US20030047295A1 - Bio-bleaching of pulp using laccase, mediator, and chain transfer agent - Google Patents
Bio-bleaching of pulp using laccase, mediator, and chain transfer agent Download PDFInfo
- Publication number
- US20030047295A1 US20030047295A1 US10/027,149 US2714901A US2003047295A1 US 20030047295 A1 US20030047295 A1 US 20030047295A1 US 2714901 A US2714901 A US 2714901A US 2003047295 A1 US2003047295 A1 US 2003047295A1
- Authority
- US
- United States
- Prior art keywords
- enzyme
- laccase
- chain transfer
- mediator
- pulp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108010029541 Laccase Proteins 0.000 title claims abstract description 46
- 238000004061 bleaching Methods 0.000 title claims abstract description 21
- 239000012986 chain transfer agent Substances 0.000 title claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 claims abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 40
- 230000008569 process Effects 0.000 claims abstract description 34
- 230000001590 oxidative effect Effects 0.000 claims abstract description 24
- 239000000203 mixture Substances 0.000 claims abstract description 22
- 229920005610 lignin Polymers 0.000 claims abstract description 18
- 239000000463 material Substances 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 108010015428 Bilirubin oxidase Proteins 0.000 claims abstract description 7
- 108010031396 Catechol oxidase Proteins 0.000 claims abstract description 7
- 102000030523 Catechol oxidase Human genes 0.000 claims abstract description 7
- 102000003425 Tyrosinase Human genes 0.000 claims abstract description 6
- 108060008724 Tyrosinase Proteins 0.000 claims abstract description 6
- 230000000694 effects Effects 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 13
- 239000007800 oxidant agent Substances 0.000 claims description 10
- HJUGFYREWKUQJT-UHFFFAOYSA-N tetrabromomethane Chemical compound BrC(Br)(Br)Br HJUGFYREWKUQJT-UHFFFAOYSA-N 0.000 claims description 10
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Natural products C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 239000011122 softwood Substances 0.000 claims description 6
- 229920001131 Pulp (paper) Polymers 0.000 claims description 5
- 239000003570 air Substances 0.000 claims description 5
- 239000011121 hardwood Substances 0.000 claims description 5
- 125000005577 anthracene group Chemical group 0.000 claims description 4
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims 2
- 239000001913 cellulose Substances 0.000 claims 2
- 239000002994 raw material Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 description 19
- 238000012546 transfer Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000002708 enhancing effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 7
- 238000004076 pulp bleaching Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 239000007844 bleaching agent Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 3
- 235000011613 Pinus brutia Nutrition 0.000 description 3
- 241000018646 Pinus brutia Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- YARKTHNUMGKMGS-LQGKIZFRSA-N chembl3193980 Chemical compound COC1=C(O)C(OC)=CC(\C=N\N=C\C=2C=C(OC)C(O)=C(OC)C=2)=C1 YARKTHNUMGKMGS-LQGKIZFRSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000002845 discoloration Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000002655 kraft paper Substances 0.000 description 2
- 239000012286 potassium permanganate Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- QGXCOCVWIKYPKW-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole-6-sulfinic acid Chemical compound OS(=O)C1=CC=C2N(CC)CSC2=C1 QGXCOCVWIKYPKW-UHFFFAOYSA-N 0.000 description 1
- HJBLUNHMOKFZQX-UHFFFAOYSA-N 3-hydroxy-1,2,3-benzotriazin-4-one Chemical compound C1=CC=C2C(=O)N(O)N=NC2=C1 HJBLUNHMOKFZQX-UHFFFAOYSA-N 0.000 description 1
- HRRVLSKRYVIEPR-UHFFFAOYSA-N 6-hydroxy-5-nitroso-1H-pyrimidine-2,4-dione Chemical compound OC1=NC(O)=C(N=O)C(O)=N1 HRRVLSKRYVIEPR-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010054320 Lignin peroxidase Proteins 0.000 description 1
- 108010059896 Manganese peroxidase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100038277 Prostaglandin G/H synthase 1 Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- BPHHNXJPFPEJOF-UHFFFAOYSA-J chembl296966 Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]S(=O)(=O)C1=CC(S([O-])(=O)=O)=C(N)C2=C(O)C(N=NC3=CC=C(C=C3OC)C=3C=C(C(=CC=3)N=NC=3C(=C4C(N)=C(C=C(C4=CC=3)S([O-])(=O)=O)S([O-])(=O)=O)O)OC)=CC=C21 BPHHNXJPFPEJOF-UHFFFAOYSA-J 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- GMJUGPZVHVVVCG-UHFFFAOYSA-N n-hydroxy-n-phenylacetamide Chemical compound CC(=O)N(O)C1=CC=CC=C1 GMJUGPZVHVVVCG-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
- D21C9/10—Bleaching ; Apparatus therefor
- D21C9/1026—Other features in bleaching processes
- D21C9/1036—Use of compounds accelerating or improving the efficiency of the processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
- D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
Definitions
- the present invention relates to the use of chain transfer agents that, in combination with laccase and a mediator, can be used to bleach pulp.
- Paper pulp is typically processed from wood through the Kraft (and other) processes.
- the process produces a pulp with a dark brown color, mostly due to the presence of lignin and lignin derivatives.
- the lignin has to be removed by a process known in the art as “bleaching.” This is typically done commercially in several stages in pulp mills, wherein lignin is first oxidized and then removed from the pulp.
- Bio-bleaching is a methodology whereby an enzyme is used to decrease the optical brightness and/or lignin content of the pulp or paper.
- the standard measure of bleaching efficiency is “Kappa number.”
- Enzymes that have most commonly been used include laccase, lignin peroxidase, and manganese peroxidase.
- An enzyme group that has received particular attention is the laccase family of enzymes, which are copper-containing enzymes that are known to be good oxidizing agents in the presence of oxygen. Laccases are found in microbes, fungi, and higher organisms.
- the oxidizing efficiency of a laccase can be improved through the use of a mediator that enhances the activity of the laccase.
- Systems that include a laccase and a mediator are known in the art as laccase-mediator systems (LMS).
- LMS laccase-mediator systems
- mediators for use in laccase-mediator systems include HBT (1-hydroxybenzotriazole), ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfinic acid)], NHA (N-hydroxyacetinilide), NHAA (N-acetyl-N-phenylhydroxylamine), HBTO (3-hydroxy-1,2,3-benzotriazin-4(3H)-one), and VIO (violuric acid).
- HBT 1-hydroxybenzotriazole
- ABTS 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfinic acid)
- NHA N-hydroxyacetinilide
- NHAA N
- the invention provides the use of chain transfer agents in standard laccase-mediator systems (LMS). These agents themselves are not necessarily mediators that enhance the activity of the enzyme. However, the chain transfer agents have been found to enhance the bleaching efficiency of certain laccase/mediator systems.
- LMS laccase-mediator systems
- the invention provides a process of bleaching a lignin-containing material.
- the process comprises the step of treating the material with an oxidative enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent.
- the invention provides a process of oxidizing a substrate comprising treating the substrate with an oxidizing enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent.
- the invention also provides a composition comprising an oxidative enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent.
- the enzyme has laccase activity.
- the enzyme is a laccase, a catechol oxidase, a monophenol monooxygenase, a bilirubin oxidase, or a mixture thereof.
- the invention further comprises adding a hydrolase, such as xylanase.
- the process of the invention can further include the addition of an oxidizing agent, such as air, oxygen, and/or hydrogen peroxide.
- the present invention discloses the use of chain transfer agents to enhance the bleach efficiency of laccase/mediator systems.
- Laccase itself can bleach pulp only to a limited extent.
- Certain mediators can enhance the activity of laccase in pulp bleaching. It has been discovered that some mediators are effective in bleaching an organic dye but are not effective in bleaching pulp. In fact, in some cases the Kappa numbers are found to increase after treating the pulp with laccase and one of these mediators.
- LMS can oxidize and degrade lignin and thereby achieve bleaching, it can also polymerize lignin under suitable reaction conditions. The oxidation, degradation, and polymerization reactions compete with one another. In this scenario, it is then possible to have an LMS that gives a high activity for the dye assay (predominant reaction being oxidation), but an increase in Kappa number (if polymerization is the major reaction).
- Vinyl polymerization can be considered to consist of four reactions: (i) initiation; (ii) propagation; (iii) chain transfer; and (iv) termination. In all four reactions, free radicals are involved.
- One common method to cut down the molecular weight of a polymer during polymer synthesis is to use chain transfer agents. These agents do not destroy the free radicals, but instead terminate a growing polymer chain, and start a new chain.
- Chain transfer agents have not been used previously in combination with mediators. In fact, the role of chain transfer in bleaching has not been previously appreciated. It is noteworthy that a chain transfer agent does not generate free radicals by itself, nor does it trap free radicals. Chain agents are described, for example, by G. Scott in Chapter 4, Antioxidants: Radical Chain-Breaking Mechanism from “Atmospheric oxidation and antioxidants”, Elsevier Publishing Co., New York, N.Y., p. 115.
- the mediator of the invention is a hardwood black liquor or a softwood black liquor.
- the chain transfer agent is anthracene, carbon tetrabromide, or a mixture thereof.
- the mediators and chain transfer agents of this invention are capable of enhancing the activities of laccase and laccase-related enzymes for the purpose of pulp bleaching.
- these enzymes include laccase enzymes of the enzyme classification EC 1.10.3.2, catechol oxidase enzymes of the enzyme classification EC 1.10.3.1, the monophenol monooxygenase enzymes of the enzyme classification EC 1.14.99.1, and bilirubin oxidase enzymes of the enzyme classification EC 1.3.3.5.
- the EC (Enzyme Commission) number is based upon the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB).
- the laccase in this invention may be derived from microbial, fungal, or other sources. It may furthermore be produced by recombinant methods, such as cultivating a host cell transformed with a recombinant DNA vector which includes a DNA sequence encoding the laccase (and DNA sequences encoding functions that permit the expression of laccase DNA sequence) in a culture medium under the conditions that permits the expression of the laccase, and recovering the enzyme from the culture.
- Another aspect of the invention provides a process for oxidizing a substrate that comprises treating the substrate with a composition comprising an enzyme exhibiting laccase activity and an enzyme enhancing agent.
- the enzyme enhancing agent can be selected from one or the above described enzyme enhancing agents.
- the enhancing agent may be present in concentrations of from about 0.01 micromolar to about 1000 micromolar, more preferably from about 0.1 micromolar to about 250 micromolar and most preferably from about 0.5 to about 100 micromolar.
- the chain transfer agent may be present in concentrations of from about 0.01 micromolar to about 1000 micromolar, more preferably from about 0.1 micromolar to about 250 micromolar and most preferably from about 0.5 to about 100 micromolar.
- the enzyme is used in amounts of from about 0.1 to 400 units (defined in Examples using ABTS as substrate) for 1 g dry pulp, more preferably from 1 to 200 units and even more preferably from about 10 to 100 units and most preferably from 20 to 50 units.
- the process of the invention can further include the step of adding an oxidizing agent, such as at least one of air, oxygen, and hydrogen peroxide.
- an oxidizing agent such as at least one of air, oxygen, and hydrogen peroxide.
- One embodiment of the invention provides a process for bleaching a lignin-containing material that comprises treating the material with an enzyme exhibiting laccase activity and an enzyme enhancing agent.
- the enhancing agent may be present in an amount of from about 0.1% to about 15% based on the weight of the dry lignin containing material, more preferably from about 0.1% to about 10% and even more preferably from about 0.5% to about 5% and most preferably from about 1% to about 4%.
- a lignin containing material is wood pulp.
- the process for bleaching a lignin-containing material can further include the step of adding an oxidizing agent, such as at least one of air, oxygen, and hydrogen peroxide.
- the specific activity was determined using ABTS (0.5 mM) as substrate.
- One unit of activity is equal to the umol of the oxidized product from ABTS per min per mg protein at pH 6.0 at 23° C.
- the activity of laccase was determined using syringaldazine as substrate.
- one unit of activity is equal to the change of 0.001 UV absorbance at A530 nm per minute per ug protein in 2 ml of 100 mM, pH 5.5 potassium phosphate buffer, and 0.5 ml of 0.25 mM syringaldazine in methanol at 23° C.
- the dried pulp was added to 80 ml of 50 mM phosphate, pH 5.5, and disintegrated in a blender. The pulp was then transferred to a 500 ml conical flask and the blender was washed with 20 ml of the same buffer. The washed buffer and the pulp were combined. The mediator was added at 1-4% (w/w, based on the dry pulp), followed by the addition of the laccase. The pH of the pulp mixture was adjusted to 5.5 if needed. The flask was covered with an aluminum foil with holes punched through and incubated at 50° C. for 16 hrs on a rotary shaker at 200 rpm.
- the filtered pulp was repulped in the alkaline solution and incubated at 70° C. for 3 hrs.
- the pH of the pulp mixture should be between 11.7-12.00 during the entire treatment. After the treatment, the pulp mixture was filtered through a Buchner funnel, and the pulp was washed with water extensively and then dried in a hood overnight.
- Each of the compounds i.e., potential mediators
- a phosphate buffer and a Chicago Blue solution A solution of a laccase was added to make up 1 ml of the final solution, containing 20 uM mediator, 20 mM buffer at pH 5.5 or 7.0, 0.1-1% laccase (v/v) and Chicago Blue solution, with absorbance at A610 m between 0.6 to 0.8.
- the change in the absorbance at A610 nm was measured immediately using a UV-VIS spectrophotometer (UV-1201, Shimadzu Scientific Instruments) after the enzyme was added. The decrease in absorbance was recorded at 30-second intervals for 5 minutes and was used to estimate the efficiency of the mediator.
- the acid-treated black liquors were prepared using the following procedure: (1) adjust pH of 20 ml of the black liquors to 1.0 using conc. HCl; (2) incubate the acidified solution at 80C for 1 hr; (3) cool down to room temperature; (4) adjust the pH to neutral (6.5-7.5) using 5 M NaOH solution.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Paper (AREA)
Abstract
Description
- This application claims the benefit of U.S. Provisional Application No. 60/318,292, filed on Sep. 10, 2001.
- The present invention relates to the use of chain transfer agents that, in combination with laccase and a mediator, can be used to bleach pulp.
- Paper pulp is typically processed from wood through the Kraft (and other) processes. The process produces a pulp with a dark brown color, mostly due to the presence of lignin and lignin derivatives. For many applications, the lignin has to be removed by a process known in the art as “bleaching.” This is typically done commercially in several stages in pulp mills, wherein lignin is first oxidized and then removed from the pulp.
- Recently, several research groups have been working with enzymes to biologically bleach pulp, referred to as “bio-bleaching.” Bio-bleaching is a methodology whereby an enzyme is used to decrease the optical brightness and/or lignin content of the pulp or paper. The standard measure of bleaching efficiency is “Kappa number.” Enzymes that have most commonly been used include laccase, lignin peroxidase, and manganese peroxidase. An enzyme group that has received particular attention is the laccase family of enzymes, which are copper-containing enzymes that are known to be good oxidizing agents in the presence of oxygen. Laccases are found in microbes, fungi, and higher organisms.
- For many applications, the oxidizing efficiency of a laccase can be improved through the use of a mediator that enhances the activity of the laccase. Systems that include a laccase and a mediator are known in the art as laccase-mediator systems (LMS). There are several known mediators for use in laccase-mediator systems. These include HBT (1-hydroxybenzotriazole), ABTS [2,2′-azinobis(3-ethylbenzothiazoline-6-sulfinic acid)], NHA (N-hydroxyacetinilide), NHAA (N-acetyl-N-phenylhydroxylamine), HBTO (3-hydroxy-1,2,3-benzotriazin-4(3H)-one), and VIO (violuric acid). In addition, there are several compounds containing NH—OH or N—O that have been found to be useful as mediators. There remains a need, however, to increase the bleaching efficiency of bio-bleaching systems.
- The invention provides the use of chain transfer agents in standard laccase-mediator systems (LMS). These agents themselves are not necessarily mediators that enhance the activity of the enzyme. However, the chain transfer agents have been found to enhance the bleaching efficiency of certain laccase/mediator systems.
- The invention provides a process of bleaching a lignin-containing material. The process comprises the step of treating the material with an oxidative enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent. In another embodiment, the invention provides a process of oxidizing a substrate comprising treating the substrate with an oxidizing enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent. The invention also provides a composition comprising an oxidative enzyme, a mediator that enhances the oxidative activity of the enzyme, and a chain transfer agent.
- In one embodiment of the invention, the enzyme has laccase activity. In another embodiment, the enzyme is a laccase, a catechol oxidase, a monophenol monooxygenase, a bilirubin oxidase, or a mixture thereof. In yet another embodiment, the invention further comprises adding a hydrolase, such as xylanase. The process of the invention can further include the addition of an oxidizing agent, such as air, oxygen, and/or hydrogen peroxide.
- The present invention discloses the use of chain transfer agents to enhance the bleach efficiency of laccase/mediator systems. Laccase itself can bleach pulp only to a limited extent. Certain mediators can enhance the activity of laccase in pulp bleaching. It has been discovered that some mediators are effective in bleaching an organic dye but are not effective in bleaching pulp. In fact, in some cases the Kappa numbers are found to increase after treating the pulp with laccase and one of these mediators.
- One likely rationalization of this surprising finding entails the nature of laccase. Whereas the LMS can oxidize and degrade lignin and thereby achieve bleaching, it can also polymerize lignin under suitable reaction conditions. The oxidation, degradation, and polymerization reactions compete with one another. In this scenario, it is then possible to have an LMS that gives a high activity for the dye assay (predominant reaction being oxidation), but an increase in Kappa number (if polymerization is the major reaction).
- The above observation reminded us of a concept in free radical chemistry and polymer science. Vinyl polymerization can be considered to consist of four reactions: (i) initiation; (ii) propagation; (iii) chain transfer; and (iv) termination. In all four reactions, free radicals are involved. One common method to cut down the molecular weight of a polymer during polymer synthesis is to use chain transfer agents. These agents do not destroy the free radicals, but instead terminate a growing polymer chain, and start a new chain.
- In view of the above reasoning, we believe the use of a chain transfer agent may solve this problem. Chain transfer agents have not been used previously in combination with mediators. In fact, the role of chain transfer in bleaching has not been previously appreciated. It is noteworthy that a chain transfer agent does not generate free radicals by itself, nor does it trap free radicals. Chain agents are described, for example, by G. Scott in Chapter 4, Antioxidants: Radical Chain-Breaking Mechanism from “Atmospheric oxidation and antioxidants”, Elsevier Publishing Co., New York, N.Y., p. 115.
- In one embodiment, the mediator of the invention is a hardwood black liquor or a softwood black liquor. In another embodiment of the invention, the chain transfer agent is anthracene, carbon tetrabromide, or a mixture thereof.
- The process disclosed herein is not confined to bio-bleaching of pulp. Other laccase-catalyzed (or laccase-facilitated) oxidation reactions or polymerization may also be enhanced with the addition of chain transfer agents. Examples of chain transfer agents can be found in “Polymer Handbook”, Second Edition, Editors J. Brandrup and E. H. Immergut, J. Wiley & Son, New York, N.Y., 1975, pages II-57 thru II-104.
- The mediators and chain transfer agents of this invention are capable of enhancing the activities of laccase and laccase-related enzymes for the purpose of pulp bleaching. In the context of this invention, these enzymes include laccase enzymes of the enzyme classification EC 1.10.3.2, catechol oxidase enzymes of the enzyme classification EC 1.10.3.1, the monophenol monooxygenase enzymes of the enzyme classification EC 1.14.99.1, and bilirubin oxidase enzymes of the enzyme classification EC 1.3.3.5. The EC (Enzyme Commission) number is based upon the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB).
- The laccase in this invention may be derived from microbial, fungal, or other sources. It may furthermore be produced by recombinant methods, such as cultivating a host cell transformed with a recombinant DNA vector which includes a DNA sequence encoding the laccase (and DNA sequences encoding functions that permit the expression of laccase DNA sequence) in a culture medium under the conditions that permits the expression of the laccase, and recovering the enzyme from the culture.
- Another aspect of the invention provides a process for oxidizing a substrate that comprises treating the substrate with a composition comprising an enzyme exhibiting laccase activity and an enzyme enhancing agent. The enzyme enhancing agent can be selected from one or the above described enzyme enhancing agents.
- The enhancing agent may be present in concentrations of from about 0.01 micromolar to about 1000 micromolar, more preferably from about 0.1 micromolar to about 250 micromolar and most preferably from about 0.5 to about 100 micromolar.
- The chain transfer agent may be present in concentrations of from about 0.01 micromolar to about 1000 micromolar, more preferably from about 0.1 micromolar to about 250 micromolar and most preferably from about 0.5 to about 100 micromolar. The enzyme is used in amounts of from about 0.1 to 400 units (defined in Examples using ABTS as substrate) for 1 g dry pulp, more preferably from 1 to 200 units and even more preferably from about 10 to 100 units and most preferably from 20 to 50 units.
- The process of the invention can further include the step of adding an oxidizing agent, such as at least one of air, oxygen, and hydrogen peroxide.
- One embodiment of the invention provides a process for bleaching a lignin-containing material that comprises treating the material with an enzyme exhibiting laccase activity and an enzyme enhancing agent. In this embodiment of the invention, the enhancing agent may be present in an amount of from about 0.1% to about 15% based on the weight of the dry lignin containing material, more preferably from about 0.1% to about 10% and even more preferably from about 0.5% to about 5% and most preferably from about 1% to about 4%. One example of a lignin containing material is wood pulp. The process for bleaching a lignin-containing material can further include the step of adding an oxidizing agent, such as at least one of air, oxygen, and hydrogen peroxide.
- The following examples are illustrative of the present invention, and are not intended to be construed in any way as limiting the scope of the invention.
- Laccase Enzyme Assay
- In the examples, two Aspergillus laccases have been used, both from Novozymes A/S (Denmark). NovoSample 51002 works best at pH 4-5 while NovoSample 51003 works best at pH 5-6. Enzyme dosage has been found to have an effect on bio-bleaching. For example, 0.1 ml of the 51003 laccase gives a modest Kappa number reduction when HBT is used, and a huge reduction when ABTS is used.
- The specific activity was determined using ABTS (0.5 mM) as substrate. One unit of activity is equal to the umol of the oxidized product from ABTS per min per mg protein at pH 6.0 at 23° C. The extinction coefficient of the oxidized ABTS is: ε(max) at 420 nm=36,000M-1 cm-1.)
- Alternatively, the activity of laccase (NS51003) was determined using syringaldazine as substrate. In this case, one unit of activity is equal to the change of 0.001 UV absorbance at A530 nm per minute per ug protein in 2 ml of 100 mM, pH 5.5 potassium phosphate buffer, and 0.5 ml of 0.25 mM syringaldazine in methanol at 23° C.
- Kappa Number
- Delignification of the pulp was measured as the change in Kappa number according to the TAPPI method T236 cm-85. A typical procedure is as follows. A known mass of paper pulp (containing lignin) is reacted with an excess of potassium permanganate in an acid solution for a specified period of time to oxidize the lignin. After the reaction, the residual permanganate is determined by titration. The Kappa number is defined as the volume (ml) of 0.1N potassium permanganate consumed by 1 g of moisture-free pulp in 0.5N sulfuric acid after a ten-minute reaction time at 25° C. under conditions such that one-half of the permanganate remains unreacted. A linear relationship with the lignin content of the pulp and the measurement of the Kappa number has already been done on samples as low as 300 mg of pulp.
- Pulp Bleaching
- A softwood Kraft pulp, Kappa number 31.0, was treated with a laccase (NS51003) under the following conditions:
Enzyme dosage 45 units/g pulp pH 5.5 Temperature 50° C. Reaction time 16 hours Pulp consistency 2% - The dried pulp was added to 80 ml of 50 mM phosphate, pH 5.5, and disintegrated in a blender. The pulp was then transferred to a 500 ml conical flask and the blender was washed with 20 ml of the same buffer. The washed buffer and the pulp were combined. The mediator was added at 1-4% (w/w, based on the dry pulp), followed by the addition of the laccase. The pH of the pulp mixture was adjusted to 5.5 if needed. The flask was covered with an aluminum foil with holes punched through and incubated at 50° C. for 16 hrs on a rotary shaker at 200 rpm.
- After the enzymatic treatment, the pulp mixture was filtered through a Buchner funnel, and the pulp was washed with water. The pulp from the control experiment was treated at the same pH and temperature as described above.
- The washed pulp was then treated with an alkaline solution under the following conditions:
Pulp 2 g Water 200 ml NaOH 240 mg H2O2 (30%) 400 ul Temperature 70° C. Reaction time 3 hours - The filtered pulp was repulped in the alkaline solution and incubated at 70° C. for 3 hrs. The pH of the pulp mixture should be between 11.7-12.00 during the entire treatment. After the treatment, the pulp mixture was filtered through a Buchner funnel, and the pulp was washed with water extensively and then dried in a hood overnight.
- The delignification of the pulp was measured as a change in Kappa Number according to TAPPI method T236 cm-85.
- In the following example, black liquor from hardwood (oak) and softwood (pine) were tested with laccase to quantify the degree of enhancement that the black liquor impart to laccase. The Chicago Blue Dye, also known as Direct Blue 1 or DB1, was used for this assay. The Chicago Blue Dye is fully described by Schneider et al. in U.S. Pat. No. 5,885,304, which is hereby incorporated by reference.
- Each of the compounds (i.e., potential mediators) was dissolved in water, or in ethanol if the potential mediator was not water soluble, and then mixed with a phosphate buffer and a Chicago Blue solution. A solution of a laccase was added to make up 1 ml of the final solution, containing 20 uM mediator, 20 mM buffer at pH 5.5 or 7.0, 0.1-1% laccase (v/v) and Chicago Blue solution, with absorbance at A610 m between 0.6 to 0.8. The change in the absorbance at A610 nm was measured immediately using a UV-VIS spectrophotometer (UV-1201, Shimadzu Scientific Instruments) after the enzyme was added. The decrease in absorbance was recorded at 30-second intervals for 5 minutes and was used to estimate the efficiency of the mediator.
- An oak black liquor and a pine black liquor (from Hercules, Inc., Durango, Ga.) were used as mediators to enhance laccase-catalyzed bleaching of Chicago Blue in solution. Both stock solutions contain 14-15% solids. Additional samples were made via acid treatment. 10
- The acid-treated black liquors were prepared using the following procedure: (1) adjust pH of 20 ml of the black liquors to 1.0 using conc. HCl; (2) incubate the acidified solution at 80C for 1 hr; (3) cool down to room temperature; (4) adjust the pH to neutral (6.5-7.5) using 5 M NaOH solution.
- The following are the results from the bleaching of the Chicago Blue dye. It is clear that the black liquor samples are good mediators for laccase as far as discoloration reaction is concerned.
ΔmA610 ΔmA610 (3 min) (3 min) Mediator (0.1 umole/ml) Usage (ugram) at pH 5.5 pH 7.0 none 0 0 ABTS 10 445 341 Oak black liquor 100 (14%) 228 370 Pine black liquor 100 (14%) 26 18 Acid-treated oak black liquor 100 (13%) 108 225 - In this example, we used the typical pulp bleaching experiment as described above. We also used laccase as the enzyme, hardwood black liquor (HWBL) as the mediator, and two chain transfer agents (anthracene and CBr4).
Laccase Mediator Chain Transfer Agent Pulp Kappa No. 0 0 0 2 g 31 100 ul 0 0 2 g 28.0 100 ul HBT, 20 mg 0 2 g 26.1 100 ul HWBL, 200 ul 0 2 g 28.0 100 ul HWBL, 200 ul anthracene, 20 mg 2 g 25.4 100 ul HWBL, 200 ul CBr4, 20 mg 2 g 24.4 - The data above clearly indicate that the black liquor alone is not effective in enhancing pulp bleaching (although the black liquor is a mediator for the discoloration reaction of Chicago Blue dye). However, in the presence of a chain transfer agent, a noticeable decrease in Kappa numbers is obtained. Thus, the combination of black liquor and a chain transfer agent is indeed an effective mediator mixture for the enhancement of pulp bleaching.
- It is to be understood that the above described embodiments are illustrative only and that modification throughout may occur to one skilled in the art. Accordingly, this invention is not to be regarded as limited to the embodiments disclosed herein.
Claims (25)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/027,149 US20030047295A1 (en) | 2001-09-10 | 2001-12-20 | Bio-bleaching of pulp using laccase, mediator, and chain transfer agent |
PCT/US2002/013049 WO2003023133A1 (en) | 2001-09-10 | 2002-04-24 | Bio-bleaching of pulp using laccase, mediator, and chain transfer agent |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US31829201P | 2001-09-10 | 2001-09-10 | |
US10/027,149 US20030047295A1 (en) | 2001-09-10 | 2001-12-20 | Bio-bleaching of pulp using laccase, mediator, and chain transfer agent |
Publications (1)
Publication Number | Publication Date |
---|---|
US20030047295A1 true US20030047295A1 (en) | 2003-03-13 |
Family
ID=26702131
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/027,149 Abandoned US20030047295A1 (en) | 2001-09-10 | 2001-12-20 | Bio-bleaching of pulp using laccase, mediator, and chain transfer agent |
Country Status (2)
Country | Link |
---|---|
US (1) | US20030047295A1 (en) |
WO (1) | WO2003023133A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040112555A1 (en) * | 2002-12-03 | 2004-06-17 | Jeffrey Tolan | Bleaching stage using xylanase with hydrogen peroxide, peracids, or a combination thereof |
WO2009069143A2 (en) * | 2007-08-12 | 2009-06-04 | Advanced Enzyme Technologies Ltd. | Synergistic composition and a process for biobleaching of ligno cellulosic pulp |
US20110263836A1 (en) * | 2008-04-22 | 2011-10-27 | Kemira Oyj | Method for reduction of light-induced yellowing of lignin-containing material |
CN112301779A (en) * | 2020-10-28 | 2021-02-02 | 天津科技大学 | Method for improving softness of bamboo pulp fibers through laccase-assisted cellulase treatment |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5885304A (en) * | 1993-06-29 | 1999-03-23 | Novo Nordisk A/S | Enhancement of laccase reactions |
US6103059A (en) * | 1993-06-16 | 2000-08-15 | Lignozym Gmbh | Process for delignification of a lignin containing pulp |
US6162260A (en) * | 1999-05-24 | 2000-12-19 | Novo Nordisk Biochem North America, Inc. | Single-bath biopreparation and dyeing of textiles |
US6242245B1 (en) * | 1997-09-26 | 2001-06-05 | Consortium für elektrochemische Industrie GmbH | Multicomponent system for modifying, degrading or bleaching lignin or lignin-containing materials, and processes for its use |
-
2001
- 2001-12-20 US US10/027,149 patent/US20030047295A1/en not_active Abandoned
-
2002
- 2002-04-24 WO PCT/US2002/013049 patent/WO2003023133A1/en not_active Application Discontinuation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6103059A (en) * | 1993-06-16 | 2000-08-15 | Lignozym Gmbh | Process for delignification of a lignin containing pulp |
US6358904B1 (en) * | 1993-06-16 | 2002-03-19 | Hans-Peter Call | Multicomponent bleaching system |
US5885304A (en) * | 1993-06-29 | 1999-03-23 | Novo Nordisk A/S | Enhancement of laccase reactions |
US6242245B1 (en) * | 1997-09-26 | 2001-06-05 | Consortium für elektrochemische Industrie GmbH | Multicomponent system for modifying, degrading or bleaching lignin or lignin-containing materials, and processes for its use |
US6162260A (en) * | 1999-05-24 | 2000-12-19 | Novo Nordisk Biochem North America, Inc. | Single-bath biopreparation and dyeing of textiles |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040112555A1 (en) * | 2002-12-03 | 2004-06-17 | Jeffrey Tolan | Bleaching stage using xylanase with hydrogen peroxide, peracids, or a combination thereof |
WO2009069143A2 (en) * | 2007-08-12 | 2009-06-04 | Advanced Enzyme Technologies Ltd. | Synergistic composition and a process for biobleaching of ligno cellulosic pulp |
WO2009069143A3 (en) * | 2007-08-12 | 2009-11-12 | Advanced Enzyme Technologies Ltd. | Synergistic composition and a process for biobleaching of ligno cellulosic pulp |
US20110263836A1 (en) * | 2008-04-22 | 2011-10-27 | Kemira Oyj | Method for reduction of light-induced yellowing of lignin-containing material |
CN112301779A (en) * | 2020-10-28 | 2021-02-02 | 天津科技大学 | Method for improving softness of bamboo pulp fibers through laccase-assisted cellulase treatment |
Also Published As
Publication number | Publication date |
---|---|
WO2003023133A1 (en) | 2003-03-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Call et al. | History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym®-process) | |
Katagiri et al. | Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system | |
Kunamneni et al. | Fungal laccase—a versatile enzyme for biotechnological applications | |
Paice et al. | Manganese peroxidase, produced by Trametes versicolor during pulp bleaching, demethylates and delignifies kraft pulp | |
Khandeparkar et al. | Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp | |
Moreira et al. | Biobleaching of oxygen delignified kraft pulp by several white rot fungal strains | |
Bajpai et al. | Biobleaching of kraft pulp | |
Addleman et al. | Production and characterization of Trametes versicolor mutants unable to bleach hardwood kraft pulp | |
JPH02264087A (en) | Delignin with oxygen and bleaching of lignocellulose by means of enzyme treatment | |
Ninawe et al. | Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32 | |
CA2103260A1 (en) | A process for the delignification of material containing lignocellulose, bleaching and treatment of white water by means of laccases with extended efficiency | |
US20040112555A1 (en) | Bleaching stage using xylanase with hydrogen peroxide, peracids, or a combination thereof | |
Böckle et al. | Mechanism of peroxidase inactivation in liquid cultures of the ligninolytic fungus Pleurotus pulmonarius | |
Saparrat et al. | Ligninolytic enzyme ability and potential biotechnology applications of the white-rot fungus Grammothele subargentea LPSC no. 436 strain | |
Monteiro et al. | Pulp bleaching using laccase from Trametes versicolor under high temperature and alkaline conditions | |
Zhao et al. | Comparative studies of lignin peroxidases and manganese-dependent peroxidases produced by selected white rot fungi in solid media | |
EP2588665B1 (en) | Bleaching of pulp | |
JP3261661B2 (en) | Method of using enzyme in processing and bleaching of paper pulp and apparatus using the same | |
JP2785877B2 (en) | Use of Aureobasidium pullulans for pulp bleaching | |
JPH04245988A (en) | Chlorine-free method for bleaching pulp | |
US20030047295A1 (en) | Bio-bleaching of pulp using laccase, mediator, and chain transfer agent | |
US20030094251A1 (en) | Enhancing laccase activity using pro-oxidants and pro-degradants | |
Virk et al. | An alkalophilic laccase from Rheinheimera species isolate: production and biobleaching of kraft pulp | |
RU2224061C2 (en) | Multicomponent system for enzymatic oxidation of substrates and enzymatic oxidation method | |
US20030089472A1 (en) | Laccase activity enhancers for pulp bleaching |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: HERCULES INCORPORATED, DELAWARE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CHENG, HUAI N.;DELAGRAVE, SIMON;GU, QU-MING;AND OTHERS;REEL/FRAME:012801/0927;SIGNING DATES FROM 20020226 TO 20020314 |
|
AS | Assignment |
Owner name: CREDIT SUISSE FIRST BOSTON, AS COLLATERAL AGENT, N Free format text: NOTICE OF GRANT OF SECURITY INTEREST;ASSIGNOR:HERCULES INCORPORATED;REEL/FRAME:013599/0200 Effective date: 20021220 |
|
AS | Assignment |
Owner name: HERCULES INCORPORATED, DELAWARE Free format text: RELEASE OF SECURITY INTEREST;ASSIGNOR:CREDIT SUISSE FIRST BOSTON, AS COLLATERAL AGENT;REEL/FRAME:013696/0681 Effective date: 20030113 |
|
AS | Assignment |
Owner name: CODEXIS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HERCULES, INC.;REEL/FRAME:013780/0826 Effective date: 20030120 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: HERCULES CORPORATION, DELAWARE Free format text: PATENT TERMINATION CS-013599-0200;ASSIGNOR:CREDIT SUISSE, CAYMAN ISLANDS BRANCH;REEL/FRAME:021901/0247 Effective date: 20081113 |
|
AS | Assignment |
Owner name: CODEXIS, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CODEXIS MAYFLOWER HOLDINGS, LLC;REEL/FRAME:066528/0932 Effective date: 20240206 |