Abstract:
New mediators for use in laccase-mediator systems (LMS) are disclosed. The mediators 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. A composition comprising an enzyme exhibiting laccase activity and an enzyme enhancing agent (also referred to as a mediator or an activating agent) is also disclosed. 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 is also disclosed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims the benefit of U.S. Provisional Application No. 60/318,291, filed on Sep. 10, 2001. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    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.  
         BACKGROUND OF THE INVENTION  
         [0003]    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.  
           [0004]    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.  
           [0005]    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.  
           [0006]    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.  
           [0007]    Thus, there is a need to identify additional mediators that activate laccase, and/or enhance the activity of enzymes that exhibit laccase activity.  
         SUMMARY OF THE INVENTION  
         [0008]    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.  
           [0009]    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:  
           [0010]    (i) a monomer, dimer, or oligomer of at least one of:  
                         
 
           [0011]    wherein:  
           [0012]    R 1  is OCH 3 , H, or another moiety;  
           [0013]    R 2  is OCH 3 , H, or another moiety;  
           [0014]    R 3  is OH, sulfonate, or another moiety;  
           [0015]    R 4  is an OH or methylene that connects to another monomer; and  
           [0016]    R 5  is SO 3 H or H;  
           [0017]    (ii) N-benzylidene benzylamine (NBBA) or a substituted NBBA having the structure:  
                         
 
           [0018]    wherein:  
           [0019]    R is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group; and  
           [0020]    R 7 is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;  
           [0021]    (iii) a derivative of cinnamic acid having the structure:  
                         
 
           [0022]    wherein:  
           [0023]    R 8  is OCH 3 , NH 2 , Cl, NH(CH 3 ) 2 ; and  
           [0024]    R 9  is OH or H;  
           [0025]    (iv) salicylic acid;  
           [0026]    (v) epigallocatechin gallate;  
           [0027]    (vi) melamine;  
           [0028]    (vii) 3,4-dihydroxybenzyl alcohol (DHBA) or a substituted DHBA;  
           [0029]    (viii) a hardwood black liquor;  
           [0030]    (ix) a hardwood black liquor that comprises the monomer, dimer, or oligomer of group (i);  
           [0031]    (x) a softwood black liquor; and  
           [0032]    (xi) a softwood black liquor that comprises monomer, dimer, or oligomer under group (i).  
           [0033]    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.  
           [0034]    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.  
           [0035]    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.  
           [0036]    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. 
       
    
    
     BREIF DESCRIPTION OF THE DRAWING  
       [0037]    [0037]FIG. 1 illustrates the bleaching of the Chicago Blue Dye (DB1) in the presence and absence of the mediator ABTS. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0038]    The invention provides newly identified enzyme enhancing agents (also referred to as mediators) that enhance the activity of enzymes exhibiting laccase activity.  
         [0039]    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).  
         [0040]    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.  
         [0041]    The enzyme enhancing agents of the invention are selected from:  
         [0042]    (i) a monomer, dimer, or oligomer of at least one of:  
                         
 
         [0043]    wherein:  
         [0044]    R 1  is OCH 3 , H, or another moiety;  
         [0045]    R 2  is OCH 3 , H, or another moiety;  
         [0046]    R 3  is OH, sulfonate, or another moiety;  
         [0047]    R 4  is an OH or methylene that connects to another monomer; and  
         [0048]    R 5  is SO 3 H or H;  
         [0049]    (ii) N-benzylidene benzylamine (NBBA) or a substituted NBBA having the structure:  
                         
 
         [0050]    wherein:  
         [0051]    R 6  is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group; and  
         [0052]    R 7  is H, OCH 3 , NH(CH 3 ) 2 , Cl, Br, an aliphatic group, or an aromatic group;  
         [0053]    (iii) a derivative of cinnamic acid having the structure:  
                         
 
         [0054]    wherein:  
         [0055]    R 8  is OCH 3 , NH 2 , Cl, NH(CH 3 ) 2 ; and  
         [0056]    R 9  is OH or H;  
         [0057]    (vi) salicylic acid;  
         [0058]    (v) epigallocatechin gallate;  
         [0059]    (vi) melamine;  
         [0060]    (vii) 3,4-dihydroxybenzyl alcohol (DHBA) or a substituted DHBA;  
         [0061]    (viii) a hardwood black liquor;  
         [0062]    (ix) a hardwood black liquor that comprises the monomer, dimer, or oligomer of group (i);  
         [0063]    (x) a softwood black liquor; and  
         [0064]    (xi) a softwood black liquor that comprises monomer, dimer, or oligomer under group (i).  
         [0065]    Without limiting the scope of the invention, the enzyme enhancing agents of the invention can be classified into four categories:  
         [0066]    I. Lignin-like Materials  
         [0067]    a. Organic soluble fraction of lignin (lignin organosolv)  
         [0068]    b. Lignin sulfonate  
         [0069]    c. Hardwood black liquor and softwood black liquor  
         [0070]    Each of these three materials is a mixture comprising monomers, dimers, and oligomers with the following structures.  
                         
 
         [0071]    In these 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.  
         [0072]    II. N-benzylidene benzylamine (NBBA) and substituted NBBA.  
                         
 
         [0073]    In this structure, 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.  
         [0074]    III. Derivatives of Cinnamic Acid  
         [0075]    a. 4-(dimethylamino)cinnamic acid  
         [0076]    b. 3-hydroxy-4-methyoxycinnamic acid  
         [0077]    c. 4-aminocinnamic acid  
         [0078]    d. 4-chlorocinnamic acid  
                         
 
         [0079]    In this structure, R 8  is OCH 3 , NH2, Cl, NH(CH 3 ) 2 , and R 9  is OH or H.  
         [0080]    IV. Other Aromatic Compounds  
         [0081]    a. salicylic acid  
         [0082]    b. epigallocatechin gallate  
         [0083]    c. melamine  
         [0084]    d. 3,4-dihydroxybenzyl alcohol (DHBA), and substituted DHBA.  
         [0085]    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.  
         [0086]    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.  
         [0087]    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.  
         [0088]    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  
         [0089]    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.  
         [0090]    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.  
       EXAMPLES  
       [0091]    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.  
         [0092]    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.)  
         [0093]    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.  
         [0094]    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                  
 
       Example 1  
     Use of the Chicago Blue assay.  
       [0095]    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.  
                         
 
         [0096]    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.  
         [0097]    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.  
       Example 2  
     Screening for Potential Mediators using the Chicago Blue Dye  
       [0098]    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                  
 
         [0099]    [0099]                                           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                    
       Example 3  
     Combination of Laccases and Xylanases using N-benzylidenebenzylamine to Enhance the Bleaching of Chicago Blue at pH 7.0.  
       [0100]    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.  
         [0101]    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.  
         [0102]    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                  
 
         [0103]    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.