U.S. patent application number 10/846123 was filed with the patent office on 2004-11-18 for enzymatic hydrolysis of a polymer comprising vinyl acetate monomer.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Borch, Kim, Fitzhenry, James William, Lund, Henrik, Pedersen, Hanne Host, Sakaguchi, Hiromichi, Sharyo, Masaki.
Application Number | 20040226672 10/846123 |
Document ID | / |
Family ID | 27222512 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040226672 |
Kind Code |
A1 |
Borch, Kim ; et al. |
November 18, 2004 |
Enzymatic hydrolysis of a polymer comprising vinyl acetate
monomer
Abstract
The invention relates to the use of certain lipolytic enzymes
such as cutinases and lipases in the manufacture of paper and paper
products from recycled paper. Examples of such enzymes are derived
from strains of Humicola, Candida, Fusarium and Pseudomonas. By use
of these enzymes, the problems relating to the so-called "stickies"
derived from waste paper are reduced.
Inventors: |
Borch, Kim; (Birkerod,
DK) ; Lund, Henrik; (Skodsborg, DK) ; Sharyo,
Masaki; (Matsudo-shi, JP) ; Sakaguchi, Hiromichi;
(Chiba city, JP) ; Pedersen, Hanne Host; (Lyngby,
DK) ; Fitzhenry, James William; (Memphis,
TN) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27222512 |
Appl. No.: |
10/846123 |
Filed: |
May 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10846123 |
May 14, 2004 |
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10152300 |
May 21, 2002 |
|
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60294539 |
May 30, 2001 |
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Current U.S.
Class: |
162/72 ; 162/147;
435/277; 435/278 |
Current CPC
Class: |
Y02W 30/64 20150501;
D21H 11/14 20130101; D21H 11/20 20130101; D21H 17/34 20130101; D21H
17/005 20130101; D21C 9/08 20130101; D21C 5/02 20130101; D21H 21/02
20130101; C12N 9/20 20130101; D21C 5/005 20130101; Y02W 30/648
20150501 |
Class at
Publication: |
162/072 ;
162/147; 435/277; 435/278 |
International
Class: |
D21C 003/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2001 |
DK |
PA 2001 00813 |
Claims
1-16. (cancelled).
17. A method for making paper comprising: a) preparing a pulp from
a material comprising recycled paper; b) treating the pulp with a
lipolytic enzyme, which is able to hydrolyze a polymer comprising
vinyl acetate monomer; and c) making paper from the treated
pulp.
18. The method of claim 17, wherein the enzyme has a) a degree of
hydrolysis by Capillary Electrophoresis of at least 0.1 after 18
hours at 45.degree. C. on a dispersed PVAc substrate, wherein the
dispersed PVAc substrate is prepared by injecting 1.5 ml of 6% PVAc
in methanol into 40 ml buffer pH 6 or 8, and wherein the molecular
weight of the PVAc is about 12800; and/or b) a hydrolytic activity
at 35.degree. C. for 4 min with a 5% (w/v) polymer preparation
comprising vinyl acetate as substrate in an emulsion consisting of
50 mM NaCl, 0.5 mM KH.sub.2PO.sub.4, 9% (V/V) Glycerol and 0.1%
(w/v) Gum Arabicum, wherein the polymer is: (i) a PVAc homopolymer
preparation and the reaction pH is 8; (ii) a vinyl acetate ethylene
copolymer preparation, and the reaction pH is 7; and/or (iii) a
vinyl acetate tert-decanoic acid, ethenyl ester copolymer
preparation, and the reaction pH is 7.
19. The method of claim 18, wherein the hydrolytic activity under
the conditions of (i) is higher than 0.2 units.
20. The method of claim 18, wherein the enzyme has a PVAc
hydrolytic activity of at least 0.7 units/ml.
21. The method of claim 18, wherein the hydrolytic activity under
the conditions of (ii) is at least 0.2 units/ml.
22. The method of claim 18, wherein the hydrolytic activity under
the conditions of (iii) is at least 0.2 units/mi.
23. The method of claim 18, wherein for the determination of degree
of hydrolysis according to a), and for the determination of
hydrolytic activity according to any one of steps (i)-(iii), the
enzyme is purified.
24. The method of claim 18, wherein for the determination of degree
of hydrolysis according to a), and for the determination of
hydrolytic activity according to any one of steps (i)-(iii), the
enzyme is desalted.
25. The method of claim 18, wherein for the determination of
hydrolytic activity according to any one of steps (i)-(iii), 200
micro-liter of an enzyme solution of an A.sub.280=0.15, or 50
micro-gram enzyme protein, is added to 15 ml of substrate
emulsion.
26. The method of claim 17, wherein the lipolytic enzyme is added
in an amount effective to reduce the amount of stickies.
27. The method of claim 17, wherein the enzyme dosage for paper
manufacturing is from 0.1 to 5000 units of PVAc hydrolytic activity
per gram of polymer comprising vinyl acetate monomer.
28. The method of claim 17, wherein the enzyme dosage per ton of
paper pulp is: (i) from about 100 to about 100,000 units according
to (i) of claim 1; or (ii) from about 0.1 to about 100,000 mg
enzyme protein.
29. The method of claim 17, wherein the enzyme is active and stable
in the presence of LAS and/or hydrogen peroxide.
30. The method of claim 17, further comprising the use of at least
one additional enzyme selected from the following group consisting
of proteases, amylases, pullulanases, lipases, hemicellulases,
endoglucanases, pectinases, and cutinases.
31. The method of claim 17, further comprising the use of at least
one anionic, non-ionic, cationic and/or zwitterionic surfactant
and/or dispersant.
32. The method of claim 17, wherein the paper material contains ink
and the lipolytic enzyme is added in an amount effective to de-ink
the paper material.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 10/152,300 filed May 21, 2002, which claims
priority or the benefit under 35 U.S.C. 119 of Danish application
no. PA 2001 00813 filed May 21, 2001 and U.S. provisional
application No. 60/294,539 filed May 30, 2001, the contents of
which are fully incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to the use of certain lipolytic
enzymes in the manufacture of paper and paper products from
recycled paper. By use of these enzymes, the problems relating to
the so-called stickies derived from waste paper are reduced.
BACKGROUND ART
[0003] Polymers comprising vinyl acetate are very commonly used as
an adhesive and coating material throughout industrial sectors
(paper, textiles etc.). However, because of their adhesive
properties these polymers often cause problems at later process
stages.
[0004] For example, in the paper industry polymers comprising vinyl
acetate are used as a binder and coating material. During recycling
these polymers tend to agglomerate together with fibres and other
substances to form so-called "stickies", which reduce the quality
of the paper product and results in a significant downtime of the
machine.
[0005] WO 00/34450, WO 01/92502 and U.S. Pat. No. 5,176,796 report
the use of certain lipases and cutinases in the manufacture of
paper, viz. for pitch control. According to the above US patent,
pitch is a natural constituent of wood, and triglyceride is a major
component thereof.
[0006] WO 01/98579 discloses the use of lipase and/or esterase for
control of the problem with stickies during recycling of paper. The
lipases designated RESINASE A2X, and NOVOCOR ADL, are included for
comparison purposes in Examples 1-3 herein.
BRIEF DESCRIPTION OF THE INVENTION
[0007] The invention relates to the use of a class of lipolytic
enzymes in the manufacture of paper from recycled paper, as well as
methods for making paper using this class of lipolytic enzymes. The
class of lipolytic enzymes is defined by reference to certain tests
for hydrolytic activity on polymers comprising vinyl acetate.
DETAILED DESCRIPTION OF THE INVENTION
[0008] Stickies
[0009] According to the Thesaurus of Pulp and Paper Terminology,
1991 (edited by Institute of Paper Science and Technology, Atlanta,
US), the term stickies designates waste paper contaminants of an
adhesive character causing sticking of paper-machine parts during
reprocessing. As examples of such contaminants, ink, tar, and latex
are mentioned.
[0010] Further examples of stickies are tacky agglomerates of
fibres, adhesives, coatings, binders and other materials, which
form in a recycled papermaking process. Accordingly, stickies are
mainly of concern for paper manufacturing processes in which
recycled paper is used.
[0011] Stickies are water insoluble and tend to agglomerate and
deposit on various parts of the paper manufacturing equipment,
thereby causing paper quality problems, breaks of the paper web,
and costly downtime periods for cleaning the equipment. For
example, stickies may deposit on the paper forming felts thereby
rendering the drainage of water from the forming paper web less
efficient, which again may give rise to problems as described
above.
[0012] Stickies have been distinguished in various ways, e.g. by
size, or by specific gravity:
[0013] Primary stickies are so small as to usually not cause any
problems, whereas secondary stickies or macro stickies are larger
and tend to deposit. Macro stickies are generally of such size as
to be retained on fine screens. Example of fine screens are those
having slots of about 50-200, 60-190, or 70-180, or 80-170, or
80-160, or of about 80-150 micro-meter.
[0014] High density and low density stickies may under certain
conditions be removable by various types of mechanical equipment,
but problems remain in particular with the so-called neutral range
density stickies, i.e. stickies of a specific gravity of about 0.9
to about 1.1, or about 0.95 to about 1.05, or about 0.98 to about
1.02.
[0015] The chemical composition of stickies from various parts of
the world may vary, mainly depending on the characteristics of the
local paper manufacturing processes. Likewise, for the same reason,
the composition of stickies may vary from factory to factory.
[0016] The following components are examples of components, one or
more of which are typically found in European stickies: Acrylic
Resins, Poly Vinyl Acetate (PVAc), Styren Butadien Resin (SBR),
Poly Ethylene (PE) and Poly Propylene (PP).
[0017] In the context of the present invention, focus is on
polymers comprising vinyl acetate, stickies being one example of
such polymers. A polymer comprising vinyl acetate monomer may be a
poly (vinyl acetate) homopolymer (PVAc) comprising vinyl acetate
monomers or it may be a copolymer comprising vinyl acetate and
other monomers such as ethylene, methyl acrylate, vinyl laurate and
t-decanoic acid, ethenyl ester. Blends and mixtures of various
polymers, wherein at least one of the polymers comprises vinyl
acetate monomers, are also included in the definition of "polymer
comprising vinyl acetate" as used herein. The same holds true for
mixtures thereof not only with other polymers, but with any other
compound.
[0018] Various ways of controlling stickies have been developed,
such as controlling the quality of the incoming recycled paper,
various mechanical screening and centrifugal methods, and
dispersion methods using various chemicals. Also the use of enzymes
has been proposed (see e.g. WO 01/98579 referred to above).
[0019] However severe problems still remain, which--according to
the invention--are diminished by the use of certain lipolytic
enzymes, superior to the enzymes previously described for this
purpose.
[0020] Lipolytic Enzymes
[0021] In the present context, the expression "lipolytic" enzyme
refers to a class of enzymes having the unifying and characteristic
feature that they are able to hydrolyse a polymer comprising vinyl
acetate monomer.
[0022] For determining whether a given enzyme falls within this
class of lipolytic enzymes, either of the following tests can be
used:
[0023] (a) a determination of the degree of hydrolysis by Capillary
Electrophoresis after 18 hours at 45.degree. C. on a dispersed PVAc
substrate, wherein the dispersed PVAc substrate is prepared by
injecting 1.5 ml of 6% PVAc in methanol into 40 ml buffer, pH 6 or
8, and wherein the molecular weight of the PVAc is about 12800;
or
[0024] (b) a determination of the hydrolytic activity of the enzyme
in question on at least one of the three following polymer
preparations all comprising vinyl acetate monomer: (i) a PVAc
homopolymer preparation, (ii) a vinyl acetate ethylene copolymer
preparation, or (iii) a vinyl acetate tert-decanoic acid, ethenyl
ester copolymer preparation. Hydrolytic activity on one, two or all
three of these substrates shows that the enzyme is a "lipolytic"
enzyme as defined herein.
[0025] For the purpose of test (a) above: If the degree of
hydrolysis determined according to Example 8 herein is at least
0.1, the enzyme in question qualifies as a lipolytic enzyme for use
according to the invention. The expression a PVAc molecular weight
of about 12800 means average molecular weight by Gel Permeation
Chromatography (GPC). In this context, "about 12800" means of an
average molecular weight of between 10000 and 20000. A buffer pH
value of 6 or 8 can be either pH 6 or pH 8. Examples of suitable
buffers are 5mM Hepes buffer (pH 8), and 5 mM MES (pH 6). In
particular embodiments, the degree of hydrolysis is at least 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, or at least 2.0.
[0026] Surprisingly, a relatively small degree of hydrolysis seems
to be required in order to reduce the stickiness of the stickies.
In particular embodiments, the degree of hydrolysis is below 10%,
or below 9%, or below 8%, or below 7%, or below 6%, or below 5%, or
below 4%. In further particular embodiments, the degree of
hydrolysis is within any range which can be derived from the above
lower and upper values.
[0027] For the testing purposes according to (b) above, the polymer
substrate is emulsified, and the reaction conditions such as pH,
temperature and incubation time are selected paying due regard to
the characteristics of the enzyme in question.
[0028] Examples of reaction temperatures are: 10, 20, 30, 35, 37,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.degree.
C.
[0029] Examples of reaction pH values are: pH 2, 3, 4, 5, 6, 7, 8,
9, 10, 11 or pH 12.
[0030] Examples of reaction times are: 30 seconds, 1 minute, 2, 3,
4, 5, 10, 20, 30, 60, 90, 120 or 180 minutes.
[0031] In a first particular embodiment the enzyme may have,
preferably has, a hydrolytic activity at 35.degree. C., pH 8 for 4
min with a 5% (w/v) poly(vinyl acetate) (PVAc) homopolymer
preparation as substrate in an emulsion consisting of 50 mM NaCl,
0.5 mM KH.sub.2PO.sub.4, 9% (v/v) Glycerol and 0.1% (w/v) Gum
Arabicum. In a particular embodiment hereof, the poly(vinyl
acetate) homopolymer preparation (also designated PVAc homopolymer)
is a homopolymer PVAc dispersion glue having a dry matter content
in the range of 46 to 50%, and a viscosity in the range of 5800 to
7200 mPa.s (Brookfield, 20.degree. C.). A preferred substrate is
the product GLUDAN.TM. 150-6.500 available from the company GLUDAN
A/S, Vesterlundvej 5-7, DK-2730 Herlev, Denmark (as used in Example
1 herein). In this first embodiment, the hydrolytic activity may be
referred to as "PVAc hydrolytic activity."
[0032] In a second particular embodiment the enzyme has a
hydrolytic activity at 35.degree. C., pH 7 for 4 min with a 5%
(w/v) vinyl acetate ethylene copolymer preparation as substrate in
an emulsion consisting of 50 mM NaCl, 0.5 mM KH.sub.2PO.sub.4, 9%
(v/v) Glycerol and 0.1% (w/v) Gum Arabicum. In a particular
embodiment hereof, the vinyl acetate ethylene copolymer is a PVAc
ethylene copolymer dispersion glue having a total solid content of
56 to 60%, and a viscosity of 6000 to 7000 mPa.s (Brookfield,
20.degree. C.). A preferred substrate is the product GLUDAN.TM.
534-6.500 available from the company GLUDAN A/S, Vesterlundvej 5-7,
DK-2730 Herlev, Denmark (as used in Example 2 herein).
[0033] In a third particular embodiment the enzyme has a hydrolytic
activity at 35.degree. C., pH 7 for 4 min with a 5% (w/v) vinyl
acetate tert-decanoic acid, ethenyl ester copolymer preparation as
substrate in an emulsion consisting of 50 mM NaCl, 0.5 mM
KH.sub.2PO.sub.4, 9% (v/v) Glycerol and 0.1% (w/v) Gum Arabicum. In
a particular embodiment hereof, the vinyl acetate tert-decanoic
acid, ethenyl ester copolymer preparation is a vinyl acetate-Veova
copolymer dispersion glue having a total solid content of 55.+-.1%,
and a viscosity of 1000 to 4000 cP (Brookfield, 23.degree. C). A
preferred substrate is the product NOREMUL.TM. VW 1501 available
from the company CHEMARK ApS, Noerregade 10A, 2. t.v., DK-4600
Koege, Denmark (as used in Example 3 herein). For the viscosity
determinations, a suitable viscosimeter is of the type Brookfield
LVF. Spindle no. 3 may be used at a speed of 60 rpm.
[0034] Under these conditions the hydrolytic activity (e.g. the
PVAc hydrolytic activity) of a lipolytic enzyme is determined by
adding 200 micro-liter of the lipolytic enzyme, dissolved in
deionised water corresponding to A.sub.280=0.15, to 15 ml of
substrate emulsion, and one unit of hydrolytic activity (e.g. PVAc
hydrolytic activity) is defined as the release of 1 micro-mole of
acetate measured by a pH-stat. Typically, A.sub.280=0.15
corresponds to about 0.10-0.40 mg enzyme protein/ml, depending on
the extinction coefficient for the enzyme in question. For most of
the enzymes used in the examples A.sub.280=0.15 may correspond to
about 0.20 to about 0.30 mg enzyme protein/ml, for example 0.25 mg
enzyme protein/ml. In a particular embodiment, the amount of enzyme
protein used in the assays (i.e. contained in the 200 micro-liter
which is added to 15 ml of substrate emulsion) is about 40 to about
60, or about 45 to about 55, or about 50 micro-gram enzyme
protein.
[0035] In a particular embodiment, the lipolytic enzyme may have,
preferably has, a PVAc hydrolytic activity higher than 0.2 units,
particularly 0.4 units or more particularly 0.6 units.
[0036] In another particular embodiment, the lipolytic enzyme has a
PVAc hydrolytic activity of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5,
12.0, 12.5, 13.0, or at least 13.5 units, per ml of the lipolytic
enzyme solution having A.sub.280 of 0.15.
[0037] In a further particular embodiment, the lipolytic enzyme has
a hydrolytic activity on the vinyl acetate ethylene copolymer
substrate of at least at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8,
0.9, 1.0, 1.1, or at least 1.2 units/ml enzyme solution having an
A.sub.280 of 0.15.
[0038] In a still further particular embodiment, the lipolytic
enzyme has a hydrolytic activity on the vinyl acetate tert-decanoic
acid, ethenyl ester copolymer substrate of at least 0.2, 0.3, 0.4,
0.5, 0.6, 0.7. 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7.
1.8, 1.9, 2.0, or at least 2.1 units/ml enzyme solution having an
A.sub.280 of 0.15.
[0039] In various other embodiments, the lipolytic enzyme:
[0040] (iv) is active in the presence of LAS ("active" means, that
the activity (measured in units/ml) in either of the assays
(i)-(iii), adding 50 or 200 ppm LAS into the reaction, is at least
50% as compared to the activity in a control experiment without
addition of LAS); in further particular embodiments the activity is
at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120 or
at least 130% as compared to the control;
[0041] (v) is stable in the presence of LAS ( "stable" means, that
the residual activity (measured in units/ml) using either of the
assays (i)-(iii), after pre-incubation of the enzyme
(A.sub.280=0.15) for 1, 2 or 3 hours, at pH 6 or 8, and a
temperature of 45.degree. C., in 50 or 200 ppm LAS (rest buffer pH
6 or 8), is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,
110, 120 or at least 130% as compared to the control);
[0042] (v) shows a higher turbidity than the control after 20
minutes at 35, 45 or 55.degree. C. and pH=8 (using 5 mM Hepes
buffer), on a PVAc substrate, wherein the substrate is prepared by
injection of 0.15 ml 2% w/v PVAc (Mw=113000) in MeOH solution into
30 ml buffer (see Tables 4-6);
[0043] (v) is tolerant to hydrogen peroxide, viz. that
substantially the same turbidity/enzyme dosage profile is obtained
with and without addition of 15 ppm hydrogen peroxide (see Table
9); and/or
[0044] (vi) is tolerant to LAS, viz. substantially the same
turbidity is obtained after 20 minutes, pH 7; 45.degree. C. with 50
and 200 ppm LAS (see Table 11).
[0045] In a particular embodiment the lipolytic enzyme is
well-defined, meaning that only one major enzyme component is
present. In the alternative, the term well-defined means that only
one major lipolytic enzyme component is present. Such well-defined,
or purified, or highly purified, lipolytic enzymes can be obtained
as is known in the art and/or described in publications relating to
the specific enzyme in question. Fractionation of a well-defined
enzyme on an appropriate Size-exclusion column reveals only one
major enzyme component which is the lipolytic enzyme in question;
or, in the alternative, reveals only one major lipolytic enzyme
component, which is the lipolytic enzyme in question.
[0046] For a peak to qualify as a major peak, the area of the peak
should correspond to at least 50, or 55, or 60, or 65, or 70, or
75, or 80, or 85, or 90, or 92, or 94, or 96, or at least 98% of
the total peak area/total lipolytic peak area.
[0047] The skilled worker will know how to select an appropriate
Size-exclusion chromatography column. He might start by
fractionating the preparation on e.g. a HiLoad26/60 Superdex75pg
column available from Amersham Biosciences UK Limited, Amersham
Place, Little Chalfont, Buckinghamshire HP7 9NA, UK. If the peaks
would not be clearly separated he would try different columns (e.g.
with an amended column particle size and/or column length), and/or
he would amend the sample volume. See Example 11 herein for further
details.
[0048] In a further particular embodiment, the lipolytic enzyme is
desalted, i.e. substantially free from low molecular weight
materials such as salts. Gel filtration chromatography is a
suitable desalting technique. A preferred column material is
Sephadex G-25, ensuring group separations of proteins/peptides
larger than Mr 5000 from molecules with a Mr less than 1000. Mr
designates relative molecular mass (molecular weight), and the
units are in Dalton. Suitable columns are PD-10 columns available
e.g. from Amersham, loaded with Sephadex G-25 M, or NAP5 columns.
The standard procedure prescribed by the supplier is used, and
desalted water (MilliQ) is used for equilibration and elution.
[0049] For determining lipolytic activity as described above, a
well-defined and/or desalted lipolytic enzyme preparation is
preferably used. Preferably the enzyme preparation is well-defined
and desalted. As described in the Materials & Methods section
herein, for carrying out the lipolytic activity test, the enzyme
preparation is first diluted to A.sub.280 of 0.15.
[0050] Examples of lipolytic enzymes as defined above are typically
found amongst enzymes classified in EC 3.1.1 Carboxylic Ester
Hydrolases according to Enzyme Nomenclature (available at
http://www.chem.qmw.ac.uk/- iubmb/enzyme, or from Enzyme
Nomenclature 1992 (Academic Press, San Diego, Calif., with
Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995),
Supplement 4 (1997) and Supplement 5 (in Eur. J. Biochem. 1994,
223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996,
237, 1-5; Eur. J. Biochem. 1997, 250; 1-6, and Eur. J. Biochem.
1999, 264, 610-650; respectively), which are enzymes capable of
hydrolysing carboxylic ester bonds. The lipolytic enzyme may have
substrate specificity with an activity such as EC 3.1.1.3
triacylglycerol lipase or EC 3.1.1.74 cutinase.
[0051] The cutinase may be derived from a fungus. Particularly, the
cutinase may be derived from a strain of Humicola, particularly H.
insolens, more particularly H. insolens strain DSM1800 (U.S. Pat.
No. 5,827,719) or from a strain of Fusarium, e.g. F. roseum
culmorum, or particularly F. solani pisi (WO 90/09446; WO 94/14964,
WO 94/03578). The fungal cutinase may also be derived from a strain
of Rhizoctonia, e.g. R. solani, or a strain of Alternaria, e.g. A.
brassicicola (WO 94/03578). The lipolytic enzyme may also be a
variant of a parent cutinase such as those described in WO
00/34450, or WO 01/92502.
[0052] SEQ ID NO:1 is the amino acid sequence of the Humicola
insolens cutinase (corresponding to the mature part of SEQ ID NO:2
of U.S. Pat. No. 5,827,719), and SEQ ID NO:2 is the amino acid
sequence of the Fusarium solani pisi according to FIG. 1D of WO
94/14964.
[0053] Another example is a lipase as classified by EC 3.1.1.3.
More particularly, a lipase derived from Pseudomonas, such as P.
putida ATCC 53552 (WO 88/09367; WO 89/09263), P. cepacia (U.S. Pat.
No. 4,876,024), P. mendocina (U.S. Pat. No. 5,389,536) P.
alcaligenes (U.S. Pat. No. 6,313,283), P. gladioli (EP 205208; EP
206390) or Pseudomonas sp. (U.S. Pat. No. 5,942,431). The lipolytic
enzyme may also be a variant of a parent lipase such as those
described in e.g. EP 0943678, U.S. Pat. No. 5,352,594 or WO
94/25578.
[0054] Another example is lipase B from Candida antarctica (U.S.
Pat. No. 5,273,898) or an enzyme having an amino acid sequence,
which has at least 60%, particularly 65%, more particularly 70%, or
75%, or 80%, or 85%, or 90%, or 92%, or 95%, or 97%, or at least
99% homology to lipase B from Candida antarctica (U.S. Pat. No.
5,273,898) such as a lipase from Hyphozyma (U.S. Pat. No.
5,856,163). This lipase has SEQ ID NO:3.
[0055] Another example is an enzyme having an amino acid sequence,
which may have at least 70% homology, particularly 75%, or 80%,
90%, 92%, 95%, 97%, or at least 99% homology to SEQ ID NO:1 or SEQ
ID NO:2.
[0056] In the context of the present invention homology is
determined as the degree of similarity between two sequences
indicating a derivation of the first sequence from the second. The
similarity is determined by means of the computer program GAP
provided in the GCG program package (Program Manual for the
Wisconsin Package, Version 8, August 1994, Genetics Computer Group,
575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and
Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453,
using GAP with the following settings for polypeptide sequence
comparison: GAP creation penalty of 3.0 and GAP extension penalty
of 0.1. In a particular embodiment, the homology is determined as
the degree of identity using the program and the settings specified
above, in which case for the present purposes "homology" may be
replaced by "identity," and "homologous" with "identical."
[0057] Paper Manufacturing Process
[0058] The present invention relates to a method for making paper
comprising: a) preparing a pulp from a material comprising recycled
paper; b) treating the pulp with a lipolytic enzyme, which is able
to hydrolyze a polymer comprising vinyl acetate monomer; c) making
paper from the treated pulp.
[0059] For the purposes of the present invention, any type of paper
making process is relevant and/or any paper making pulp can be
applied, as long as it comprises recycled paper. In particular
embodiments, the amount of pulp based on recycled paper relative to
the total amount of pulp is at least 2%, or at least 4%, or at
least 6%, or at least 8%, or at least 10%, or at least 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or at least 95%.
In further particular embodiments, the amount of pulp based on
recycled paper relative to the total amount of pulp is not higher
than 25%, or not higher than 30%, or 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95 or not higher than 98%. In still further
particular embodiments, the amount of pulp based on recycled paper
relative to the total amount of pulp lies within any interval which
can be derived from the above upper and lower values. Dry weight is
preferably used for determining the amount of pulp.
[0060] The part of the pulp which is not derived from recycled
paper may be derived from mechanical pulping, chemical pulping, and
any mixtures thereof such as chemi-mechanical pulping,
thermo-mechanical pulping, chemi-thermo-mechanical pulping etc.
[0061] For preparing a pulp from a material comprising recycled (or
waste) paper, also known as secondary fibres, the material is
mixed, dispersed, or suspended in water. This is the process known
as pulping. Thereby at least part of the ink and other contaminants
such as glue, adhesives, coatings etc. is released from the fibres.
A papermaking pulp often comprises both recycled paper and fresh
so-called virgin pulp. The pulp may have a high (above 18%), medium
(7-18%), or low (below 7%) consistency. In particular embodiments,
the method and the use of the invention are operated at a high, a
medium or a low pulp consistency.
[0062] The source of recycled fibre can be any of the grades of
recycled furnishes known in the art or mixtures thereof, as well as
mixtures of recycled fibres with virgin fibres. Major grades of
recycled fibre furnishes are for instance MOW (mixed office waste),
SOW (sorted office waste), ONP (old newsprint), WM (waste
magazines) and OCC (old corrugated containers).
[0063] The lipolytic enzymes for use in the method and use of the
invention are disclosed in the section herein headed "Lipolytic
Enzymes."
[0064] The expression "treating the pulp" basically means adding
the enzyme in question to the pulp. The treatment takes place at
suitable conditions. The conditions of the treatment may be varied
depending on particular paper manufacturing process parameters or
requirements, and on the characteristics of the enzyme in question,
such as pH-activity and -stability range, temperature activity and
-stability range, rate of hydrolysis etc. Examples of treatment
conditions are described in the section herein headed "Process
Conditions." In particular embodiments, the conditions are suitable
to reduce the amount of stickies, and/or to obtain a de-inking (see
below).
[0065] Making paper from the treated pulp comprises forming the
enzyme-treated fibres into paper or paperboard products.
[0066] Over and above steps a)-c) further, optional, steps may be
included, for example one or more of the following steps:
[0067] d) de-inking, e.g. by pulping the fibres in the presence of
an aqueous alkaline solution, optionally containing a peroxide
compound, such as hydrogen peroxide;
[0068] e) separation of the fibres from the contaminants, e.g. by
screening (coarse and/or fine);
[0069] f) centrifugal cleaning;
[0070] g) flotation, e.g. using one or more surfactants;
[0071] h) washing, e.g. using one or more surfactants;
[0072] i) dispersion; e.g. using one or more surfactants;
and/or
[0073] j) inactivation of the enzymes, if required, e.g. by a heat
treatment step.
[0074] Any number of these additional steps may be included, and
the sequence need not be as indicated a-b-c-d-e-f-g-h-i-j. In a
particular embodiment, step d) and/or j) are included. The
lipolytic enzymes as defined herein may advantageously be used also
in the de-inking step d).
[0075] The enzyme may be introduced prior to the pulping, during a
pulping stage, during or before, preferably right before, a stock
preparation stage, or after a flotation or a de-inking stage. In
particular embodiments, it is introduced in the paper machine
headbox, or in the paper machine white water.
[0076] The process may be a closed loop process (at least a part of
the water being re-used, e.g. at least 10%, 15, 20, 25, 30, 40, 50,
60, 70, or at least 80% of the water being re-used (vol/vol)).
[0077] In further particular embodiments of the method and the use
of the invention, the enzyme serves the purpose of controlling
stickies. Controlling stickies means that the amount of stickies is
reduced as compared to a control not treated with enzymes. In
preferred embodiments, the amount of stickies in a sample treated
with the enzyme in question is maximum 95%, or 90%, or 80%, or 75%,
or 70%, or 60%, or 50%, or 40%, or 30%, or maximum 20% (w/w), as
compared to the control. Any of the methods disclosed in Examples
1-6 of WO01/98579 may be used for this determination.
[0078] In still further embodiments of the method and the use of
the invention, the enzyme serves de-inking purposes. The enzyme can
be applied to de-ink recycled paper with printing made by any
printing method, e.g. off set printing, gravure printing, and
letterpress printing. The effect of the enzyme is to improve the
whiteness or brightness of and/or reduce the amount of ink in the
resulting paper. This can be measured as generally known in the
art, e.g. as disclosed in JP patent no. 96014078.
[0079] Glycerides present in the virgin pulp tend to agglomerate
with a polymer comprising vinyl acetate monomer from the recycled
paper pulp in the stickies. Thus in another example the method of
the present invention may comprise a lipolytic enzyme capable of
hydrolyzing a polymer comprising vinyl acetate monomer and a lipase
capable of hydrolyzing the ester bonds in a glyceride, such as
those classified by EC 3.1.1.3.
[0080] Accordingly, the method and the use according the invention
may be carried out with a combination of enzymes in order to make
the enzymatic process effective against a broader range of
contaminants, such as proteinaceous impurities, starch-containing
impurities, triglyceride containing impurities as well as
contaminants containing hemi-celluloses and pectins. The lipolytic
enzymes for use according to the invention can thus be combined
with at least one additional enzyme, selected from amongst
proteases, amylases, pullulanases, lipases, hemicellulases,
endoglucanases, cutinases, and pectinases. These enzymes may be
wild-type enzymes, or mutants or variants thereof having the
relevant enzyme activity, i.e. catalyzing at least one of the
reactions indicated at the following web-site for the relevant
enzyme class: http://www.expasy.ch/en- zyme/.
[0081] Examples of suitable proteases are the ALCALASE, ESPERASE,
SAVINASE, NEUTRASE and DURAZYM proteases. Other preferred
serine-proteases are proteases from Nocardiopsis, Aspergillus,
Rhizopus, Bacillus alcalophilus, B. cereus, B. natto, B. vulgatus,
B. mycoide, and subtilisins from Bacillus, especially proteases
from the species Nocardiopsis sp. and Nocardiopsis dassonvillei
such as those disclosed in WO 88/03947, especially proteases from
the species Nocardiopsis sp., NRRL 18262, and Nocardiopsis
dassonvillei, NRRL 18133. Yet other preferred proteases are the
serine proteases from mutants of Bacillus subtilis, e.g. those
disclosed in WO 91/00345 and EP 415296. Examples of suitable
amylases are the BAN, AQUAZYM, TERMAMYL, and AQUAZYM Ultra
amylases. An example of a lipase is the RESINASE A2X lipase. An
example of a hemicellulase is the PULPZYME HC hemicellulase.
Examples of endoglucanases are the NOVOZYM 613, 342, and 476 enzyme
products. An example of a pectinase is the NOVOZYME 863 enzyme
product. An example of a pullulanase is the PROMOZYM pullulanase.
The enzymes written in capitals are commercial enzymes available
from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd,
Denmark.
[0082] Particularly the method of the present invention may be used
on a polymer comprising vinyl acetate monomer present in and/or on
paper. More particularly, it may be used to hydrolyze a polymer
comprising vinyl acetate monomer in a papermaking pulp of recycled
paper to diminish the problem with stickies. Alternatively, it may
be used to remove labels, such as mailing- and bottle-labels. In
another example the method of the present invention may be for
stickies control during recycling of paper. The method of the
present invention may be carried out with stirring or mechanical
agitation.
[0083] Process Conditions
[0084] For the method for making paper and the use in the
manufacture of paper of at least one lipolytic enzyme as defined
herein, the lipolytic enzyme in question need not be as pure as
described above. First of all, it need not be a well-defined enzyme
preparation. Secondly, the enzyme preparation may not be a desalted
enzyme preparation.
[0085] In a particular embodiment, however, the enzyme preparation
is well-defined. This is mainly because a well-defined preparation
is advantageous, e.g. as regards consistency and reproducibility of
results.
[0086] The process conditions will be a function of the enzyme(s)
applied, the reaction time and the conditions given. Generally, the
enzyme is dosed in a sufficient amount to control the stickies
present in the paper pulp. However, it is contemplated that the
enzyme(s) may be dosed in total amount from 0.1 to 5000 units per
gram of polymer comprising vinyl acetate monomer, particularly from
0.5 to 1000 units per gram of polymer comprising vinyl acetate
monomer, more particularly from 1 to 500 units per gram of polymer
comprising vinyl acetate monomer wherein one unit refers to the
PVAc hydrolytic activity as described above.
[0087] In particular embodiments, the dosage of the lipolytic
enzyme is from about 100 to about 100,000 units per ton of paper
pulp. In particular embodiments, these units refer to (i) PVAc
hydrolytic activity; (ii) activity on vinyl acetate ethylene
copolymer; or (iii) activity on vinyl acetate tert-decanoic acid,
ethenyl ester copolymer (using the methods specified herein). In a
preferred embodiment, the units refer to (i) PVAc hydrolytic
activity. Further examples of particular dosage ranges (all "from
about" and "to about," and in units per ton paper pulp) are the
following: 200-100,000; 400-100,000; 600-100,000; 800-100,000;
1000-100,000; 1200-100,000; 1500-100,000; 2000-100,000; 100-90,000;
100-80,000; 100-70,000; 100-60,000; 100-50,000; 100-40,000;
100-30,000; 100-20,000; 100-10,000; as well as any combination of
the upper and lower values here indicated.
[0088] In other particular embodiments, the dosage of the lipolytic
enzyme is from about 0.1 mg enzyme protein to about 100,000 mg
enzyme protein per ton of paper pulp. Enzyme protein may be
determined as described in Example 11 herein. Further examples of
particular dosage ranges (all "from about" and "to about," and in
units per ton paper pulp) are the following: 0.5-100,000;
1-100,000; 5-100,000; 10-100,000; 20-100,000; 40-100,000;
60-100,000; 80-100,000; 100-100,000; 150-100,000; 200-100,000;
500-100,000; 0.1-90,000; 0.1-80,000; 0.1-70,000; 0.1-60,000;
0.1-50,000; 0.1-40,000; 0.1-30,000; 0.1-20,000; 0.1-10,000;
0.1-8,000; 0.1-6,000; 0.1-4,000; 0.1-2,000; 0.1-1,000; as well as
any combination of the upper and lower values here indicated.
[0089] The enzymatic treatment may be carried out at a temperature
of from about 10 to about 100.degree. C. Further examples of
temperature ranges (all "from about" and "to about") are the
following: 20-100, 30-100, 35-100, 37-100, 40-100, 50-100, 60-100,
70-100, 10-90, 10-80, 10-70, 10-60, and 30-60.degree. C., as well
as any combination of the upper and lower values here
indicated.
[0090] The enzymatic treatment may be carried out at a pH of from
about 2 to about 12. Further examples of pH ranges (all "from
about" and "to about") are the following: 3-12, 4-12, 5-12, 6-12,
7-12, 8-12, 9-12, 2-11, 2-10, 2-9, 2-8, 4-10, 5-8 as well as any
combination of the upper and lower values here indicated.
[0091] A suitable duration of the enzymatic treatment may be in the
range from a few seconds to several hours, e.g. from about 30
seconds to about 48 hours, or from about 1 minute to about 24
hours, or from about 1 minute to about 18 hours, or from about 1
minute to about 12 hours, or from about 1 minute to 5 hours, or
from about 1 minute to about 2 hours, or from about 1 minute to
about 1 hour, or from about 1 minute to about 30 minutes.
[0092] Additives
[0093] Various additives over and above the lipolytic enzyme can be
used in the process or use of the invention. For example, talc,
clay and other inorganic particles may be used to render the
stickies less tacky, and dispersants, surfactants, polymers, and
solvents may be used to reduce the size of the stickies and/or keep
them dispersed or suspended.
[0094] Surfactants and/or dispersants are often present in and/or
added to a papermaking pulp. Thus the method of the present
invention may be carried out in the presence of an anionic,
non-ionic, cationic and/or zwitterionic surfactant and/or
dispersant conventionally used in a paper-making pulp. An enzyme
capable of hydrolyzing a polymer comprising vinyl acetate monomer
may be an enzyme, which is active and stable in the presence of an
anionic, non-ionic, cationic and/or zwitterionic surfactant and/or
dispersant conventionally used in a papermaking pulp or during
washing of textiles.
[0095] Examples of anionic surfactants are carboxylates, sulphates,
sulphonates or phosphates of alkyl, substituted alkyl or aryl.
Examples of non-ionic surfactants are polyoxyethylene compounds,
such as alcohol ethoxylates, propoxylates or mixed
ethoxy-/propoxylates, polyglycerols and other polyols, as well as
certain block-copolymers. Examples of cationic surfactants are
water-soluble cationic polymers, such as quartenary ammonium
sulphates and certain amines, e.g. epichlorohydrin/dimethylamine
polymers (EPI-DMA) and cross-linked solutions thereof, polydiallyl
dimethyl ammonium chloride (DADMAC), DADMAC/Acrylamide copolymers,
and ionene polymers, such as those disclosed in U.S. Pat. Nos.
5,681,862; and 5,575,993. Examples of zwitterionic or amphoteric
surfactants are betains, glycinates, amino propionates, imino
propionates and various imidazolin-derivatives. Also the polymers
disclosed in U.S. Pat. No. 5,256,252 may be used.
[0096] Also according to the invention, surfactants such as the
above, including any combination thereof may be used in a paper
making process together with at least one lipolytic enzyme as
defined herein, and included in a composition together with such
enzyme. The amount of each surfactant in such composition may
amount to from about 8 to about 40% (w/w) of the composition. In
particular embodiments the amount of each surfactant is from about
10 to about 38, or from about 12 to about 36, or from about 14 to
about 34, or from about 16 to about 34, or from about 18 to about
34, or from about 20 to about 34, or from about 22 to about 34, or
from about 24 to about 34, or from about 26 to about 34, or from
about 28 to about 32% (w/w).
[0097] In another particular embodiment, each of the above ranges
refers to the total amount of surfactants.
[0098] The compositions are preferably additives for the
manufacture of paper. In a particular embodiment, they are
non-build compositions, i.e. they do not contain builders. Examples
of builders are certain phosphates, zeolites etc. conventionally
used for detergent compositions.
[0099] The compositions may comprise at least one additional enzyme
selected from the following group of enzymes: Proteases, amylases,
pullulanases, lipases, hemicellulases, endoglucanases, cutinases,
and pectinases; as well as any combination thereof.
[0100] The compositions may be stabilized using the formulations
described in e.g. U.S. Pat. Nos. 5,356,800; 5,780,283.
[0101] The invention also relates to:
[0102] (I) a method for hydrolyzing a polymer comprising vinyl
acetate monomer by treating with a cutinase; a method according to
the above wherein the cutinase is derived from a fungus; a method
according to the above wherein the cutinase is derived from
Humicola insolens or Fusarium solani pisi; a method for hydrolyzing
a polymer comprising vinyl acetate monomer by treating with a
lipase derived from Pseudomonas; a method for hydrolyzing a polymer
comprising vinyl acetate monomer by treating with a lipase having
an amino acid sequence, which has at least 60% homology to lipase B
from Candida antarctica SEQ ID NO. 3; a method for hydrolyzing a
polymer comprising vinyl acetate monomer by treating with a
lipolytic enzyme having an amino acid sequence, which has at least
70% homology to SEQ ID NO.1 or SEQ ID NO.2;
[0103] (II) a method for hydrolyzing a polymer comprising vinyl
acetate monomer treating the polymer with an enzyme, which has a
hydrolytic activity at 35.degree. C., pH 8 for 4 min with 5% (w/v)
poly (vinyl acetate) (PVAc) homopolymer as substrate in a emulsion
consisting of 50 mM NaCl, 0.5 mM KH.sub.2PO.sub.4, 9% (v/v)
Glycerol and 0.1% (w/v) Gum Arabicum; and
[0104] (III) use of an enzyme in the manufacture of paper, which
enzyme has
[0105] a) a degree of hydrolysis by Capillary Electrophoresis of at
least 0.1 after 18 hours at 45.degree. C. on a dispersed PVAc
substrate, wherein the dispersed PVAc substrate is prepared by
injecting 1.5 ml of 6% PVAc in methanol into 40 ml buffer pH 6 or
8, and wherein the molecular weight of the PVAc is about 12800;
and/or
[0106] b) a hydrolytic activity at 35.degree. C. for 4 min with a
5% (w/v) polymer preparation comprising vinyl acetate as substrate
in an emulsion consisting of 50 mM NaCl, 0.5 mM KH.sub.2 PO.sub.4,
9% (v/v) Glycerol and 0.1% (w/v) Gum Arabicum, wherein the polymer
is:
[0107] (i) a PVAc homopolymer preparation and the reaction pH is
8;
[0108] (ii) a vinyl acetate ethylene copolymer preparation, and the
reaction pH is 7; and/or
[0109] (iii) a vinyl acetate tert-decanoic acid, ethenyl ester
copolymer preparation, and the reaction pH is 7;
[0110] as well as the use of above (III), wherein the hydrolytic
activity under the conditions of (i) is higher than 0.2 units,
preferably higher than 0.4 units, still more preferably higher than
0.6 units; wherein the enzyme has a PVAc hydrolytic activity of at
least 0.7 units/ml; wherein the hydrolytic activity under the
conditions of (ii) is at least 0.2 units/ml; wherein the hydrolytic
activity under the conditions of (iii) is at least 0.2 units/ml;
wherein for the determination of degree of hydrolysis according to
a), and for the determination of hydrolytic activity according to
any one of steps (i)-(iii), the enzyme is well-defined; wherein for
the determination of degree of hydrolysis according to a), and for
the determination of hydrolytic activity according to any one of
steps (i)-(iii), the enzyme is desalted; wherein for the
determination of hydrolytic activity according to any one of steps
(i)-(iii), 200 micro-liter of an enzyme solution of A.sub.280=0.15,
or 50 micro-gram enzyme protein, is added to 15 ml of substrate
emulsion; for stickies control and/or for the manufacture or paper
prepared at least partly on the basis of recycled paper; wherein
for the manufacture of paper the enzyme dosage is from 0.1 to 5000
units of PVAc hydrolytic activity per gram of polymer comprising
vinyl acetate monomer; wherein the enzyme dosage per ton of paper
pulp is: (i) from about 100 to about 100,000 units according to (i)
of (III); or (ii) from about 0.1 to about 100,000 mg enzyme
protein; wherein the enzyme is active and stable in the presence of
LAS and/or hydrogen peroxide: further comprising the use of at
least one additional enzyme, which additional enzyme is selected
from the following group of enzymes: Proteases, amylases,
pullulanases, lipases, hemicellulases, endoglucanases, cutinases,
and pectinases; and/or further comprising the use of at least one
anionic, non-ionic, cationic and/or zwitterionic surfactant and/or
dispersant.
[0111] The invention described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims. In the
case of conflict, the present disclosure including definitions will
control.
[0112] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
[0113] Materials and Methods
[0114] Enzymes
[0115] A) Cutinase from Humicola insolens according to U.S. Pat.
No. 5,827,719.
[0116] B) Thermostable variant of A).
[0117] C) Thermostable variant of A) according to WO 00/34450.
[0118] D) Cutinase from Fusarium solani pisi according to WO
94/14964.
[0119] E) Lipase B from Candida antarctica according to U.S. Pat.
No. 5,273,898.
[0120] F) Lipase from Pseudomonas cepacia according to U.S. Pat.
No. 4,876,024.
[0121] G) Lipase from Pseudomonas sp. according to U.S. Pat. No.
5,942,431.
[0122] H) Phospholipase from Porcine Pancreas.
[0123] I) NOVOCOR ADL lipase.
[0124] J) RESINASE A 2X lipase.
[0125] K) Variant of J) with lipase and phospholipase activity
according to WO 00/32758.
[0126] L) Ferulic acid esterase from Aspergillus niger.
[0127] The NOVOCOR ADL and RESINASE A 2X lipases are commercial
enzyme products available from Novozymes A/S, Krogshoejvej 36,
DK-2880 Bagsvaerd, Denmark.
[0128] The variants of the Humicola insolens cutinase shown in the
below Table A were prepared as described in WO 00/34450 and WO
01/92502 and tested for lipolytic activity as described in the
following. The test results for two of these variants are indicated
in the examples as B) and C).
1TABLE A R51P E6N/Q + L138I A14P + E47K E47K E179N/Q E6N/Q + E47K +
R51P A14P + E47K + E179N/Q E47K + E179N/Q E47K + D63N E6N/Q +
E10N/Q + A14P + E47K + R51P + E179N/Q E6N/Q + A14P + E47K + R51P +
E179N/Q E6Q + A14P + E47K + R51P + E179Q Q1P + L2V + S11C + N15T +
F24Y + L46I + E47K E6Q + A14P + E47K + R51P + E179Q + S48E + A88H +
N91H + R189V E6Q + A14P + E47K + R51P + E179Q + N44D + A130V E6Q +
A14P + E47K + R51P + E179Q + Q1C + L2V + G120D E6Q + A14P + E47K +
R51P + E179Q + A88L + R189A E6Q + A14P + E47K + R51P + E179Q + S48E
+ L66I + A88L + I169A + R189H E6Q + A14P + E47K + R51P + E179Q +
A88V + S116K + S119P + Q139R + I169V + R189V E6Q + A14P + E47K +
R51P + E179Q + A88V + R189A E6Q + A14P + E47K + R51P + E179Q + S48K
+ A88H + I169G + R189H E6Q + A14P + E47K + R51P + E179Q + Q1L + L2Q
+ A4V + S11T E6Q + A14P + E47K + R51P + E179Q + T164S E6Q + A14P +
E47K + R51P + E179Q + L174F E6Q + A14P + E47K + R51P + E179Q + H49Y
E6Q + A14P + E47K + R51P + E179Q + G8D + N15D + A16T E6Q + A14P +
E47K + R51P + E179Q + A130V E6Q + A14P + E47K + R51P + E179Q + Q1C
+ L2V E6Q + A14P + E47K + R51P + E179Q + G8D + N15D + S48E + A88H +
N91H + A130V + R189V E6Q + A14P + E47K + R51P + E179Q + G8D + N15D
+ T29M + S48E + A88H + N91H + A130V + R189V E6Q + A14P + E47K +
R51P + E179Q + G8D + N15D + T29I + S48E + A88H + N91H + A130V +
R189V E6Q + A14P + E47K + R51P + E179Q + G8D + N15D + T29C + S48E +
A88H + N91H + A130V + R189V E6Q + A14P + E47K + R51P + E179Q + G8D
+ N15D + S48E + A88H + N91H + A130V + L174F + I178V + R189V E6Q +
A14P + E47K + R51P + E179Q + G8D + N15D + S48E + A88H + N91H +
A130V + T166M + I168F + R189V E6Q + A14P + E47K + R51P + E179Q +
G8D + N15D + S48E + A88H + N91H + A130V + T166I + L167P + R189V E6Q
+ A14P + E47K + R51P + E179Q + G8D + N15D + V38H + S48E + A88H +
N91H + A130V + I169T + R189V E6Q + A14P + E47K + R51P + E179Q + G8D
+ N15D + V38H + S48E + A88H + N91H + A130V + R189V E6Q + A14P +
E47K + R51P + E179Q + G8D + N15D + T29M + S48E + A88H + N91H +
A130V + T166I + L167P + R189V
[0129] Reagents/Substrates
[0130] 0.025 M NaOH
[0131] 5 mM MES Buffer pH 6-6.5
[0132] 0.98 g MES 2-[N-Morpholino]Ethanesulfonic Acid Hydrate
[0133] 0.44 g CaCl.sub.2, 2 H.sub.2O
[0134] 0.246 g MgSO.sub.4, 7 H.sub.2O
[0135] Milli Q water added up to 1 liter
[0136] pH adjusted with weak NaOH or HCl
[0137] 5 mM Hepes Buffer pH 7-9
[0138] 1.19 g Hepes
(N-[2-Hydroxyethyl]Piperazine-N'-[2-Ethanesulfonic Acid])
[0139] 0.44 g CaCl.sub.2, 2 H.sub.2O
[0140] 0.246 g MgSO.sub.4, 7 H.sub.2O
[0141] Milli Q water added up to 1 liter
[0142] pH adjusted with weak NaOH or HCl
[0143] 6% w/v Poly (Vinyl Acetate) PVAc in MeOH Solution
[0144] 6 g PVAc Mw.apprxeq.12800 (GPC) (Aldrich Chemical Company.
Inc. Cat. No. 43,043-9) dissolved in approx. 80 ml MeOH at approx.
50.degree. C. Let cool.
[0145] MeOH is added up to 100 ml
[0146] 2% w/v Poly (Vinyl Acetate) PVAc in MeOH Solution
[0147] 2 g PVAc Mw.apprxeq.113000 (Aldrich Chemical Company. Inc.
Cat. No. 18.948-0) dissolved in approx. 80 ml MeOH at approx.
50.degree. C. Let cool.
[0148] MeOH is added up to 100 ml
[0149] LAS (LAS Nansa 1169 A, dodecyl benzene sulphonate sodium
salt, Albright & Wilson).
[0150] Emulsion Reagent
[0151] 17.9 g NaCl
[0152] 0.41 g KH.sub.2PO.sub.4
[0153] 540 ml Glycerol
[0154] 6.0 g Gum Arabicum
[0155] NaCl, KH.sub.2PO.sub.4, Glycerol and 400 ml Dl water are
mixed before Gum Arabicum is added under strong stirring. Stirring
is continued until it is completely dissolved. Dl water is added up
to 1 l.
[0156] Substrate Emulsion for Examples 1-3
[0157] 15 g PVAc homo-or copolymer (Gludan, Denmark) (example 1,
and examples 2-3, respectively)
[0158] 50 ml Emulsion reagent
[0159] 235 ml Dl water
[0160] The solution is stirred for about 30 min until all the
substrate is dissolved.
[0161] Equipment
[0162] Titrator: Radiometer Titralab (Vit 90)
[0163] Electrode: Orion 8103 Ross--Semi micro Combination pH
electrode, glass body.
[0164] Turbidimeter: HACH Turbidimeter 2100AN with a USEPA filter
Cat. 30312-00, including a special glass (sample cells cat. No.
20849-00) fitting the apparatus.
[0165] Water bath: Water bath including a magnetic stirrer (oblong
stir bars, 15 mm)
[0166] Water bath: Water bath with temperature control.
[0167] Filters: 0.22 micro-m filters (Millex-GV single use filters
by Millipore)
[0168] Capillary Electrophoresis:
[0169] Hewlett Packard 3D CE with Chemstation software
[0170] Agilent buffer: Agilent Basic Anion Buffer for HPCE, Part.
No. 5064-8209
[0171] Agilent capillar:
[0172] Agilent, Fused silica, CE ext. Light path cap. Inner
diameter 50 micro-m, effective length 104 cm, total length 112.5
cm, Part. no. G1600-64232
[0173] Tensiometer: KSV Sigma 70 Model 6000, equipped with a
platinum Wilhelmy plate (dim. 10 mm.times.20 mm), temperature
regulation and stirring of solution
EXAMPLES
Examples 1-3
[0174] Polymer preparations comprising vinyl acetate monomer is
hydrolysed at a constant pH under release of acetate by an enzyme.
The release of acetate as a function of time is measured by
neutralization with base by pH-stat titration.
[0175] The Titrator (see Equipment) is turned on, the water bath is
set to 35.degree. C. and the electrode is calibrated using regular
calibration buffers. Fifteen ml of substrate emulsion is added to
each beaker and allowed to heat for 3 min to obtain the right
temperature (the substrate should be stirred at all times prior to
use to avoid precipitation). The pH is adjusted with a freshly made
diluted NaOH solution (25 mM). Enzyme solutions (in de-ionised
water) are prepared on the basis of highly purified and desalted
enzymes corresponding to Enzymes A)-L). The solutions correspond to
an absorbance of A.sub.280=0.15, and 200 micro-liter thereof is
added to the substrate emulsion and the titration is begun, using
25 mM NaOH. After 4 min the titration is stopped. The average
consumption in ml per minute of NaOH is calculated for the period
2-4 minutes, and this value is used for the below unit
calculation.
[0176] 1 unit enzyme is defined as the amount of enzyme that
releases 1 micro mole titratable acetate per min by hydrolysis of
the substrate under the given conditions. Units/ml as indicated in
the below tables refers to ml of the enzyme solution of
A.sub.280=0.15.
Example 1
[0177] Hydrolysis of PVAc Homopolymer
[0178] The ability of different lipolytic enzymes to degrade a PVAc
homopolymer preparation was measured as described above. Before
addition of the enzyme to the substrate emulsion, pH was adjusted
to 8. The results are shown in table 1.
2TABLE 1 PVAc hydrolytic activity Type of enzyme Enzyme (U/ml)
Cutinase A) H. insolens 2.9 B) Thermostable variant of A) 1.8 C)
Thermostable variant of A) 3.0 D) F. solani pisi 1.0 Lipase B from
C. antarctica E) C. antarctica 13.6 Lipase from Pseudomonas F) P.
cepacia 0.6 G) P. sp. 0.2 Other lipases H) Phospholipase from 0
Porcine Pancreas I) NOVOCOR ADL lipase 0 J) RESINASE A 2X lipase 0
K) Variant of J) 0 L) Ferulic acid esterase from 0 A. niger
Example 2
[0179] Hydrolysis of a Vinyl Acetate Copolymer
[0180] The ability of lipolytic enzymes to degrade a vinyl acetate
copolymer preparation consisting of vinyl acetate and ethylene was
measured as described above. Before addition of the enzyme to the
substrate emulsion, pH was adjusted to 7. The results are shown in
table 2.
3TABLE 2 Hydrolytic activity Type of enzyme Enzyme (U/ml) Cutinase
A) H. insolens 1.2 B) Thermostable variant of A) 0.6 C)
Thermostable variant of A) 0.5 Lipase B from C. antarctica G) C.
antarctica 0.3 Other lipases J) RESINASE A 2X lipase 0
Example 3
[0181] Hydrolysis of a Latex-Based Vinyl Acetate Copolymer
[0182] The ability of lipolytic enzymes to degrade a vinyl acetate
copolymer preparation consisting of vinyl acetate and tert-Decanoic
acid, ethenyl ester was measured as described above. Before
addition of the enzyme to the substrate emulsion, pH was adjusted
to 7. The results are shown in table 3.
4TABLE 3 Hydrolytic Type of enzyme Enzyme activity (U/ml) Cutinase
A) H. insolens 1.9 B) Thermostable variant of A) 1.9 C)
Thermostable variant of A) 2.1 Other lipases J) RESINASE A 2X
lipase 0
Example 4-10
[0183] In examples 4-10 pure Poly (vinyl acetate), abbreviated
PVAc, is used as a substrate. PVAc is not soluble in water but in
methanol. When small amounts of PVAc solubilized in methanol is
injected into water, the PVAc momentarily precipitates in extremely
small particles, and the liquid becomes turbid. These particles
tend to agglomerate into much bigger particles due to their
stickiness, resulting in a decreased turbidity. With enzymes
showing lipolytic activity (e.g. PVAc hydrolytic activity), this
agglomeration can be prevented. Accordingly, the effect of a
lipolytic enzyme is measured directly as the turbidity of the PVAc
dispersion as a function of time. Turbidity is measured in
NTU-units (Nephelometric Turbidity Units). A low number indicates
that the sample is clear, and a high number that the sample is
turbid (i.e. the enzyme works to decrease stickiness). In these
examples, the enzyme dosage is given in ppm (w/w) enzyme
protein.
Example 4
[0184] Ability to Agglomerate PVAC
[0185] Add a stir bar and 30 ml of buffer to the glass. Put the
glass in the water bath, and await adjustment to the prescribed
temperature. The speed of the magnetic stirrer is kept constant.
Remove an amount of buffer corresponding to the volume of the
enzyme solution to be added (to make sure the volume is constant).
Add the enzyme. Inject 0.15 ml 2% w/v PVAc (Mw.apprxeq.13000) in
MeOH solution, and start the timer. After stirring for 10 seconds,
the time=0 turbidity is determined. Measure the turbidity as a
function of time.
[0186] The results using 10 ppm of enzyme B), at 35, 45 or
55.degree. C., respectively, and pH=8 (using 5 mM Hepes buffer) are
shown in below Tables 4-6, respectively.
5TABLE 4 Turbidity Turbidity (NTU) Time (NTU) Blank 10 ppm enzyme
(min) 35.degree. C. B) 35.degree. C. 0 118 1 110 2 97 3 87 4 80 8
31 0 134 1 126 2 120 3 117 4 115 5 113 6 113 13 109 18 107 37 106
60 105 120 97
[0187]
6TABLE 5 Turbidity Turbidity (NTU) Time (NTU) Blank 10 ppm enzyme
(min) 45.degree. C. B) 45.degree. C. 0 107 1 104 2.5 81 4 71 6 59 8
49 10 37 12 32 13 29 0 118 1.5 112 4 108 7 106 10.5 105 53 103 60
103
[0188]
7TABLE 6 Time Turbidity (NTU) Turbidity (NTU) 10 ppm (min) Blank
55.degree. C. enzyme B) 55.degree. C. 0 101 1 98 3 77 5 67 7 60 9
53 11 49 13 44 15 40 17 37 20 33 22 31 0 135 2 132 3 132 6 132 11
130 13 130 20 129 75 125
[0189] Table 7 shows Dose-Response figures for two lipolytic
enzymes at 45.degree. C., pH=8 using 5 mM Hepes buffer. The
turbidity was measured after 20 minutes. The figures are average of
three experiments.
8TABLE 7 Enzyme Turbidity Turbidity dosage (NTU) (NTU) (ppm) Enzyme
B) Enzyme C) 0 11 0.5 45 1 57 1.5 88 2 91 3.5 112 5 115 10 120 0 11
10 43 50 51
[0190] Table 8 shows the effect of a lipolytic enzyme versus pH
(45.degree. C.; turbidity after 20 minutes; enzyme dosage 10 ppm of
enzyme B). The figures are average of three experiments.
9TABLE 8 Turbidity (NTU) Turbidity (NTU) pH Blank 10 ppm enzyme B)
6 8.6 44.3 6.5 10.5 118 7 10.5 114.7 8 12.3 130 9 19 129 10 12.6
127.5
Example 5
[0191] Tolerance Towards Hydrogen Peroxide
[0192] The same setup as in example 4 was used. Turbidity is
measured after 20 minutes, but here 15 ppm of hydrogen peroxide was
added at constant level to different enzyme dosage levels of enzyme
B); temperature 48.degree. C.; pH=8.5 using 5 mM Hepes buffer. The
results are shown in Table 9 below.
10TABLE 9 Dosage Turbidity (NTU) Turbidity (NTU) Enzyme with 15 ppm
without B) (ppm) peroxide peroxide 0 19 12 0.1 37 32 0.5 82 86 1
118 114 2.5 123 130
[0193] In another experiment a constant enzyme dosage level of 0.5
ppm of enzyme B) was combined with various levels of
hydrogenperoxide (0-1000 ppm). The results are shown in Table 10
below.
11 TABLE 10 Dosage of Turbidity peroxide (ppm) (NTU) 0 93 15 86 200
102 600 101 1000 92
Example 6
[0194] Tolerance Towards LAS (Linear Alkylbenzene Sulfonate)
[0195] The same setup as in Example 4 was used. Buffer 5 mM Hepes
pH=7; 45.degree. C.; the turbidity was measured after 20 minutes.
Two different levels of LAS were added to the assay: 50 and 200
ppm. The results are shown in Table 11 below.
12 TABLE 11 Turbidity (NTU) Blank pH 7, 50 ppm LAS 116 Blank pH 7,
200 ppm LAS 133 0.5 ppm Enzyme B; pH 7, 50 ppm 226 LAS 0.5 ppm
Enzyme B; pH 7, 200 ppm 220 LAS
Example 7
[0196] Measurement of Degree of Hydrolysis of Vinyl Acetate
Homopolymer.
[0197] The degree of hydrolysis (deacetylation) of a polymer
comprising vinyl acetate monomer can be measured by detection of
acetate released into the solution. Acetate in small concentrations
can be detected and quantified by Capillary Electrophoresis
analysis.
[0198] 40 ml of 5 mM Hepes buffer, pH=8 is heated to 50.degree. C.
in closed Erlenmeyer flasks on the heated water bath. The enzyme is
dosed into the buffer in amounts according to the below table. Then
6% w/v PVAc in methanol (Mw.apprxeq.12800) is injected into the
buffer in amounts according to the table below. Samples are taken
after 18 hours and filtered through the 0.22 micro-m filters. The
amount of acetate in these samples is detected by Capillary
electrophoresis, using Agilent buffer and Agilent capillar is used
(see equipment list).
[0199] Program: Temperature 30.degree. C., Voltage -30 kV. Flush 4
min with buffer. Inject sample at 50 mbar, 6 sec., inject buffer at
50 mbar, 10 sec. Elution for 20 min. Detection signal: 350/20
nm.
[0200] Reference signal: 275/10 nm.
[0201] An acetate standard curve 0-450 ppm acetate is run in
parallel. The acetate peaks are integrated, and the amounts in the
samples are determined relative to the standard curve. The degree
of hydrolysis of the PVAc is determined by calculating the actual
amount of vinyl acetate monomer in the flask from the beginning.
Vinyl Acetate has Mw of 86 g/mol.
[0202] For Experiment no.1-6 with injection of 1.5 ml PVAc
solution, the calculations are as follows: 1 0.0015 liter * 60 g
VAc / liter 86 g / mol * 0.0415 liter = 2.522 * 10 - 2 mol /
liter
[0203] The results are shown in table 12 below. A degree of
hydrolysis of 1% will result in an acetate concentration of
2.522.times.10.sup.-4 mol/liter=15.1 mg/liter=15.1 ppm acetate. For
Experiments 1-6, the degree of hydrolysis (%) is calculated
relative to 1% hydrolysis corresponding to 15.1 ppm Acetate. For
Experiments 7-12, the calculations give 1% hydrolysis corresponding
to 29.2 ppm acetate.
13TABLE 12 Experiment PVAc Degree of hydrolysis No. injected Enzyme
dosage After 18 hours 1 1.5 ml 10 ppm enzyme B) 1.9 2 1.5 ml 10 ppm
enzyme B) + 1.4 1 ppm enzyme J) 3 1.5 ml 10 ppm enzyme B) + 1.6 10
ppm enzyme J) 4 1.5 ml 1 ppm enzyme J) 0 5 1.5 ml 10 ppm enzyme J)
0 6 1.5 ml Blank 0 7 3.0 ml 10 ppm enzyme B) 1.1 8 3.0 ml 10 ppm
enzyme B) + 1.3 1 ppm enzyme J) 9 3.0 ml 10 ppm enzyme B) + 1.3 10
ppm enzyme J) 10 3.0 ml 1 ppm enzyme J) 0 11 3.0 ml 10 ppm enzyme
J) 0 12 3.0 ml Blank 0
Example 8
[0204] Measurement of Degree of Hydrolysis of Vinyl Acetate
Homopolymer at Different pH and Temperatures.
[0205] This example is run exactly as example 7, but with different
conditions, viz. pH=6 using 5 mM MES buffer or pH=8 using 5 mM
Hepes buffer at 35.degree. C., 45.degree. C. and 55.degree. C. The
results are shown in Table 13 below.
14TABLE 13 Degree of Experi- hydrolysis ment PVAc After 18 No.
injected Temperature pH Enzyme dosage hours 1 1.5 ml 35.degree. C.
8 Blank 0 2 1.5 ml 35.degree. C. 8 10 ppm enzyme C) 1.4 3 1.5 ml
35.degree. C. 8 10 ppm enzyme B) 2.0 4 1.5 ml 35.degree. C. 6 Blank
0 5 1.5 ml 35.degree. C. 6 10 ppm enzyme C) 1.0 6 1.5 ml 35.degree.
C. 6 10 ppm enzyme B) --* 7 1.5 ml 45.degree. C. 8 Blank 0 8 1.5 ml
45.degree. C. 8 10 ppm enzyme C) 1.4 9 1.5 ml 45.degree. C. 8 10
ppm enzyme B) 1.5 10 1.5 ml 45.degree. C. 6 Blank 0 11 1.5 ml
45.degree. C. 6 10 ppm enzyme C) 1.1 12 1.5 ml 45.degree. C. 6 10
ppm enzyme B) 1.4 13 1.5 ml 55.degree. C. 8 Blank 0 14 1.5 ml
55.degree. C. 8 10 ppm enzyme C) 2.2 15 1.5 ml 55.degree. C. 8 10
ppm enzyme B) 3.0 16 1.5 ml 55.degree. C. 6 Blank 0 17 1.5 ml
55.degree. C. 6 10 ppm enzyme C) 1.3 18 1.5 ml 55.degree. C. 6 10
ppm enzyme B) 1.5 *experimental error, result discarded
Example 9
[0206] Prevention of Deposition of PVAc Agglomerates on Metal
[0207] PVAc agglomerates have a high tendency to precipitate and
stick to different surfaces like metals and hydrophobic materials
used for e.g. forming felts, pressing felts etc. The lipolytic
enzymes described herein when added to the PVAc containing solution
will prevent the formation of these stickies (the agglomerates do
not stick to glass surfaces).
[0208] 200 ml buffer 5 mM Hepes pH=8 is heated to 45.degree. C. 10
ppm enzyme B) is added to one beaker. A beaker without enzyme
addition is run in parrallel as an enzyme blank. Agitation is
carried out by glass coated magnets. A metal surface is dipped into
the solutions. 4 ml 2% w/v PVAC (Mw.apprxeq.113000) in methanol
solution is injected into the solutions. The experiment is stopped
after 10 minutes.
[0209] A photo of the metal surfaces is appended as FIG. 1. The rod
to the left relates to the experiment with added enzyme B, and the
rod to the right relates to the control experiment without added
enzyme. The surface of the left rod appears clean, while the PVAc
solution stays turbid. In contrast, the control experiment (rod to
the right) shows PVAc agglomerates adhering to the metal surface,
and the surrounding solution becomes clear.
Example 10
[0210] Prevention of PVAc Deposition on Metal Surface
[0211] The deposition of agglomerates on metal surfaces can be
quantified as the mass uptake on a well defined surface. For this
purpose a tensiometer equipped with a Wilhelmy plate can be
applied. The tensiometer is operated to measure the weight increase
of the Wilhelmy plate after slowly dipping the plate totally into a
solution of PVAc every 2 minutes. The weight after 10 minutes is
determined.
[0212] The Wilhelmy plate is cleaned by dipping in ethanol and
glowing in a gas burner flame. The clean plate is placed on the
tensiometer electronic balance. 100 ml buffer 5 mM Hepes pH=8 is
heated to the desired temperature (35.degree. C. or 45.degree. C.)
in the tensiometer beaker. Enzyme is added.
[0213] 2 ml of 2% w/v PVAc (Mw.apprxeq.113000) in methanol solution
is injected. The up/down movement of the Wilhelmy plate is started.
After 10 minutes the weight of the plate is determined. A low
weight uptake indicates prevention of PVAc deposition. The results
obtained at a temperature of 35 and 45.degree. C., respectively,
are shown in Tables 13 and 14, respectively.
15TABLE 13 Dosage Dosage Mass uptake Enzyme B) Enzyme C) (mg) (ppm)
(ppm) t = 10 min 0 127 2 114 5 67 6 8 7.5 8 10 7 0 127 10 129 50 66
100 22
[0214]
16TABLE 14 Dosage Dosage Mass uptake Enzyme B) Enzyme C) (mg) (ppm)
(ppm) t = 10 min 0 32 0.5 12 1 4.3 2 4.5 5 5.4 10 5.7 0 32 100
26
Example 11
[0215] Determination of Purity of Lipolytic Enzymes
[0216] The protein concentration of an enzyme sample is determined
with a BCA protein assay kit from PIERCE (identical to PIERCE cat.
No.23225). The sodium salt of Bicinchoninic acid (BCA) is a stable,
water-soluble compound capable of forming an intense purple complex
with cuprous ions (Cu.sup.1+) in an alkaline environment. The BCA
reagent forms the basis of the BCA protein assay kit capable of
monitoring cuprous ions produced in the reaction of protein with
alkaline Cu.sup.2+ (Biuret reaction). The colour produced from this
reaction is stable and increases in a proportional fashion with
increasing protein concentrations (Smith, P. K., et al. (1985),
Analytical Biochemistry, vol. 150, pp. 76-85). The BCA working
solution is made by mixing 50 parts of reagent A with 1 part
reagent B (Reagent A is PIERCE cat. No. 23223, contains BCA and
tartrate in an alkaline carbonate buffer; reagent B is PIERCE cat.
No. 23224, contains 4% CuSO.sub.4 *5H.sub.2O). 300 ml sample is
mixed with 3.0 ml BCA working solution. After 30 minutes at
37.degree. C., the sample is cooled to room temperature and
A.sub.490 is read as a measure of the protein concentration in the
sample. Dilutions of Bovine serum albumin (PIERCE cat. No. 23209)
are included in the assay as a standard.
[0217] If the lipolytic enzyme is on solid form, the product is
first dissolved/suspended in 20 volumes of 100 mM H.sub.3BO.sub.3,
10 mM 3,3'-dimethylglutaric acid, 2 mM CaCl.sub.2, pH 6 (Buffer A)
for at least 15 minutes at 5.degree. C., and if the enzyme at this
stage is a suspension, the suspension is filtered through a 0.45
micro-m filter to give a clear solution. The solution is from this
point treated as a liquid lipolytic enzyme.
[0218] If the lipolytic enzyme is a liquid, the product is first
dialysed in a 6-8000 Da cut-off SpectraPor dialysis tube (cat.no.
132 670 from Spectrum Medical Industries) against 100 volumes of
Buffer A+150 mM NaCl (Buffer B) for at least 5 hours at 5.degree.
C., to remove formulation chemicals that could give rise to a high
viscosity, which is detrimental to the size-exclusion
chromatography.
[0219] The dialysed lipolytic enzyme is filtered through a 0.45
micro-m filter if a precipitate was formed during the dialysis. The
protein concentration in the dialysed enzyme product is determined
with the above described protein concentration assay and the enzyme
product is diluted with Buffer B, to give a sample ready for
size-exclusion chromatography with a protein concentration of 5
mg/ml. If the enzyme product has a lower than 5 mg/ml protein
concentration after dialysis, it is used as is.
[0220] A 300 ml HiLoad26/60 Superdex75pg column (Amersham Pharmacia
Biotech) is equilibrated in Buffer B (Flow: 1 ml/min). 1.0 ml of
the enzyme sample is applied to the column and the column is eluted
with Buffer B (Flow: 1 ml/min). 2.0 ml fractions are collected from
the outlet of the column, until the sample has eluted from the
column. The collected fractions are analysed for lipolytic
activity. A protein peak with activity in one or more of the assays
described herein is defined as a lipolytic enzyme peak. The purity
of a lipolytic enzyme peak is calculated as the protein amount in
the peak divided with the total protein amount in all identified
peaks. In a particular embodiment the purity refers to protein
amount in the peak divided with the total protein amount in all
identified lipolytic peaks.
Sequence CWU 1
1
3 1 194 PRT Humicola insolens 1 Gln Leu Gly Ala Ile Glu Asn Gly Leu
Glu Ser Gly Ser Ala Asn Ala 1 5 10 15 Cys Pro Asp Ala Ile Leu Ile
Phe Ala Arg Gly Ser Thr Glu Pro Gly 20 25 30 Asn Met Gly Ile Thr
Val Gly Pro Ala Leu Ala Asn Gly Leu Glu Ser 35 40 45 His Ile Arg
Asn Ile Trp Ile Gln Gly Val Gly Gly Pro Tyr Asp Ala 50 55 60 Ala
Leu Ala Thr Asn Phe Leu Pro Arg Gly Thr Ser Gln Ala Asn Ile 65 70
75 80 Asp Glu Gly Lys Arg Leu Phe Ala Leu Ala Asn Gln Lys Cys Pro
Asn 85 90 95 Thr Pro Val Val Ala Gly Gly Tyr Ser Gln Gly Ala Ala
Leu Ile Ala 100 105 110 Ala Ala Val Ser Glu Leu Ser Gly Ala Val Lys
Glu Gln Val Lys Gly 115 120 125 Val Ala Leu Phe Gly Tyr Thr Gln Asn
Leu Gln Asn Arg Gly Gly Ile 130 135 140 Pro Asn Tyr Pro Arg Glu Arg
Thr Lys Val Phe Cys Asn Val Gly Asp 145 150 155 160 Ala Val Cys Thr
Gly Thr Leu Ile Ile Thr Pro Ala His Leu Ser Tyr 165 170 175 Thr Ile
Glu Ala Arg Gly Glu Ala Ala Arg Phe Leu Arg Asp Arg Ile 180 185 190
Arg Ala 2 199 PRT Fusarium solani pisi 2 Gly Arg Thr Thr Arg Asp
Asp Leu Ile Asn Gly Asn Ser Ala Ser Cys 1 5 10 15 Ala Asp Val Ile
Phe Ile Tyr Ala Arg Gly Ser Thr Glu Thr Gly Asn 20 25 30 Leu Gly
Thr Leu Gly Pro Ser Ile Ala Ser Asn Leu Glu Ser Ala Phe 35 40 45
Gly Lys Asp Gly Val Trp Ile Gln Gly Val Gly Gly Ala Tyr Arg Ala 50
55 60 Thr Leu Gly Asp Asn Ala Leu Pro Arg Gly Thr Ser Ser Ala Ala
Ile 65 70 75 80 Arg Glu Met Leu Gly Leu Phe Gln Gln Ala Asn Thr Lys
Cys Pro Asp 85 90 95 Ala Thr Leu Ile Ala Gly Gly Tyr Ser Gln Gly
Ala Ala Leu Ala Ala 100 105 110 Ala Ser Ile Glu Asp Leu Asp Ser Ala
Ile Arg Asp Lys Ile Ala Gly 115 120 125 Thr Val Leu Phe Gly Tyr Thr
Lys Asn Leu Gln Asn Arg Gly Arg Ile 130 135 140 Pro Asn Tyr Pro Ala
Asp Arg Thr Lys Val Phe Cys Asn Thr Gly Asp 145 150 155 160 Leu Val
Cys Thr Gly Ser Leu Ile Val Ala Ala Pro His Leu Ala Tyr 165 170 175
Gly Pro Asp Ala Arg Gly Pro Ala Pro Glu Phe Leu Ile Glu Lys Val 180
185 190 Arg Ala Val Arg Gly Ser Ala 195 3 317 PRT Candida
antarctica 3 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys
Ser Val Leu 1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Pro
Ser Ser Val Ser Lys 20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly
Thr Thr Gly Pro Gln Ser Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu
Ser Thr Gln Leu Gly Tyr Thr Pro Cys 50 55 60 Trp Ile Ser Pro Pro
Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr 65 70 75 80 Glu Tyr Met
Val Asn Ala Ile Thr Ala Leu Tyr Ala Gly Ser Gly Asn 85 90 95 Asn
Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln 100 105
110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu
115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly
Pro Leu 130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln
Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn
Ala Gly Gly Leu Thr Gln Ile 165 170 175 Val Pro Thr Thr Asn Leu Tyr
Ser Ala Thr Asp Glu Ile Val Gln Pro 180 185 190 Gln Val Ser Asn Ser
Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys 195 200 205 Asn Val Gln
Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His 210 215 220 Ala
Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230
235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile
Thr 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu
Gln Lys Val 260 265 270 Ala Ala Ala Ala Leu Leu Ala Pro Ala Ala Ala
Ala Ile Val Ala Gly 275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu
Met Pro Tyr Ala Arg Pro Phe 290 295 300 Ala Val Gly Lys Arg Thr Cys
Ser Gly Ile Val Thr Pro 305 310 315
* * * * *
References