US20030096394A1 - Laccase activity enhancers - Google Patents
Laccase activity enhancers Download PDFInfo
- Publication number
- US20030096394A1 US20030096394A1 US10/027,166 US2716601A US2003096394A1 US 20030096394 A1 US20030096394 A1 US 20030096394A1 US 2716601 A US2716601 A US 2716601A US 2003096394 A1 US2003096394 A1 US 2003096394A1
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- enzyme
- och
- acid
- monomer
- dimer
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- Abandoned
Links
- 108010029541 Laccase Proteins 0.000 title claims abstract description 57
- 230000000694 effects Effects 0.000 title claims abstract description 44
- 239000003623 enhancer Substances 0.000 title 1
- 102000004190 Enzymes Human genes 0.000 claims abstract description 81
- 108090000790 Enzymes Proteins 0.000 claims abstract description 81
- 230000002708 enhancing effect Effects 0.000 claims abstract description 29
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 28
- 230000008569 process Effects 0.000 claims abstract description 28
- 230000001747 exhibiting effect Effects 0.000 claims abstract description 21
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- 239000000758 substrate Substances 0.000 claims abstract description 12
- 230000001590 oxidative effect Effects 0.000 claims abstract description 5
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 33
- 239000000178 monomer Substances 0.000 claims description 26
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- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 claims description 20
- PCYGLFXKCBFGPC-UHFFFAOYSA-N 4-(hydroxymethyl)benzene-1,2-diol Chemical compound OCC1=CC=C(O)C(O)=C1 PCYGLFXKCBFGPC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000539 dimer Substances 0.000 claims description 18
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- 125000003118 aryl group Chemical group 0.000 claims description 14
- 125000001931 aliphatic group Chemical group 0.000 claims description 13
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- 229920005610 lignin Polymers 0.000 claims description 11
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 10
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- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 7
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- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 claims description 4
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- 102000003425 Tyrosinase Human genes 0.000 claims description 4
- 108060008724 Tyrosinase Proteins 0.000 claims description 4
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- 229920002678 cellulose Polymers 0.000 claims 2
- 239000001913 cellulose Substances 0.000 claims 2
- 239000002994 raw material Substances 0.000 claims 1
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Images
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
- D21C3/00—Pulping cellulose-containing materials
- D21C3/22—Other features of pulping processes
- D21C3/222—Use of compounds accelerating the pulping processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
-
- 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
-
- 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 enhancement of enzyme activity and the activation of enzymes. More specifically, the present invention relates to mediators, enhancing agents, or activating agents that are useful in enhancing the activity of enzymes having laccase activity.
- Laccases 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. Laccase enzymes are used for many applications, including pulp bleaching, treatment of pulp waste water, de-inking, industrial color removal, bleaching laundry detergents, oral care teeth whiteners, and as catalysts or facilitators for polymerization and oxidation reactions. Commercial enzymes are often produced from fungal sources.
- oxidizing efficiency of a laccase can be improved through the use of a mediator, also known as an enhancing agent.
- a mediator also known as an enhancing agent.
- Systems that include a laccase and a mediator are known in the art as laccase-mediator systems (LMS).
- LMS laccase-mediator systems
- the same compounds can also be used to activate or initiate the action of laccase.
- mediators for use in a laccase-mediator system 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-acetyl-N-phenylhydroxylamine
- HBTO 3-hydroxy-1,2,3-benzotriazin-4(3H)-one
- VIO violuric acid
- the present invention provides mediators for use in laccase-mediator systems (LMS).
- LMS laccase-mediator systems
- the mediators of the invention enhance the activity of enzymes exhibiting laccase activity and can be used, for example, to bleach a dye in solution, to oxidize an appropriate compound, or to initiate vinyl or phenol polymerizations.
- the invention provides a composition comprising an enzyme exhibiting laccase activity and an enzyme enhancing agent (also referred to as a mediator or an activating agent).
- an enzyme enhancing agent also referred to as a mediator or an activating agent.
- the enzyme enhancing or activating agent is selected from:
- R 1 is OCH 3 , H, or another moiety
- R 2 is OCH 3 , H, or another moiety
- R 3 is OH, sulfonate, or another moiety
- R 4 is an OH or methylene that connects to another monomer
- R 5 is SO 3 H or H
- N-benzylidene benzylamine or a substituted NBBA having the structure:
- R is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;
- R 7 is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;
- R 8 is OCH 3 , NH 2 , Cl, NH(CH 3 ) 2 ;
- R 9 is OH or H
- the invention also 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 invention further 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 invention provides a process for enhancing the activity of an enzyme exhibiting laccase activity that comprises adding an enzyme enhancing agent to the enzyme.
- 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.
- FIG. 1 illustrates the bleaching of the Chicago Blue Dye (DB1) in the presence and absence of the mediator ABTS.
- the invention provides newly identified enzyme enhancing agents (also referred to as mediators) that enhance the activity of enzymes exhibiting laccase activity.
- the mediators of this invention are capable of enhancing the activities of laccase and laccase-related enzymes, i.e., enzymes exhibiting laccase activity.
- the enzymes exhibiting laccase enzyme activity include the laccase enzymes of enzyme classification EC 1.10.3.2, the catechol oxidase enzymes of enzyme classification EC 1.10.3.1, the monophenol monooxygenase enzymes of enzyme classification EC 1.14.99.1, the bilirubin oxidase enzymes of enzyme classification EC 1.3.3.5, and the ascorbate oxidase enzymes of enzyme classification EC 1.10.3.3.
- the EC (Enzyme Commission) number is based upon the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB).
- the enzymes exhibiting laccase activity may be derived from microbial, fungal, or other sources. These enzymes can also be produced by recombinant methods that are well known to those skilled in the art, such as cultivating a host cell transformed with a recombinant DNA vector that includes a DNA sequence encoding a laccase (and, optionally, DNA sequences that permit the expression of the laccase DNA sequence) in a culture medium under conditions that permit the expression of the laccase, and recovering the enzyme from the culture.
- the composition of the invention can further include a hydrolase, such as xylanase.
- the enzyme enhancing agents of the invention are selected from:
- R 1 is OCH 3 , H, or another moiety
- R 2 is OCH 3 , H, or another moiety
- R 3 is OH, sulfonate, or another moiety
- R 4 is an OH or methylene that connects to another monomer
- R 5 is SO 3 H or H
- N-benzylidene benzylamine or a substituted NBBA having the structure:
- R 6 is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;
- R 7 is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;
- R 8 is OCH 3 , NH 2 , Cl, NH(CH 3 ) 2 ;
- R 9 is OH or H
- the enzyme enhancing agents of the invention can be classified into four categories:
- Each of these three materials is a mixture comprising monomers, dimers, and oligomers with the following structures.
- R 1 and R 2 are OCH 3 , H, or other moieties;
- R 3 is OH, sulfonate, or other moieties;
- R 4 is either an OH group or a methylene that connects to another monomer;
- R 5 is SO 3 H or H.
- N-benzylidene benzylamine (NBBA) and substituted NBBA.
- R 6 and R 7 are H or OCH 3 , NH(CH 3 ) 2 , Cl, Br, aliphatic or aromatic groups, substituted at the 2 and 4 positions.
- R 8 is OCH 3 , NH2, Cl, NH(CH 3 ) 2 , and R 9 is OH or H.
- DHBA 3,4-dihydroxybenzyl alcohol
- substituted DHBA d. 3,4-dihydroxybenzyl alcohol (DHBA), and substituted DHBA.
- the mediators of the invention can be used, for example, for pulp delignification and bleaching. Laccase itself can bleach pulp only to a limited extent. The use of the mediators as disclosed herein enhances the activity of laccase in pulp bleaching through delignification.
- Other applications that may use the present invention include: polymerization of vinyl monomers or phenolic compounds; oxidation of materials containing hydroxy, amine, mercaptan, olefin, and aromatic moieties; microbial control in personal care products; bleaching of hair; treatment of waste water, particularly waste water in pulp mills; and bleaching in laundry detergents.
- 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 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 0.001 to 50 units (defined in the Examples using ABTS as substrate) in 1 ml of the reaction solution, preferably from about 0.01 to 20 units and even more preferably from 0.1 to 10 units and most preferably from 2 to ⁇ 3 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 aspect 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 A530nm 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.
- test 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 nm 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 enzyme enhancing agent N-benzylidenebenzylamine was used as a mediator for a laccase to enhance the bleaching of Chicago Blue at pH 7.0 in the presence of a laccase and a xylanase.
- the activity of the xylanase is 1000 units per ml enzyme solution.
- One xylanase unit is defined as the amount of enzyme which, under standard conditions (pH 9.0, 50° C., 30 minutes of incubation), release one micromole of the dye from a dyed RBB xylan.
- N-Benzylidenebenzylamine was dissolved in ethanol and then mixed with a phosphate buffer and a Chicago Blue solution.
- a solution of laccase (NS51003, Novozymes A/S) and xylanase (Pulpzyme HC, Novozymes A/S) was added to make 1 ml of the final solution, containing 20 uM of the mediator, 20 mM buffer at pH 7.0 and Chicago Blue solution, with absorbance at A610 nm between 0.6 to 0.8.
- the change in the absorbance was measured immediately using a UV spectrophotometer (UV-1201, Shimadzu Scientific Instruments). The decrease in absorbance was recorded after 3 minutes and was used to estimate the results of bleaching.
- mediators of the invention also include mediators which are functionally equivalent to the mediators specifically recited herein, such equivalents having minor structural variations such as the addition of a methyl or ethyl substituent or the formation of a methyl ester from a carboxylic acid. Accordingly, this invention is not to be regarded as limited to the embodiments disclosed herein.
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Abstract
Description
- This application claims the benefit of U.S. Provisional Application No. 60/318,291, filed on Sep. 10, 2001.
- The present invention relates to the enhancement of enzyme activity and the activation of enzymes. More specifically, the present invention relates to mediators, enhancing agents, or activating agents that are useful in enhancing the activity of enzymes having laccase activity.
- Laccases 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. Laccase enzymes are used for many applications, including pulp bleaching, treatment of pulp waste water, de-inking, industrial color removal, bleaching laundry detergents, oral care teeth whiteners, and as catalysts or facilitators for polymerization and oxidation reactions. Commercial enzymes are often produced from fungal sources.
- For many applications, the oxidizing efficiency of a laccase can be improved through the use of a mediator, also known as an enhancing agent. Systems that include a laccase and a mediator are known in the art as laccase-mediator systems (LMS). The same compounds can also be used to activate or initiate the action of laccase.
- There are several known mediators for use in a laccase-mediator system. 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.
- Functional groups and substituents have large effects on mediator efficiency. Even within the same class of compounds, a substituent can change the laccase specificity towards a substrate, thereby increasing or decreasing mediator efficiency greatly. In addition, a mediator may be effective for one particular application but unsuitable for another application. Thus, there is a need to discover efficient mediators for specific applications. One such application is the bleaching of pulp, wherein it is also important that the mediators are not unduly expensive or hazardous. Other applications of the laccase-mediator system are given below.
- Thus, there is a need to identify additional mediators that activate laccase, and/or enhance the activity of enzymes that exhibit laccase activity.
- The present invention provides mediators for use in laccase-mediator systems (LMS). The mediators of the invention enhance the activity of enzymes exhibiting laccase activity and can be used, for example, to bleach a dye in solution, to oxidize an appropriate compound, or to initiate vinyl or phenol polymerizations.
- The invention provides a composition comprising an enzyme exhibiting laccase activity and an enzyme enhancing agent (also referred to as a mediator or an activating agent). The enzyme enhancing or activating agent is selected from:
-
- wherein:
- R1 is OCH3, H, or another moiety;
- R2 is OCH3, H, or another moiety;
- R3 is OH, sulfonate, or another moiety;
- R4 is an OH or methylene that connects to another monomer; and
- R5 is SO3H or H;
-
- wherein:
- R is H, OCH3, NH(CH3)2, Cl, Br, an aliphatic group, or an aromatic group; and
- R7is H, OCH3, NH(CH3)2, Cl, Br, an aliphatic group, or an aromatic group;
-
- wherein:
- R8 is OCH3, NH2, Cl, NH(CH3)2; and
- R9 is OH or H;
- (iv) salicylic acid;
- (v) epigallocatechin gallate;
- (vi) melamine;
- (vii) 3,4-dihydroxybenzyl alcohol (DHBA) or a substituted DHBA;
- (viii) a hardwood black liquor;
- (ix) a hardwood black liquor that comprises the monomer, dimer, or oligomer of group (i);
- (x) a softwood black liquor; and
- (xi) a softwood black liquor that comprises monomer, dimer, or oligomer under group (i).
- The invention also 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 invention further 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 addition, the invention provides a process for enhancing the activity of an enzyme exhibiting laccase activity that comprises adding an enzyme enhancing agent to the enzyme.
- 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.
- FIG. 1 illustrates the bleaching of the Chicago Blue Dye (DB1) in the presence and absence of the mediator ABTS.
- The invention provides newly identified enzyme enhancing agents (also referred to as mediators) that enhance the activity of enzymes exhibiting laccase activity.
- The mediators of this invention are capable of enhancing the activities of laccase and laccase-related enzymes, i.e., enzymes exhibiting laccase activity. The enzymes exhibiting laccase enzyme activity include the laccase enzymes of enzyme classification EC 1.10.3.2, the catechol oxidase enzymes of enzyme classification EC 1.10.3.1, the monophenol monooxygenase enzymes of enzyme classification EC 1.14.99.1, the bilirubin oxidase enzymes of enzyme classification EC 1.3.3.5, and the ascorbate oxidase enzymes of enzyme classification EC 1.10.3.3. The EC (Enzyme Commission) number is based upon the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB).
- The enzymes exhibiting laccase activity may be derived from microbial, fungal, or other sources. These enzymes can also be produced by recombinant methods that are well known to those skilled in the art, such as cultivating a host cell transformed with a recombinant DNA vector that includes a DNA sequence encoding a laccase (and, optionally, DNA sequences that permit the expression of the laccase DNA sequence) in a culture medium under conditions that permit the expression of the laccase, and recovering the enzyme from the culture. The composition of the invention can further include a hydrolase, such as xylanase.
- The enzyme enhancing agents of the invention are selected from:
-
- wherein:
- R1 is OCH3, H, or another moiety;
- R2 is OCH3, H, or another moiety;
- R3 is OH, sulfonate, or another moiety;
- R4 is an OH or methylene that connects to another monomer; and
- R5 is SO3H or H;
-
- wherein:
- R6 is H, OCH3, NH(CH3)2, Cl, Br, an aliphatic group, or an aromatic group; and
- R7 is H, OCH3, NH(CH3)2, Cl, Br, an aliphatic group, or an aromatic group;
-
- wherein:
- R8 is OCH3, NH2, Cl, NH(CH3)2; and
- R9 is OH or H;
- (vi) salicylic acid;
- (v) epigallocatechin gallate;
- (vi) melamine;
- (vii) 3,4-dihydroxybenzyl alcohol (DHBA) or a substituted DHBA;
- (viii) a hardwood black liquor;
- (ix) a hardwood black liquor that comprises the monomer, dimer, or oligomer of group (i);
- (x) a softwood black liquor; and
- (xi) a softwood black liquor that comprises monomer, dimer, or oligomer under group (i).
- Without limiting the scope of the invention, the enzyme enhancing agents of the invention can be classified into four categories:
- I. Lignin-like Materials
- a. Organic soluble fraction of lignin (lignin organosolv)
- b. Lignin sulfonate
- c. Hardwood black liquor and softwood black liquor
-
- In these structures, R1 and R2 are OCH3, H, or other moieties; R3 is OH, sulfonate, or other moieties; R4 is either an OH group or a methylene that connects to another monomer; R5 is SO3H or H.
-
- In this structure, R6 and R7 are H or OCH3, NH(CH3)2, Cl, Br, aliphatic or aromatic groups, substituted at the 2 and 4 positions.
- III. Derivatives of Cinnamic Acid
- a. 4-(dimethylamino)cinnamic acid
- b. 3-hydroxy-4-methyoxycinnamic acid
- c. 4-aminocinnamic acid
-
- In this structure, R8 is OCH3, NH2, Cl, NH(CH3)2, and R9 is OH or H.
- IV. Other Aromatic Compounds
- a. salicylic acid
- b. epigallocatechin gallate
- c. melamine
- d. 3,4-dihydroxybenzyl alcohol (DHBA), and substituted DHBA.
- The mediators of the invention can be used, for example, for pulp delignification and bleaching. Laccase itself can bleach pulp only to a limited extent. The use of the mediators as disclosed herein enhances the activity of laccase in pulp bleaching through delignification. Other applications that may use the present invention include: polymerization of vinyl monomers or phenolic compounds; oxidation of materials containing hydroxy, amine, mercaptan, olefin, and aromatic moieties; microbial control in personal care products; bleaching of hair; treatment of waste water, particularly waste water in pulp mills; and bleaching in laundry detergents.
- 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 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 0.001 to 50 units (defined in the Examples using ABTS as substrate) in 1 ml of the reaction solution, preferably from about 0.01 to 20 units and even more preferably from 0.1 to 10 units and most preferably from 2 to −3 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 aspect 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 aspect 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.
- 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=36,000 M−1cm−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 A530nm 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 assay results for three samples of laccase enzyme are shown in the following table. Samples NS51002 and NS51003 were obtained from Novozymes A/S (Denmark). Unless otherwise stated, the results given in the examples were based on the enzyme NS51003.
Activity Protein Specific activity (umol/min. con. (umol/min. Name Source Density ml) (mg/ml) mg protein) NS51002 Aspergillus sp., 1.08 895 25 36 Novozymes NS51003 Aspergillus sp., 1.04 750 20 38 Novozymes - In the following example, several compounds were tested to identify enzyme enhancing agents that enhance the activity of an enzyme that exhibits laccase activity. The Chicago Blue Dye, also known as Direct Blue 1 or DB 1, was used to identify these compounds. 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, and has the following formula.
- Each of the test 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 nm 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.
- As a reference point, the decrease in absorbance for the Chicago Blue dye in the presence and the absence of the ABTS mediator is shown in FIG. 1.
- Experiments were conducted at pH levels of 5.5 and 7.0. The results of the Chicago Blue Assay at pH 5.5 and pH 7.0 are provided in the following tables below. The last column gives the decrease in absorbance at 610 nm. The larger the decrease in absorbance at 610 nm, the better is the ability to discolor the dye.
TABLE 1 Results of the Chicago Blue Assay at pH 5.5 Appearance ΔmA610 Potential Mediator (0.1 micromole/ml) in water (3 min) None clear 0 4-(Dimethylamino)cinnamic acid clear, yellow 519 3,5-Dimethoxy-4-hydroxycinnamic acid clear 491 4-Hydroxy-3-methoxycinnamic acid (ferulic acid) clear 441 Hardwood black liquor (150 ul, 12%) yellow 425 Lignin, organosolv. (20 ug/ml) clear, brown 390 Melamine cloudy 128 3-Hydroxy-4-methoxycinnamic acid clear 94 4-Aminocinnamic acid clear 43 4-Hydroxycinnamic acid clear 42 N-benzylidene benzylamine clear 28 Lignosulfonic acid. (25 ug/ml) Light brown 24 4-Chlorocinnamic acid cloudy 13 Salicylic acid clear 11 Softwood black liquor (150 ul, 12%) yellow 10 2-Chlorocinnamic acid cloudy 9 3-Chlorocinnamic acid cloudy 8 3-Hydroxycinnamic acid clear 7 4-Methoxycinnamic acid cloudy 6 Isatin clear 6 2-Hydroxycinnamic acid clear 5 3,4,5-Trimethoxycinnamic acid cloudy 5 3-Methoxycinnamic acid clear 4 Cinnamic acid clear 3 2-Aminopyridine light brown 3 Veratryl alcohol clear 3 Ethyl Salicylate cloudy 2 3,4-Dimethoxycinnamic acid clear 0 4- Nitrocinnamic acid cloudy 0 -
TABLE 2 Results of the Chicago Blue Assay at pH 7.0 Appearance ΔmA610 Potential Mediator (0.1 micromole/ml) in water (3 min) None clear 0 Hardwood black liquor (150 ul, 12%) yellow 358 Lignin, organosolv (20 ug/ml) clear, yellow 218 Ligninsulfonic acid (20 ug/ml) clear 18 2-Aminopyridine light brown 2 N-Benzylidenebenzylamine clear 2 - In the following example, the enzyme enhancing agent N-benzylidenebenzylamine was used as a mediator for a laccase to enhance the bleaching of Chicago Blue at pH 7.0 in the presence of a laccase and a xylanase.
- The activity of the xylanase (Pulpzyme HC, Novozymes A/S) is 1000 units per ml enzyme solution. One xylanase unit is defined as the amount of enzyme which, under standard conditions (pH 9.0, 50° C., 30 minutes of incubation), release one micromole of the dye from a dyed RBB xylan.
- N-Benzylidenebenzylamine was dissolved in ethanol and then mixed with a phosphate buffer and a Chicago Blue solution. A solution of laccase (NS51003, Novozymes A/S) and xylanase (Pulpzyme HC, Novozymes A/S) was added to make 1 ml of the final solution, containing 20 uM of the mediator, 20 mM buffer at pH 7.0 and Chicago Blue solution, with absorbance at A610 nm between 0.6 to 0.8. After the enzyme was added, the change in the absorbance was measured immediately using a UV spectrophotometer (UV-1201, Shimadzu Scientific Instruments). The decrease in absorbance was recorded after 3 minutes and was used to estimate the results of bleaching. The results are shown below.
Concentration Laccase Xylanase mA610 Mediator (μM) (%, v/v) (%, v/v) (3 min) None 0 0.5 0.5 71 N-Benzylidenebenzylamine 20 0.5 0 11 N-Benzylidenebenzylamine 20 0 0.5 112 N-Benzylidenebenzylamine 20 0.5 0.5 163 - 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. For example, a person of skill in the art will recognize that the mediators of the invention also include mediators which are functionally equivalent to the mediators specifically recited herein, such equivalents having minor structural variations such as the addition of a methyl or ethyl substituent or the formation of a methyl ester from a carboxylic acid. Accordingly, this invention is not to be regarded as limited to the embodiments disclosed herein.
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WO2007095398A2 (en) | 2006-02-14 | 2007-08-23 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
WO2009045627A2 (en) | 2007-10-03 | 2009-04-09 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
WO2009088949A1 (en) | 2008-01-03 | 2009-07-16 | Verenium Corporation | Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them |
CN101957315A (en) * | 2010-09-11 | 2011-01-26 | 大连工业大学 | Method for measuring lignin content in black liquor |
US9663899B2 (en) * | 2015-08-26 | 2017-05-30 | Solenis Technologies, L.P. | Method for making lignocellulosic paper and paper product |
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DK77393D0 (en) * | 1993-06-29 | 1993-06-29 | Novo Nordisk As | ENZYMER ACTIVATION |
EP0781328B1 (en) * | 1994-09-27 | 2002-12-04 | Novozymes A/S | Enhancers such as acetosyringone |
EP0905306A1 (en) * | 1997-09-26 | 1999-03-31 | Consortium für elektrochemische Industrie GmbH | Multicomponent system for modifying, decomposing or bleaching lignin or lignin-containing materials and method of using this system |
US6200786B1 (en) * | 1999-09-08 | 2001-03-13 | Givaudan S.A. | Process for the preparation of nootkatone by laccase catalysis |
-
2001
- 2001-12-20 US US10/027,166 patent/US20030096394A1/en not_active Abandoned
-
2002
- 2002-06-07 WO PCT/US2002/018540 patent/WO2003023043A1/en not_active Application Discontinuation
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EP2548955A1 (en) | 2006-02-14 | 2013-01-23 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
EP2548956A1 (en) | 2006-02-14 | 2013-01-23 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
EP2548954A1 (en) | 2006-02-14 | 2013-01-23 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
WO2009045627A2 (en) | 2007-10-03 | 2009-04-09 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
EP2708602A2 (en) | 2007-10-03 | 2014-03-19 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
WO2009088949A1 (en) | 2008-01-03 | 2009-07-16 | Verenium Corporation | Transferases and oxidoreductases, nucleic acids encoding them and methods for making and using them |
CN101957315A (en) * | 2010-09-11 | 2011-01-26 | 大连工业大学 | Method for measuring lignin content in black liquor |
US9663899B2 (en) * | 2015-08-26 | 2017-05-30 | Solenis Technologies, L.P. | Method for making lignocellulosic paper and paper product |
US20170218570A1 (en) * | 2015-08-26 | 2017-08-03 | Solenis Technologies, L.P. | Method for Making Lignocellulosic Paper and Paper Products |
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