U.S. patent application number 10/274411 was filed with the patent office on 2003-07-03 for oxidizing enzymes in the manufacture of paper materials.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Borch, Kim, Franks, Neal, Lund, Henrik, Luo, Jing, Xu, Hui.
Application Number | 20030124710 10/274411 |
Document ID | / |
Family ID | 27222551 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124710 |
Kind Code |
A1 |
Borch, Kim ; et al. |
July 3, 2003 |
Oxidizing enzymes in the manufacture of paper materials
Abstract
The use of fatty acid oxidizing enzymes in the manufacture of
paper materials, such as paper, linerboard, corrugated paperboard,
tissue, towels, corrugated containers and boxes. Examples of fatty
acid oxidizing enzymes are oxygenases classified as EC 1.13.11.
including any of the sub-classes thereof, such as lipoxygenase, EC
1.13.11.12. The effect of these enzymes is that the deposition of
pitch is reduced, and bleaching and de-inking effects are also
observed on the paper pulp and the resulting paper material. The
fatty acid oxidizing enzyme can be used in combination with a
substrate, with proteases, lipases, xylanases, cutinases,
oxidoreductases, cellulases, endoglucanases amylases, mannanases,
steryl esterases, and/or cholesterol esterases; or with surfactants
and other adjuvants.
Inventors: |
Borch, Kim; (Birkerod,
DK) ; Franks, Neal; (Raleigh, NC) ; Lund,
Henrik; (Skodsborg, DK) ; Xu, Hui; (Wake
Forest, NC) ; Luo, Jing; (Raleigh, NC) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27222551 |
Appl. No.: |
10/274411 |
Filed: |
October 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60336894 |
Oct 29, 2001 |
|
|
|
Current U.S.
Class: |
435/262 ;
162/70 |
Current CPC
Class: |
C12N 9/52 20130101; D21H
21/02 20130101; Y02W 30/648 20150501; D21C 5/027 20130101; D21C
9/08 20130101; D21C 5/005 20130101; C12N 9/0069 20130101; Y02W
30/64 20150501; C12Y 113/11012 20130101; D21C 9/10 20130101 |
Class at
Publication: |
435/262 ;
162/70 |
International
Class: |
D21C 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 2001 |
DK |
2001 01551 |
Claims
1. A process for manufacturing a paper material, which process
comprises the step of treating a papermaking pulp and/or process
water with a fatty acid oxidizing enzyme.
2. The process of claim 1, further comprising the steps of forming
and drying the enzyme-treated pulp.
3. The process of claims 1, wherein the enzyme-treatment results in
reduced deposition of pitch.
4. The process of claim 1, wherein the enzyme-treatment results in
bleaching of the paper material.
5. The process of any one of claims 1-4, in which the papermaking
pulp comprises a chemical pulp.
6. The process of any one of claims 1-4, wherein the papermaking
pulp comprises pulp from recycled printed paper material.
7. The process of claim 6, wherein the enzyme-treatment results in
bleaching of the paper material.
8. The process of claim 6, wherein the enzyme-treatment results in
de-inking of the paper material.
9. The process of any one of claims 1-4, wherein a substrate for
the enzyme is added before or during the enzyme-treatment step.
10. The process of any one of claims 1-9, further comprising
treating the papermaking pulp and/or process water with an
additional enzyme selected from the group consisting of a lipase,
cutinase, oxidoreductase, cellulase, amylase, mannanase, steryl
esterase, cholesterol esterase activity, and combinations
thereof.
11 The process of claim 10, wherein the oxidoreductase enzyme has
laccase activity, peroxidase activity or both laccase and
peroxidase activity.
12. The process of claim 10, wherein the additional enzyme has
lipase activity.
13. The process of any one of claims 10-12, wherein the treatment
with the additional enzyme occurs before, concomitantly with,
and/or after the treatment with the fatty acid oxidizing
enzyme.
14. The process of any one of claims 1-13, wherein the fatty acid
oxidizing enzyme is a lipoxygenase.
15. A process for reducing pitch deposition, which process
comprises the step of treating a papermaking pulp and/or process
water with a fatty acid oxidizing enzyme, wherein the
enzyme-treatment results in reduced deposition of pitch.
16. The process of claim 15, wherein the fatty acid oxidizing
enzyme is a lipoxygenase.
17. A process for bleaching a paper material, comprising the step
of treating a papermaking pulp and/or process water with a fatty
acid oxidizing enzyme, wherein the enzyme-treatment results in
bleaching of the paper material.
18. The process of claim 17, wherein the fatty acid oxidizing
enzyme is a lipoxygenase.
19. A process for de-inking a printed paper material, which process
comprises the step of treating a papermaking pulp and/or process
water with a fatty acid oxidizing enzyme, wherein the
enzyme-treatment results in de-inking of the paper material.
20. The process of claim 19, wherein the fatty acid oxidizing
enzyme is a lipoxygenase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims, under 35 U.S.C. 119, the benefit of
Danish application no. PA 2001 01551, filed Oct. 23, 2001, and U.S.
provisional application No. 60/336,894, filed Oct. 29, 2001, the
contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to the use of a fatty acid
oxidizing enzyme in the manufacture of a paper material, as well as
a process for the manufacture of a paper material, the process
comprising a step in which papermaking pulp and/or papermaking
process water is treated with a fatty acid oxidizing enzyme.
BACKGROUND ART
[0003] It is well-known to use enzymes in the manufacture of paper
materials. Examples of enzymes used for this purpose are proteases,
lipases, xylanases, amylases, cellulases, as well as various
oxidizing enzymes such as oxidoreductases (phenol oxidizing
enzymes), for example laccases and peroxidases.
[0004] The effects of these enzymes are wide-spread, e.g. control
of various deposits such as pitch, strength-improvement, de-inking,
drainage improvement, tissue softening, bleaching etc.
[0005] In a papermaking process, dissolved and colloidal substances
(DCS) are dispersed into the process water during the pulp and
paper production. The DCS are often referred to as wood pitch or
wood resin. Pitch causes problems in paper machines by sticking to
the rollers and causing spots or holes in the paper material.
[0006] Wood contains about 1 to 10% of pitch or extractives in
addition to its main components cellulose, hemicellulose and
lignin. Major components of pitch are fatty acids, triglycerides,
sterols, steryl esters and so-called resin acids, e.g. abietic
acid.
[0007] WO 00/53843 discloses certain steryl esterase enzyme
preparations and their use in the manufacture of paper to hydrolyze
the steryl ester part of pitch.
[0008] U.S. Pat. No. 6,066,486 discloses an enzyme preparation
comprising a cholesterol esterase derived from Pseudomonas fragi,
and the use thereof to hydrolyze pulp resin.
[0009] JP 2000080581 discloses the use of certain peroxidases for
the decomposition of abietic acid during pulping or paper making
processes.
[0010] X. Zhang; Pulp & Paper Canada, 101:3 (2000), page 59-62,
discloses studies of the ability of e.g. laccase to remove
dissolved and colloidal substances.
[0011] Also Karlsson et al.: Reactivity of Trametes laccases with
fatty and resin acids; Appl. Microbiol. Biotechnol. (2001)
55:317-320 discloses experiments in which laccases were used to
treat a model pitch preparation.
[0012] However, none of the references cited above disclose the use
of a fatty acid oxidizing enzyme as defined herein for the
manufacture of a paper material.
SUMMARY OF THE INVENTION
[0013] The present inventors surprisingly found that certain
oxidizing enzymes, viz. fatty acid oxidizing enzymes, are
advantageous in the manufacture of paper materials. An important
effect of these enzymes is that the deposition of pitch is reduced.
Furthermore, these enzymes have a bleaching effect on the paper
pulp and the resulting paper material. And finally, a de-inking
effect has been observed, too.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Paper and Pulp
[0015] By the term a "paper-making process" is meant a process,
wherein the pulp is suspended in water, mixed with various
additives and then passed to equipment in which the paper,
cardboard, tissue, towel etc. is formed, pressed and dried.
[0016] The term "paper material" refers to products, which can be
made out of pulp, such as paper, linerboard, corrugated paperboard,
tissue, towels, corrugated containers or boxes.
[0017] The term "a papermaking pulp" or "pulp" means any pulp which
can be used for the production of a paper material. For example,
the pulp can be supplied as a virgin pulp, or can be derived from a
recycled source. The papermaking pulp may be a wood pulp, a
non-wood pulp or a pulp made from waste paper. A wood pulp may be
made from softwood such as pine, redwood, fir, spruce, cedar and
hemlock or from hardwood such as maple, alder, birch, hickory,
beech, aspen, acacia and eucalyptus. A non-wood pulp may be made,
e.g., from bagasse, bamboo, cotton or kenaf. A waste paper pulp may
be made by re-pulping waste paper such as newspaper, mixed office
waste, computer print-out, white ledger, magazines, milk cartons,
paper cups etc.
[0018] In a particular embodiment, the papermaking pulp to be
treated comprises both hardwood pulp and softwood pulp.
[0019] The wood pulp to be treated may be mechanical pulp (such as
ground wood pulp, GP), chemical pulp (such as Kraft pulp or sulfite
pulp), semichemical pulp (SCP), thermomechanical pulp (TMP),
chemithermomechanical pulp (CTMP), or bleached
chemithermomechanical pulp (BCTMP).
[0020] Mechanical pulp is manufactured by the grinding and refining
methods, wherein the raw material is subjected to periodical
pressure impulses. TMP is thermomechanical pulp, GW is groundwood
pulp, PGW is pressurized groundwood pulp, RMP is refiner mechanical
pulp, PRMP is pressurized refiner mechanical pulp and CTMP is
chemithermimechanical pulp.
[0021] Chemical pulp is manufactured by alkaline cooking whereby
most of the lignin and hemicellulose components are removed. In
Kraft pulping or sulphate cooking sodium sulphide or sodium
hydroxide are used as principal cooking chemicals. In these types
of pulp, as a result of the alkaline cooking, the triglyceride part
of pitch will be hydrolysed into fatty acids and glycerol. Fatty
acid oxidizing enzymes are particularly useful in the treatment of
such pulps, because, as the designation tells, these enzymes will
catalyze the further degradation of the fatty acids resulting from
the alkaline hydrolysis of the triglycerides.
[0022] The Kraft pulp to be treated may be a bleached Kraft pulp,
which may consist of softwood bleached Kraft (SWBK, also called
NBKP (Nadel Holz Bleached Kraft Pulp)), hardwood bleached Kraft
(HWBK, also called LBKP (Laub Holz Bleached Kraft Pulp and)) or a
mixture of these.
[0023] The pulp to be used in the process of the invention is a
suspension of mechanical or chemical pulp or a combination thereof.
For example, the pulp to be used in the process of the invention
may comprise 0%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,
70-80%, 80-90%, or 90-100% of chemical pulp. In a particular
embodiment, a chemical pulp forms part of the pulp being used for
manufacturing the paper material. In the present context, the
expression "forms part of" means that in the pulp to be used in the
process of the invention, the percentage of chemical pulp lies
within the range of 1-99%. In particular embodiments, the
percentage of chemical pulp lies within the range of 2-98%, 3-97%,
4-96%, 5-95%, 6-94%, 7-93%, 8-92%, 9-91%, 10-90%, 15-85%, 20-80%,
25-75%, 30-70%, 40-60%, or 45-55%.
[0024] In a particular embodiment of the use and the process of the
invention, the chemical pulp is a Kraft pulp, a sulfite pulp, a
semichemical pulp (SCP), a thermomechanical pulp (TMP), a
chemithermomechanical pulp (CTMP), a bleached chemithermomechanical
pulp (BCTMP). In particular embodiments the Kraft pulp is bleached
Kraft pulp, for example softwood bleached Kraft (SWBK, also called
NBKP (Nadel Holz Bleached Kraft Pulp)), hardwood bleached Kraft
(HWBK, also called LBKP (Laub Holz Bleached Kraft Pulp and)) or a
mixture thereof.
[0025] Process Conditions
[0026] The process of the invention is particularly applicable to
the oxidation and hydrolysis of compounds constituting the pitch
during a pulping or paper-making process, e.g. to avoid pitch
troubles.
[0027] The process of the invention may be applied to any
pitch-containing pulp, especially to pulps with a considerable
content of linoleic acid or other unsaturated free fatty acids.
[0028] In the case of paper and pulp processing, the process
according to the invention can be carried out at any pulp
production stage. The enzyme can be added to any holding tank, e.g.
to a pulp storing container (storage chest), storage tower, mixing
chest or metering chest. The enzyme treatment can be performed
before the bleaching of pulp, in connection with the pulp bleaching
process or after the bleaching. When carried out in connection with
pulp bleaching the enzyme preparation may be added together with
bleaching chemicals such as chlorine, chlorine dioxide. Applying
oxygen gas, hydrogen peroxide or ozone or combinations thereof may
also carry out the bleaching of pulp. The enzyme preparation may
also be added together with these substances. Preferably the enzyme
preparation is added prior to bleaching. The enzyme can also be
added to the circulated process water (white water) originating
from bleaching and process water (brown water) originating from the
mechanical or chemimechanical pulping process. In a particular
embodiment of a Kraft pulping process, the enzyme is added during
the brown-stock washing.
[0029] In the present context, the term "process water" comprises
i.a. 1) water added as a raw material to the paper manufacturing
process; 2) intermediate water products resulting from any step of
the process for manufacturing the paper material; as well as 3)
waste water as an output or by-product of the process. In a
particular embodiment, the process water is, has been, is being, or
is intended for being circulated (re-circulated), i.e. re-used in
another step of the process. The term "water" in turn means any
aqueous medium, solution, suspension, e.g. ordinary tap water, and
tap water in admixture with various additives and adjuvants
commonly used in paper manufacturing processes. In a particular
embodiment the process water has a low content of solid (dry)
matter, e.g. below 20%, 18%, 16%, 14%, 12%, 10%, 8%, 7%, 6%, 5%,
4%, 3%, 20% or below 1% dry matter.
[0030] The use and process of the invention does not include the
use of the lipoxygenase derived from Magnaporthe salvinii as
described in Example 2 of PCT/DK02/00251 for bleaching dye for pulp
industry in waste water.
[0031] The process of the invention may be carried out at
conventional conditions in the paper and pulp processing. The
process conditions will be a function of the enzyme(s) applied, the
reaction time and the conditions given.
[0032] The enzyme of the invention should be added in an effective
amount. By the term "effective amount" is meant the amount
sufficient to achieve the desired and expected effect, such as
oxidizing pitch components, obtaining a desired bleaching and/or
de-inking etc.
[0033] In a particular embodiment, the dosage of the fatty acid
oxidizing enzyme and additional enzymes, if any, is from about 0.1
mg enzyme protein to about 100.000 mg enzyme protein (of each
enzyme) per ton of paper pulp.
[0034] In further particular embodiments, the amount of the fatty
acid oxidizing enzyme and additional enzymes, if any, is in the
range of 0.00001-20; or 0.0001-20 mg of enzyme (calculated as pure
enzyme protein) per gram (dry weight) of lignocellulosic material,
such as 0.0001-10 mg/g, 0.0001-1 mg/g, 0.001-1 mg/g, 0.001-0.1, or
0.01-0.1 mg of enzyme per gram of lignocellulosic material. Again,
these amounts refer to the amount of each enzyme.
[0035] The enzymatic treatment can be done at conventional
consistency, e.g. 0.5-10 % dry substance. In particular
embodiments, the consistency is within the range of 0.5-45; 0.5-40;
0.5-35; 0.5-30; 0.5-25; 0.5-20; 0.5-15; 0.5-10; 0.5-8; 0.5-6; or
0.5-5% dry substance.
[0036] 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. A
typical temperature is from about 20 to 90.degree. C., or 20 to
95.degree. C., preferably from about 40 to 70.degree. C., or 40 to
75.degree. C.
[0037] 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. A typical
pH range is from about 2 to 11, preferably within the range from
about 4 to 9.5, or 6 to 9.
[0038] 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. A typical
reaction time is from about 10 minutes to 3 hours, 10 minutes to 10
hours, preferably 15 minutes to 1 hour, or 15 minutes to 2
hours.
[0039] Molecular oxygen from the atmosphere will usually be present
in sufficient quantity, if required. Therefore, the reaction may
conveniently be carried out in an open reactor, i.e. at atmospheric
pressure.
[0040] Various additives over and above the fatty acid oxidizing
enzyme and additional enzymes, if any, can be used in the process
or use of the invention. Surfactants and/or dispersants are often
present in, and/or added to a papermaking pulp. Thus the process
and use 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 papermaking pulp.
Examples of anionic surfactants are carboxylates, sulphates,
sulphonates or phosphates of alkyl, substituted alkyl or aryl.
Fatty acids are examples of alkyl-carboxylates. Examples of
non-ionic surfactants are polyoxyethylene compounds, such as
alcohol ethoxylates, propoxylates or mixed ethoxy-/propoxylates,
poly-glycerols 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 co-polymers,
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.
[0041] Also according to the invention, surfactants such as the
above, including any combination thereof may be used in a paper
making process together with a fatty acid oxidizing 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).
[0042] In another particular embodiment, each of the above ranges
refers to the total amount of surfactants.
[0043] Enzymes
[0044] EC-numbers may be used for classification of enzymes, e.g.
lipase EC-number for enzymes having lipase activity, etc. Reference
is made to the Recommendations (1992) of the Nomenclature Committee
of the International Union of Biochemistry and Molecular Biology,
Academic Press Inc., 1992.
[0045] It is to be understood that the term enzyme, as well as the
various enzymes and enzyme classes mentioned herein, encompass
wild-type enzymes, as well as any variant thereof that retains the
activity in question. Such variants may be produced by recombinant
techniques. The wild-type enzymes may also be produced by
recombinant techniques, or by isolation and purification from the
natural source.
[0046] In a particular embodiment the enzyme in question is
well-defined, meaning that only one major enzyme component is
present. This can be inferred e.g. by fractionation on an
appropriate Size-exclusion column. Such well-defined, or purified,
or highly purified, enzyme can be obtained as is known in the art
and/or described in publications relating to the specific enzyme in
question.
[0047] Fatty Acid Oxidizing Enzyme
[0048] The term "a" fatty acid oxidizing enzyme means at least one
of such enzymes. The term "at least one" means one, two, three,
four, five, six or even more of such enzymes.
[0049] In the present context, a fatty acid oxidizing enzyme is an
enzyme which hydrolyzes the substrate linoleic acid more
efficiently than the substrate syringaldazine. "More efficiently"
means with a higher reaction rate. This can be tested using the
method described in Example 2, and calculating the difference
between (1) absorbancy increase per minute on the substrate
linoleic acid (absorbancy at 234 nm), and (2) absorbancy increase
per minute on the substrate syringaldazine (absorbancy at 530 nm),
i.e. by calculating the Reaction Rate Difference
(RRD)=(d(A.sub.234)/dt-d(A.sub.530)/dt). If the RRD is above zero,
the enzyme in question qualifies as a fatty acid oxidizing enzyme
as defined herein. If the RRD is zero, or below zero the enzyme in
question is not a fatty acid oxidizing enzyme.
[0050] In particular embodiments, the RRD is at least 0.05, 0.10,
0.15, 0.20, or at least 0.25 absorbancy units/minute.
[0051] In a particular embodiment of the method of Example 2, the
enzymes are well-defined. Still further, for the method of Example
2 the enzyme dosage is adjusted so as to obtain a maximum
absorbancy increase per minute at 234 nm, or at 530 nm. In
particular embodiments, the maximum absorbancy increase is within
the range of 0.05-0.50; 0.07-0.4; 0.08-0.3; 0.09-0.2; or 0.10-0.25
absorbancy units pr. min. The enzyme dosage may for example be in
the range of 0.01-20; 0.05-15; or 0.10-10 mg enzyme protein per
ml.
[0052] In the alternative, a "fatty acid oxidizing enzyme" may be
defined as an enzyme capable of oxidizing unsaturated fatty acids
more efficiently than syringaldazine. The activity of the enzyme
could be compared in a standard oximeter setup as described in
Example 1 of the present application at pH 6 and 30.degree. C.
including either syringaldazine or linoleic acid as substrates.
[0053] In a particular embodiment, the fatty acid oxidizing enzyme
is defined as an enzyme classified as EC 1.11.1.3, or as EC
1.13.11.--. EC 1.13.11.--means any of the sub-classes thereof,
presently forty-nine: EC 1.13.11.1-EC 1.13.11.49. EC 1.11.1.3 is
designated fatty acid peroxidase, and EC 1.13.11.--is designated
oxygenases acting on single donors with incorporation of two atoms
of oxygen.
[0054] In a further particular embodiment, the EC 1.13.11.--enzyme
is classified as EC 1.13.11.12, EC 1.13.11.31, EC 1.13.11.33, EC
1.13.11.34, EC 1.13.11.40, EC 1.13.11.44 or EC 1.13.11.45,
designated lipoxygenase, arachidonate 12-lipoxygenase, arachidonate
15-lipoxygenase, arachidonate 5-lipoxygenase, arachidonate
8-lipoxygenase, linoleate diol synthase, and linoleate 11
-lipoxygenase, respectively).
[0055] In a further particular embodiment, the fatty acid oxidizing
enzyme is a lipoxygenase (LOX), classified as EC 1.13.11.12, which
is an enzyme that catalyzes the oxygenation of polyunsaturated
fatty acids, especially cis,cis-1,4-dienes, e.g. linoleic acid and
produces a hydroperoxide. But also other substrates may be
oxidized, e.g. monounsaturated fatty acids.
[0056] Microbial lipoxygenases can be derived from, e.g.,
Saccharomyces cerevisiae, Thermoactinomyces vulgaris, Fusarium
oxysporum, Fusarium proliferatum, Thermomyces lanuginosus,
Pyricularia oryzae, and strains of Geotrichum. The preparation of a
lipoxygenase derived from Gaeumannomyces graminis is described in
Examples 3-4 of WO 02/20730. The expression in Aspergillus oryzae
of a lipoxygenase derived from Magnaporthe salvinii is described in
Example 2 of PCT/DK02/00251, and this enzyme can be purified using
standard methods, e.g. as described in Example 4 of WO
02/20730.
[0057] Lipoxygenase (LOX) may also be extracted from plant seeds,
such as soybean, pea, chickpea, and kidney bean. Alternatively,
lipoxygenase may be obtained from mammalian cells, e.g. rabbit
reticulocytes.
[0058] Lipoxygenase activity may be determined
spectrophotometrically at 25.degree. C. by monitoring the formation
of hydroperoxides. For the standard analysis, 10 micro liters
enzyme was added to a 1 ml quartz cuvette containing 980 micro
liter 25 mM sodium phosphate buffer (pH 7.0) and 10 micro liter of
substrate solution (10 mM linoleic acid dispersed with 0.2%(v/v)
Tween 20 (should not be kept for extended time periods)). The
enzyme was typically diluted sufficiently to ensure a turn-over of
maximally 10% of the added substrate within the first minute. The
absorbance at 234 nm was followed and the rate was estimated from
the linear part of the curve. The cis-trans-conjugated
hydro(pero)xy fatty acids were assumed to have a molecular
extinction coefficient of 23,000 M.sup.-1 cm.sup.-1.
[0059] The fatty acid oxidizing enzyme may also be applied together
with a substrate for the enzyme capable of enhancing the enzymatic
effect. Suitable substrates are hydrolyzed oils such as oils from
soybeans (rich in linoleic acid) or tall oil. Fatty acid substrates
may be released from the added oil by lipolytic enzymes or produced
during the Kraft pulping or sulphate cooking.
[0060] In particular embodiments the substrate is a compound with
1,4-pentadien structure, e.g. with cis,cis-1,4-pentadien structure,
i.e. compounds having at least one such element in its structural
formula. Examples of such substrates are unsaturated fatty acids,
e.g. palmitoleic acid, oleic acid, linoleic acid, linolenic acid,
and arachidonic acid, as well as their salts and esters, e.g.
methyl- and ethyl-esters.
[0061] In further particular embodiments the substrate is linoleic
acid; linoleic acid methyl or ethyl ester; linolenic acid, or
linolenic acid methyl or ethyl ester.
[0062] To explore the effect of adding a substrate for the fatty
acid oxidizing enzyme in question, the following method may be
used: The spectrum of 10 mM abietic acid (emulsified in 0.2% Tween
20) is recorded. Characteristic peaks are observed around 200 nm
and around 250 nm. In a first experiment, a fatty acid oxidizing
enzyme is added to the abietic acid emulsion. In a second
experiment, a substrate for the fatty acid oxidizing enzyme is also
added. The enzyme is e.g. a lipoxygenase derived from M. salvinii
as described above, and the substrate is e.g. linoleic acid. The
degradation of abietic acid is followed spectrophotometrically, and
the peaks around 200 nm and around 250 nm decrease more rapidly
when linoleic acid is added together with the lipoxygenase.
[0063] In particular embodiments of the above method, and of the
process of the invention, the substrate, e.g. linoleic acid, is
added in an amount of 5-10000 ppm (mg/l), or 10-9000, 10-8000,
25-7500, 30-7000, 50-6000, 50-5000, 50-4000, 75-3000, 75-2500,
80-2000, 90-1500, 100-1000, 150-800, or 200-700 ppm. In Example 4,
333 ppm of linoleic acid was used together with a fatty acid
oxidizing enzyme.
[0064] In further particular embodiments of the above method, and
of the process of the invention, the fatty acid oxidizing enzyme is
used in an amount of 0.005-50 ppm (mg/l), or 0.01-40, 0.02-30,
0.03-25, 0.04-20, 0.05-15, 0.05-10, 0.05-5, 0.05-1, 0.05-0.8,
0.05-0.6, or 0.1-0.5 ppm. The amount of enzyme refers to mg of a
well-defined enzyme preparation.
[0065] In the process of the invention, the fatty acid oxidizing
enzyme may be applied alone or together with an additional enzyme.
The term "an additional enzyme" means at least one additional
enzyme, e.g. one, two, three, four, five, six, seven, eight, nine,
ten or even more additional enzymes.
[0066] The term "applied together with" (or "used together with")
means that the additional enzyme may be applied in the same, or in
another step of the process of the invention. The other process
step may be upstream or downstream in the paper manufacturing
process, as compared to the step in which the papermaking pulp or
process water is treated with a fatty acid oxidizing enzyme.
[0067] In particular embodiments the additional enzyme is an enzyme
which has protease, lipase, xylanase, cutinase, oxidoreductase,
cellulase, endoglucanase, amylase, mannanase, steryl esterase,
and/or cholesterol esterase activity. Examples of oxidoreductase
enzymes are enzymes with laccase, and/or peroxidase activity. In a
preferred embodiment, the additional enzyme is lipase.
[0068] The term "a step" of a process means at least one step, and
it could be one, two, three, four, five or even more process steps.
In other words the fatty acid oxidizing enzyme of the invention may
be applied in at least one process step, and the additional
enzyme(s) may also be applied in at least one process step, which
may be the same or a different process step as compared to the step
where the fatty acid oxidizing enzyme is used.
[0069] The term "enzyme preparation" means a product containing at
least one fatty acid oxidizing enzyme. The enzyme preparation may
also comprise enzymes having other enzyme activities, preferably
lipolytic enzymes or enzymes having oxidoreductase activity, most
preferably lipolytic enzymes. In addition to the enzymatic activity
such a preparation preferably contains at least one adjuvant.
Examples of adjuvants, which are used in enzyme preparations for
the paper and pulp industry are buffers, polymers, surfactants and
stabilizing agents.
[0070] Additional Enzymes
[0071] Any enzyme having protease, lipase, xylanase, cutinase,
oxidoreductase, cellulase endoglucanase, amylase, mannanase, steryl
esterase, and/or cholesterol esterase activity can be used as
additional enzymes in the use and process of the invention. Below
some non-limiting examples are listed of such additional enzymes.
The enzymes written in capitals are commercial enzymes available
from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
The activity of any of those additional enzymes can be analyzed
using any method known in the art for the enzyme in question,
including the methods mentioned in the references cited.
[0072] Examples of cutinases are those derived from Humicola
insolens (U.S. Pat. No. 5,827,719); 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 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), or variants thereof such as
those described in WO 00/34450, or WO 01/92502.
[0073] Examples of proteases are the ALCALASE, ESPERASE, SAVINASE,
NEUTRASE and DURAZYM proteases. Other proteases are derived 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, and mutants thereof, e.g. those disclosed in WO 91/00345
and EP 415296.
[0074] Examples of amylases are the BAN, AQUAZYM, TERMAMYL, and
AQUAZYM Ultra amylases. An example of a lipase is the RESINASE A2X
lipase. An example of a xylanase is the PULPZYME HC hemicellulase.
Examples of endoglucanases are the NOVOZYM 613, 342, and 476 enzyme
products.
[0075] Examples of mannanases are the Trichoderma reesei
endo-beta-mannanases described in Stahlbrand et al, J. Biotechnol.
29 (1993), 229-242.
[0076] Examples of steryl esterases. peroxidases, laccases, and
cholesterol esterases are disclosed in the references mentioned in
the background art section hereof. Further examples of
oxidoreductases are the peroxidases and laccases disclosed in EP
730641; WO 01/98469; EP 719337; EP 765394; EP 767836; EP 763115;
and EP 788547. In the present context, whenever an oxidoreductase
enzyme is mentioned that requires or benefits from the presence of
acceptors (e.g. oxygen or hydrogenperoxide), enhancers, mediators
and/or activators, such compounds should be considered to be
included. Examples of enhancers and mediators are disclosed in EP
705327; WO 98/56899; EP 677102; EP 781328; and EP 707637. If
desired a distinction could be made by defining an oxidoreductase
enzyme system (e.g. a laccase, or a peroxidase enzyme system) as
the combination of the enzyme in question and its acceptor, and
optionally also an enhancer and/or mediator for the enzyme in
question.
[0077] These are particular embodiments of the present invention:
Use of a fatty acid oxidizing enzyme for reducing the deposition of
pitch in the paper making process. A process for reducing
deposition of pitch in the paper making process, wherein the
process comprises treating the pulp and/or process water with an
enzyme preparation comprising a fatty acid oxidizing enzyme;
preferably a process wherein the pulp is a mechanical pulp or a
chemical pulp or a combination thereof; such as a chemical pulp.
The process as described above, wherein the enzyme is classified in
EC 1.13.11, preferably 1.13.11.12, preferably wherein the enzyme is
derived from a strain of the genus Magnaporthaceae, preferably M.
salvinii or the genus Gaeumannomyces, preferably G. graminis. The
process described above, wherein the treatment is carried out by
adding a substrate for the enzyme, preferably linoleic acid. The
process described above, wherein the enzyme preparation comprises a
lipolytic enzyme and/or a further oxidoreductase. The process
described above wherein the treatment is carried out at a
temperature is in the range 20-90.degree. C., preferably
40-70.degree. C., and/or at a pH in the range 2-11, preferably
4-9.5, more preferably 6-9, and/or wherein treatment is carried out
in 10 minutes to 3 hours, preferably 15 minutes to 1 hour; and/or
wherein the enzyme is added in a concentration in the range of
0.0001-20 mg/g, preferably 0.0001-10 mg/g, more preferably 0.001-1
mg/g and most preferably 0.01-0.1 mg/g. In one embodiment of the
above process the enzyme preparation is added in the storage chest
or mixing chest before the paper machine.
[0078] 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.
[0079] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
EXAMPLES
Example 1
[0080] Measurement of the Activity of Fatty Acid Oxidizing Enzymes
on Linoleic Acid
[0081] An "Oxi 3000 Oximeter" (WTW, Weilheim, Germany) with a
TriOxmatic 300 oxygen electrode and a standard reaction volume of 4
ml was used.
[0082] 10 mg linoleic acid (10 ml 60% linoleic acid) was dissolved
in 1 ml ethanol, and 2 micro liter Tween 20 was added. From this
stock substrate solution 50 micro liter was added into a reaction
beaker containing 3.85 ml buffer solution (Britton-Robinson: 100 mM
of Phosphoric-, Acetic- and Boric acid; pH adjusted with NaOH) with
a small stir bar allowing the solution to be mixed well, and the
oxygen electrode was inserted into the reaction beaker. 100 micro
liter purified enzyme solution was added, viz. (a) lipoxygenase
derived from Magnaporthe salvinii at a concentration of approx. 0.4
mg/ml; or (b) lipoxygenase derived from Gaeumannomyces. graminis at
a concentration of approx. 0.76 mg/ml (which means approximately
0.02 mg/ml in the final reaction). These lipoxygenases were
prepared as previously described. The temperature was 25.degree. C.
The concentration of dissolved oxygen (mg/l) is measured and
plotted as a function of time (min.). The enzymatic activity is
calculated as the slope of the linear part of the curve (mg/l/min.)
after addition of the enzyme. The baseline was corrected by
subtraction when relevant, meaning that if the curve showing oxygen
concentration as a function of time had a slope of above about 0.05
mg oxygen/ml/min before addition of the fatty acid oxidizing enzyme
(i.e. the control), this value was subtracted from the sample slope
value.
[0083] Table 1 below shows the results of the experiments.
1 TABLE 1 Fatty Acid Oxidizing Enzyme (a) LOX from M. salvinii (b)
LOX from G. graminis pH mgO.sub.2/ml/min mgO.sub.2/ml/min 2 0.0 0.0
4 0.4 0.1 5 0.7 0.4 6 1.1 0.4 7 1.0 0.4 8 0.7 0.5 9 0.8 0.4 10 0.7
0.4 11 0.6 0.2
Example 2
[0084] Fatty Acid Oxidizing Enzymes
[0085] Four enzymes, viz. two laccases and two lipoxygenases were
tested as described below. The laccase derived from Polyporus
pinsitus had a MW by SDS-Page of 65 kDa, a pl by IEF of 3.5, and an
optimum temperature at pH 5.5 of 60.degree. C. The laccase derived
from Coprinus cinereus had a MW by SDS-Page of 67-68 kDa, a pl by
IEF of 3.5-3.8, and an optimum temperature at pH 7.5 of 65.degree.
C. The enzymes were prepared and purified as described in WO
96/00290 and U.S. Pat. No. 6,008,029. The two lipoxygenases were
derived from Magnaporthe salvinii and Gaeumannomyces graminis, and
they were prepared as described previously.
[0086] The enzyme dosage was adjusted to ensure maximum absorbancy
increase per minute at 234 nm /530 nm, viz. in the range of
0.1-0.25 absorbancy units pr. min.
[0087] Substrate solution: 11.65 mg linoleic acid (60% Sigma), as
well as 12.5 ml 0.56 mM Syringaldazine (Sigma) in ethanol was mixed
with deionized water to a total volume of 25 ml.
[0088] 50 microliter of the enzyme preparation to be tested was
transferred to a quartz cuvette containing 900 microliter phosphate
buffer (50 mM, pH 7.0) and 50 microliter of the substrate solution
The cuvette was placed in a spectrofotometer, thermostated at
23.degree. C., and the absorbancies at 234 nm and 530 nm were
measured as a function of time. The absorbancy at 530 nm is
indicative of degradation of syringaldazine, whereas the absorbancy
at 234 nm is indicative of degradation of linoleic acid. The
absorbancy increase as a function of time is calculated on the
basis of minutes 2 to 4 of the reaction time, i.e. d(A.sub.234)/dt,
as well as d(A.sub.530)/dt.
[0089] The results are shown in Table 2 below. Of these four
enzymes, only the two lipoxygenases qualify as a fatty acid
oxidizing enzyme as defined herein. This is because RRD=Reaction
Rate Difference=(dA.sub.234/dt-dA.su- b.530/dt) is above zero only
for these two enzymes.
2TABLE 2 dA.sub.530/dt dA.sub.234/dt dA.sub.234/dt - dA.sub.530/dt
Enzyme (units/min) (units/min) (units/min) Polyporus pinsitus 0.20
0.002* -0.20 laccase Magnaporthe salvinii 0.0001* 0.13 0.13
lipoxygenase Coprinus cinereus 0.17 -0.001* -0.17 laccase
Gaeumannomyces -0.03* 0.21 0.21 graminis lipoxygenase *this is
equivalent to zero activity (analytical inaccuracy)
Example 3
[0090] Reduction of Pitch with a Fatty Acid Oxidizing Enzyme
[0091] A model pitch is prepared as follows:
[0092] 50% Linoleic Acid 60% (Sigma L-1626).
[0093] 20% Abietic Acid (Sigma A9424).
[0094] 20% Oleic Acid (Merck 471).
[0095] 5% Cholesterol-Linoleate (Sigma C-0289).
[0096] 5% Olive Oil (Sigma 0-1500).
[0097] Mixed for 30 minutes at 65.degree. C. Stored in refrigerator
for no longer than 30 days.
[0098] Preparation of 0.1% pitch suspension:
[0099] 50 mg model pitch
[0100] 1 ml ethanol.
[0101] 1 ml 0.1 M NaOH.
[0102] 48 ml buffer (50 mM borate pH 9.0) Mixed for 10 minutes at
room temperature.
[0103] Circular paper pieces (diameter=5.5-6 mm; Multicopy 80
g/m.sup.2) are transferred to the wells of two 96-well
microtiterplates (ID 269620 from NUNC) designated A and B. Two
other similar microtiterplates C and D are also used, but without
paper pieces. 100 microliter of the 0.1% pitch suspension is added
into each of the wells of each of these four microtiterplates. A
lipoxygenase derived from Magnaporthe salvinii as described
previously is used as the fatty acid oxidizing enzyme, and it is
added to the wells of microtiterplates A and C to obtain an
in-well-concentration of 10 ppm. A similar amount of buffer (50 mM
borate pH 9.0) is added to the wells of microtiterplates B and D.
The microtiterplates are then incubated during shaking (600 rpm)
for 30 minutes. After 30 minutes, 20 microliter of the
enzyme-treated pitch suspension is transferred onto a second set of
microtiterplates corresponding to microtiterplates A-D (Corning
Inc. Costar UV plate 96 well No. 3635) each containing 200
microliter ethanol per well (solubilizing the pitch components).
Abietic acid, a major component of the pitch, absorps strongly at
about 255 nm. Accordingly, A.sub.255 is indicative of the amount of
pitch remaining in the suspension. A.sub.255 is determined as the
average of 8 identical experiments, and the amount of pitch
adsorbed onto the paper is estimated based on the variation in
A.sub.255 measured in the pitch suspensions obtained after
incubation with and without paper present (after 11.times. dilution
in ethanol).
[0104] The results are shown in a table like the below Table 3. The
basic (blind) adsorption of pitch onto the paper in the absence of
a fatty acid oxidizing enzyme may be calculated as the ratio D/B.
The effect of the enzyme (the sample) as regards the adsorption of
pitch to the paper may be calculated as the ratio C/A. One way of
showing hat the enzyme has caused a reduction in the deposition of
pitch is if (C/A-D/B) is below zero. Alternatively, the enzyme
effect may be calculated as ((C-A)-(D-B)), and if this value is
below zero, this would be another way of showing the effect of the
enzyme on the deposition of pitch. Other solid materials than paper
may also be tested, e.g. metal, and textile (Style 400 cotton). The
above ways of showing reduction in pitch deposition are applicable
by analogy as regards deposition on the other solid materials.
[0105] Of course, the assay-pH (i.e. buffer), and the
assay-temperature is selected paying regard to the characteristics
of the fatty acid oxidizing enzyme in question, e.g. an assay pH of
around 4, 5, 6, 7, 8, 9, 10, or 11; and an assay-temperature of
around 10, 15, 20, 25, 30, 37, 40, 50, 60, 70, 80, 90 or 95.degree.
C.
3 TABLE 3 With M. salvinii Without M. salvinii A.sub.255
lipoxygenase lipoxygenase With paper A B Without paper C D
Example 4
[0106] Bleaching Paper with a Fatty Acid Oxidizing Enzyme
[0107] Unbleached Kraft Pulp derived from Eucalyptus grandis was
used. The pulp was repulped at 4% consistency in a pulper
manufactured by Loretzen and Wettre. Repulping was done in buffer
(Britton-Robinson) at pH=9.0.
[0108] Britton Robinson buffer:
[0109] 100 mM Phosphoric acid (85%) 6.28 ml
[0110] 100 mM Acetic acid (100%) 5.72 ml
[0111] 100 mM Boric acid 6.18 g
[0112] Dem. water up to 1000 ml
[0113] The pH was adjusted to 9.0 by addition of sodium
hydroxide.
[0114] After repulping the pulp slurry was diluted to 1%
consistency by addition of buffer and pH was readjusted to pH
=9.0.
[0115] Treatments with a fatty acid oxidizing enzyme were carried
out in beakers containing 3 g dry pulp i.e. 300 ml pulp slurry. The
treatments were carried out at 25.degree. C. in a water bath with
agitation by magnetic stir bars, 500 rpm. 333 ppm linoleic acid was
added to all beakers. The fatty acid oxidizing enzyme used was a
purified lipoxygenase derived from Gaeumannomyces graminis prepared
as described previously. The amount of enzyme used appears from
Tables 4 and 5 below. The enzyme treatment was carried out for 2
hours. Two beakers were run for each condition.
[0116] After two hours the enzyme reaction was stopped by addition
of 5 ml (fixed amount) of NaOH (27.65% solution), this raises pH to
>12, and deactivates the enzyme.
[0117] The content of the beaker was transferred quantitatively to
a 1000 ml beaker using 700 ml deionised water. This pulp suspension
was poured onto a Buchner funnel (15 cm diameter) with a filter
paper. A paper sheet was formed by sucking the water out. The paper
sheet was removed from the funnel and separated from the filter
paper. The sheet was pressed in a sheet press manufactured by
Lorentzen and Wettre. The sheet was pressed in a sandwich of metal
plate, 2 blotting papers, 2 filter papers, the sheet, 2 filter
papers, 2 blotting papers, metal plate at 0,4 MPa for 5.5 min. Wet
papers were replaced by dry ones and the pressing repeated at 0.4
MPa for 2 min. The sheets were air dried overnight.
[0118] The brightness of the sheets was measured using a Macbeth
Color-Eye 7000 reflectometer. The brightness was recorded at 600nm.
4 measurements were done at each sheet. The results obtained are
shown in Table 4 below.
[0119] The Kappa Number, which describes the degree of
delignification of a pulp, was also determined for each sheet using
the method described in Tappi Test Methods T236 (Tappi Press). The
amounts used for each determination was 1/4 of that described in
the standard method. The dry matter content of the sheets was
determined to calculate the Kappa no. The results obtained are
shown in Table 5 below.
4 TABLE 4 Brightness Brightness Reflection at Reflection at 600 nm
600 nm Brightness Sheet 1 Sheet 2 Reflection at Average of 4
Average of 4 600 nm LOX [mg/l] determinations Determinations
Average 0 46.93 46.60 46.77 0.1 49.69 50.23 49.96 1.3 48.51 47.15
47.83 3.2 49.16 50.12 49.64 6.3 48.56 52.56 50.56
[0120]
5TABLE 5 Kappa No. Kappa No. Sheet 1 Sheet 2 Average of 3 Average
of 3 Kappa No. LOX [mg/l] determinations determinations Average 0
18.44 18.43 18.44 0.1 14.80 14.68 14.74 1.3 14.94 14.93 14.94 3.2
14.65 14.42 14.54 6.3 14.83 14.40 14.61
Example 5
[0121] De-Inking Old Newsprint with a Fatty Acid Oxidizing
Enzyme
[0122] 200 g shredded old newsprint was placed in a Hobart Mixer
together with 1500 ml of water. The water bath temperature was set
at 45.degree. C. Mixing occurred at low speed for about 0.5-1
minutes. Then 3.6 kg/ton (7 lb/ton) of surfactant and 2 mg (10
mg/ton pulp) of a lipoxygenase derived from Magnaporthe salvinii
prepared as described previously was added to the mixer, following
which 500 ml of water was added to Hobart and mixed well. The mixer
was run on low speed for 30 minutes. The pulper temperature was set
at 45.degree. C., and the pH at 7.
[0123] Half of the pulp was transferred from the mixer to a
container and diluted to 10 l. Stirring took place for 2
minutes.
[0124] Feed pads: 30 ml of pulp was measured from the mixer and
diluted to 300 ml with water and mixed well. The pulp was filtered
through a Wattman#40 filter paper under vacuum. The pad was dried
at 90.degree. C. (195.degree. F.) for 10 minutes.
[0125] Regular washing of pads: 900 ml of pulp slurry was measured
from the container, poured slowly onto an 80 mesh sieve and shaken
slowly until all free water drained. All pulp was removed and put
into a 1000 ml beaker, which was filled with water up to the 900 ml
line. The pulp was slowly stirred. 300 ml of the pulp slurry was
measured and filtered through a Wattman#40 filter paper under
vacuum. The pad was dried at 90.degree. C. (195.degree. F.) for 10
minutes.
[0126] Hyper-washing of pads: 900 ml of pulp slurry was measured
from the container, poured slowly onto an 80 mesh sieve and shaken
slowly until all free water drained. The pulp was rinsed with
faucet water for 3 minutes, removed and put into a 1000 ml beaker,
which was filled with water up to the 900 ml line. The pulp was
slowly stirred. 300 ml of the pulp slurry was measured and filtered
through a Wattman #40 filter paper to make a filter pad. The pads
were dried on a speed dryer at 90.degree. C. (195.degree. F.) for
10 minutes.
[0127] The brightness of the pads was determined by a Macbeth color
eye using a Tappi standard method (T452).
[0128] Comparative experiments were conducted as described above
with two commercial enzymes, viz. the lipase RESINASE A 2.times.,
and the cellulase DENIMAX L, both commercially available from
Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. These
enzyme preparations were used in an amount of 0.51 kg per ton pulp
(1 lb/t).
[0129] The results are shown in Table 6 below.
6 TABLE 6 Brightness Enzyme Feed Pads Washed Pads Hyper-washed Pads
Control (no enzyme) 38.5 41.7 45.9 Fatty acid oxidizing 38.1 44.0
48.2 enzyme RESINASE A 2X 40.6 42.9 45.9 (lipase) DENIMAX L 37.6
39.0 44.2 (cellulase)
* * * * *