.degree. C. when
glycogen was used as substrate. At 50.degree. C., the reactivity of
glycogen was similar to that of amylopectin; below 50.degree. C.,
amylopectin was a better substrate than glycogen.
[0494] 6. The pH optimum range for the lyase was between pH 3.5 and
pH 7.0; the optimal pH was 3.8. The buffers used in the pH tests
were glycine-HCl (pH 2.2-3.6); NaOAc--HOAc (pH 3.5-5.5); Mes-NaOH
(pH5.5-6.7); Mops-NaOH (pH 6.0-8.0) and bicine-NaOH (pH 7.69.0).
All buffers used were 40 mM.
[0495] 7. At a final concentration of 2 mM, p-chloromercuribenzoic
acid (PCMB) inhibited the lyase activity by 96%, indicating the
--SH group(s) is essential for the enzymatic activity.
[0496] 7. Further Studies
[0497] 7.1 Effect of Alcohols in Increasing the Activity and
Stability of the Lyase Purified from the Fungal Infected Algae.
[0498] 1-propanol, 2-propanol and 1-butanol were tested at the
following concentrations (0%, 1%, 5% and 10%). The optimal
concentration of 1-propanol was 5% which increased the AF yield by
34% after 6 days of incubation; the optimal concentration for
2-propanol was 1% which increased the AF yield by 20% after 10 days
incubation; the optimal concentration for 1-butanol was 5% which
increased the AF yield by 52% after 3-day incubation.
[0499] Ethanol was tested at the following concentrations (0, 1, 3,
5, 7, 9, 11, 13, 15%). The optimal concentration for 7 days
incubation was 5% which increased the AF yield by 12%. For 10 days
incubation the optimal concentration was 3% which increased AF
yield by 16%.
[0500] The effect of 1-propanol:
41 1-propanol Reaction time (days) concentraction 0 1 3 6 10 (v/v)
AF yield (.mu.mol) 0% 0 84 261 451 689 1% 0 80 280 530 803 5% 0 115
367 605 853 10% 0 107 307 456 583
[0501] 7.2 Effect of Different Reaction Media Upon the Production
of AF by the Lyase Purified from the Fungal Infected Algae and the
Fugnal Lyase from M. costata and M. vulgars.
[0502] 2.1. The lyase from the fungal infected algae.
[0503] The results (see table below) indicate that the best
reaction medium is 5 mM of HOAc--NaOAc (pH 3.9) (BACE for short)
and containing mM concentrations of Na.sub.2-EDTA. The production
of AF using either pure water or 0.85% NaCl as reaction medium
decreased the yield. Inclusion of 0.85 % of NaCl in BACE also
decreased the AF yield.
42 Reaction Time (days) Reaction 0 1 3 8 Media AF yield (.mu.mol)
BACE 0 229 498 575 Water 0 46 128 217 NaCl (0.85%) 0 123 239 249
BACE + NaCl (0.85%) 0 153 281 303
[0504] 2.2. The following buffers: Mes-NaOH, Mops-NaOH. Hepes-NaOH,
and Bicine-NaOH were the optimal reaction media for the lyase from
M. costata and M. vulgaris. In the HOAc--NaOAc buffer, the lyase
was unstable and therefore use of this buffer system caused a
decrease in AF yield.
[0505] 7.3. The Effect of Endoamylases and Debranching Enzymes Upon
the AF Production.
[0506] 3.1. The Effect of Endoamylase
[0507] The starch used for AF production may first be liquified
either by endoamylases, or by acid hydrolysis.
[0508] Endoamylase degraded starch is more suitable as substrate
for the lyase as compared to native starch. Starch has a limited
solubility at the temperature used for the lyase-catalyzed
reaction. Treatment of starch with endoamylases led to increased
glucose yied. It was found that a reducing matter of around 10-15%
(on a dry mater basis) was most suitable as substrate for the lyase
with respect to AF yield and further treatment with the endoamylase
to a reducing matter of 19% was no longer suitable for the
lyase.
[0509] 3.2. The Effect of Pullulanase and Isoamylase
[0510] As seen from the results below, both the isoamylase and the
pullulanase increased AF yield by up to 50% at pH 4.5 and 5.0. The
reaction system consisted of the lyase from the fungal affected red
algae with or without the addition of isoamylase or pulluanase
(MegaZyme Ltd.). Amylopectin was used as substrate. The AF produced
in the presence of only the lyase was expressed as 100%.
43 The pH of the reaction medium Enzymes added 3.5 4.5 5.0 Lyase
only 100 100 100 Lyase + isoamylase 136 152 150 Lyase + pullulanase
132 158 155
[0511] 4. The Relative Degrading Rates of the Fungal Lyase Towards
Various Substrates
[0512] 4.1. The Lyase from M. costata.
[0513] The activity observed with maltotetraose is expressed as
100%.
44 Substrate concentration 2 mg/ml 4 mg/ml 10 mg/ml Maltose 0.5 1.6
2.2 Maltotriose 40.6 58.6 56.0 Maltotetraose 100 100 100
Maltopentaose 107.1 100.1 99.7 Maltohexaose 86.6 98.2 95.9
Maltoheptaose 82.2 81.5 75.7 Dextrin 10* --** -- 68.3 Dextrin 15*
-- -- 61.1 Dextrin 20* -- -- 46.6 Soluble Starch -- -- 92.9
Amylopectin -- -- 106.5 glycogen -- -- 128.5 *the number indicates
the contents of the reducing matter on a dry weight basis. **not
determined.
[0514] 4.2. The Lyase from M. vulgaris Lyase.
[0515] The activity observed for maltotetraose is treated as 100%.
The final concentration of all substrates was 10 mg ml.sup.-1.
45 Substrates Activity (%) Maltose 10.1 Maltotriose 49.8
Maltotetraose 100.0 Maltopentaose 79.3 Maltohexaose 92.4
Maltoheptaose 73.9 Dextrin 10 62 Dextrin 15 45 Dextrin 20 37
Soluble starch 100.5 Amylopectin 139.9 Glycogen 183.3
[0516] The lyase from M. costata and M. vulgaris was unable to
degrade the following sugars.
[0517] Trehalose, panose, nigerose, nigerotetraose, glucose,
isomaltose, alpha-, beta and gama-cyclodextrins, pullulalans and
non-reducing end blocked p-nitrophenyl .alpha.-D-maltoheptaoside as
there was no AF detectable on a TLC plates after these substrates
had been incubated for 48 h with the fungal lyase.
[0518] 7.5. pH and Temperature Optimum for the Lyase Catalyzed
Reaction.
46 GL sources Optimal pH Optimal pH range Optimal temperature M.
costata 6.5 5.5-7.5 37 C.; 40 C.sup.a M. vulgaris 6.4 5.9-7.6 43
C.; 48 C.sup.a Fungal infected 3.8 3.7-4.1 40 C.; 45 C.sup.a
Gracilariopsis lemaneiformis .sup.aParameters determined using
glycogen as substrate; other parameters determined using
amylopectin as substrate.
[0519] 7.6. The Stabilizing Effect of Glycogen on the Lyase from
the Fungal Infected Gracilariopsis lemaneiformis.
[0520] The results indicate that at higher temperatures the
reaction rates were higher when glycogen was used as substrate
instead of amylopectin.
[0521] Reaction Temperature
47 Reaction temperature Substrates 25 C. 30 C. 45 C. Amylopectin
0.818.sup.a 1.133.sup.a 1.171.sup.a Glycogen 0.592.sup.a
0.904.sup.a 1.861.sup.a The ratio of relative 72.4 79.8 158.9
reaction rates between Glycogen and Amylopectin (%) .sup.athe
relative reaction rates.
[0522] 7.7. The Molecular Masses and pI Values of the Lyases
[0523] The molecular masses of the lyases from the fungal infected
G. lemaneiformis, both forms of lyase from apparent fungal free G.
lemaneiformis, from M. costata and M. vulgaris were estimated to
110,000.+-.10,000 daltons using SDS-PAGE on a gradient gel
(8-25%).
[0524] The pI of the lyase from the fungal infected G.
lemaneiformis was around 3.9. For the lyase from M. vuglaris, the
pI was around pH 4.6 and the pI for the lyase from M. costata was
around 5.0. These values were obtained by isoelectric focusing on a
gel with a pH gradient from 3 to 9.
[0525] The pI values deduced from the amino acid compositions
are:
[0526] The lyase from the fungal infected G. lemaneiformis: 4.58
and for the lyase from M. costata: 6.30.
[0527] 7.8. Immunological Test of the Lyase by Western
Blotting.
[0528] The results showed that the antibodies to the algal lyase
could recognize the fungal lyase both in cell-free extracts and in
purified form, as revealed by Western blottings. The antibodies to
the algal lyase purified form the algae collected from China also
recognized the lyase from the algae collected from Sant Cruz,
Calif.
48 Reactivity with the antibodies against the GL from the fungal GL
sources infected G. lemaneiformis Fungal infected Strong G.
lemaneiformis G. lemaneiformis from Califonia Strong both form of
GL M. costata medium M. vulgaris medium
[0529] 7.9. Reversible and Irreversible Inhibitors of the Fungal
Lyase
[0530] 9.1. The Reversible Inhibitors, Glucose and Maltose.
[0531] At a substrate concentration of 10 mg/ml, the activity for
the M. costata lyase decreased by 19.3 % in the presence of 0.1 M
glucose when amylopectin was used as substrate; the activity was
not affected when glycogen was used as substrate. In the presence
of 0.1 M of maltose the activity decreased by 48.8% and 73.4%,
respectively for glycogen and armylopectin.
49 Substrates Inhibitors Concentrations Glucose Maltose Amylopectin
1% (2%) 19.3% (7%) 73.4% (67.2%) Glycogen 1% (2%) 0.000 (-) 48.8%
(49.7%)
[0532] It seems that the inhibition by 0.1 M glucose is competitive
as increasing the substrate from 1% to 2% decreased the inhibition
from 19.3 to 7%, whereas the inhibition by 0.1 M maltose is
non-competitive as the increase of substrate did not significantly
affect the inhibition degree.
[0533] For the M. vulgaris lyase, 0.1 M glucose and maltose did
also inhibit the reaction when either amylopectin or glycogen was
used as substrate.
50 Substrates Glucose Maltose Amylopectin (1%) 28% 80% Glycogen
(1%) 5% 57%
[0534] 9.2. The Reversible Inhibitor Deoxyjirimycin
[0535] At a final substrate concentration of 2%, the activity was
decreased to 10.4% for the algal lyase and the M.costata lyase in
the presence of 25 .mu.M of deoxyjirmycin, using amylopectin as
substrate. At 100 .mu.M, the activity of both lyases was completely
lost.
[0536] 9.3. Irreversible Inhibitor: PCMB
[0537] Under the same assay conditions and in the presence of 2 mM
PCMB, the activity decreased by 60% for the M. costata lyase and
98% for the lyase from the fungal infected red algae. This means
that the fungal lyase was much less sensitive to heavy metal
inhibition.
[0538] 7.10. Examples of Laboratory Scale Production of AF
[0539] 10.1. Production of AF Using Dextrin as Substrate
[0540] The reactor contained 1000 g dextrins (obtained by treatment
of starch with Termamyl to a final reducing matter of 10%) in a
final volume of 4.6 liter (HOAC--NaOAC, pH 3.9, containing 5 mM
Na.sub.2-EDTA). The reaction was initiated by adding 3 mg lyase
purified from fungal infected algae. The reaction was performed at
room temperature. At day 19, another batch of lyase (4 mg) was
added.
51 Reaction time (days) 0 1 7 13 19 24 31 AF produced (grams) 0 18
116 195 264 500 668
[0541] 10.2. Using .sup.14C-Starch for the Production of
.sup.14C-AF
[0542] The uniformly labelled .sup.14C-starch (340 .mu.Ci obtained
from Sigma) was vaccum-dried to remove the ethanol it contained and
then dissolved in 2 ml water. The reaction was initiated by adding
20 .mu.l lyase purified from the fungal infected algae and 20 .mu.l
pullulanase (MegaZyme Ltd.) The reaction was performed overnight at
30.degree. C. At the end of the reaction, the reaction mixture was
filtered using a filter with a molecular mass cut off of 10,000 to,
remove the enzymes and unreacted starch molecules.
[0543] The filtrate was applied on a Ca.sub.2 carbohydrate column
(Chrompack) using a Waters HPLC. Water was used as eluent. The flow
rate was 0.5 ml/min. AF was efficiently separated from glucose and
maltosaccharides. The pooled AF fractions were freeze-dried and
totally 140 .mu.Ci .sup.14C-AF was obtained.
[0544] These findings relate to an even further aspect of the
present invention, namely the use of a reagent that can increase
the hydrophobicity of the reaction medium (preferably an alcohol)
to increase the stability and activity of the lyase according to
the present invention. This increased stability leads to a
increased AF yield.
[0545] Other modifications of the present invention will be
apparent to those skilled in the art without departing from the
scope of the invention.
Sequence CWU 1
1
* * * * *