Patent Publication Number: US-9896667-B2

Title: Laccase variants with improved properties

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2015/057917, filed Apr. 11, 2015, designating the United States of America and published in English as International Patent Publication WO 2015/155363 A1 on Oct. 15, 2015, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 14164498.9, filed Apr. 11, 2014. 
     STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS ASCII TEXT FILE 
     Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII text version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. 
     TECHNICAL FIELD 
     This application relates to laccase variants and uses thereof as eco-friendly biocatalysts in various industrial processes. 
     BACKGROUND 
     Laccases (EC 1.10.3.2) are enzymes having a wide taxonomic distribution and belonging to the group of multicopper oxidases. Laccases are eco-friendly catalysts that use molecular oxygen from air to oxidize various phenolic and non-phenolic lignin-related compounds as well as highly recalcitrant environmental pollutants, and produce water as the only side-product. These natural “green” catalysts are used for diverse industrial applications including the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, and used as bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. Another large proposed application area of laccases is biomass pretreatment in biofuel and in the pulp and paper industries. 
     Laccase molecules are usually monomers consisting of three consecutively connected cupredoxin-like domains twisted in a tight globule. The active site of laccases contains four copper ions: a mononuclear “blue” copper ion (T1 site) and a three-nuclear copper cluster (T2/T3 site) consisting of one T2 copper ion and two T3 copper ions. 
     Laccases may be isolated from different sources such as plants, fungi or bacteria and are very diverse in primary sequences. However, they have some conserved regions in the sequences and certain common features in their three-dimensional structures. A comparison of sequences of more than 100 laccases has revealed four short conservative regions (no longer than 10 aa each), which are specific for all laccases. [7, 8]  One cysteine and ten histidine residues form a ligand environment of copper ions of the laccase active site present in these four conservative amino acid sequences. 
     The best studied bacterial laccase is CotA laccase. CotA is a component of the outer coat layers of  bacillus  endospore. It is a 65-kDa protein encoded by the CotA gene. [1]   
     CotA belongs to a diverse group of multi-copper “blue” oxidases that includes the laccases. This protein demonstrates high thermostability, and resistance to various hazardous elements in accordance with the survival abilities of the endospore. 
     Recombinant protein expression in easily cultivatable hosts can allow higher productivity in a shorter time and reduces the costs of production. The versatility and scaling-up possibilities of the recombinant protein production opened up new commercial opportunities for their industrial uses. Moreover, protein production from pathogenic or toxin-producing species can take advantage of safer or even GRAS (generally recognized as safe) microbial hosts. In addition, protein engineering can be employed to improve the stability, activity and/or specificity of an enzyme; thus, tailor made enzymes can be produced to suit the requirement of the users or of the process. 
     Enzyme productivity can be increased by the use of multiple gene copies, strong promoters and efficient signal sequences, properly designed to address proteins to the extracellular medium, thus simplifying downstream processing. 
     Recombinant protein yield in bacterial hosts is often limited by the inability of the protein to fold into correct 3D-structure upon biosynthesis of the polypeptide chain. This may cause exposure of hydrophobic patches on the surface of the protein globule and result in protein aggregation. Mechanisms of heterologous protein folding in vivo are poorly understood, and foldability of different proteins in bacteria is unpredictable. 
     Yield of soluble active protein can sometimes be improved by changing cultivation conditions. In addition, there are examples when protein yield was improved by introducing single-point mutations in the protein sequence. However, no rationale has been identified behind finding suitable mutations. 
     Heterologous expression of laccase in  Escherichia coli  has often been used as a strategy to get around the problem of obtaining laccases that are not easily producible in natural hosts. The recombinant expression of  Bacillus subtilis  CotA in  E. coli  has allowed its deep characterization, structure solving, and functional evolution. [1, 2, 3]  However, very often, the production yield is low, due to a strong tendency of this enzyme to form aggregates that renders the protein irreversibly inactive. [4]  This tendency has been attributed to the fact that, in nature, CotA laccase is integrated in a spore coat structure via interaction with other protein components, and it is likely that correct laccase folding is enhanced by interaction with other proteins. When this laccase is recombinantly expressed as an individual polypeptide, those supporting interactions are missing and many miss-folded proteins form aggregates in bacterial cells. When expressed in higher microorganisms such as yeast, misfolded laccase molecules are, for a large part, degraded. 
     There is a need in the art for means and methods for improving the yield of laccases in heterologous expression systems. This is particularly true for bacterial laccases, such as CotA laccases. 
     BRIEF SUMMARY 
     This disclosure addresses this need in that it provides variant laccases with improved properties. More in particular, the disclosure relates to a polypeptide with laccase activity comprising an amino acid sequence that is at least 75% identical to the amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises a small amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine at an amino acid position corresponding to position 149 in SEQ ID NO: 1. 
     A threonine residue at a position corresponding to amino acid 149 of SEQ ID NO: 1 is preferred. 
     In addition, the disclosure provides improved nucleic acids, vectors and compositions encoding the variant laccase enzymes according to the disclosure. 
     The disclosure also provides recombinant heterologous expression systems such as host cells comprising a nucleic acid, a vector or a composition according to the disclosure. 
     Also provided herein are methods for producing a polypeptide according to the disclosure, comprising the steps of:
         a. culturing a recombinant host cell comprising a polynucleotide according to the disclosure under conditions suitable for the production of the polypeptide, and   b. recovering the polypeptide obtained, and   c. optionally purifying the polypeptide.       

     The disclosure also relates to the use of a polypeptide according to the disclosure in an application selected from the group consisting of pulp delignification, degrading or decreasing the structural integrity of lignocellulosic material, textile dye bleaching, wastewater detoxification, xenobiotic detoxification, production of a sugar from a lignocellulosic material and recovering cellulose from a biomass. 
     The disclosure also relates to a method for improving the yield of a polypeptide with laccase activity in a heterologous expression system comprising the step of altering the amino acid of that polypeptide at a position corresponding to position 149 in SEQ ID NO: 1 to a small amino acid residue. 
     Preferred embodiments of these aspects will be described in more detail below. For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the disclosure may include embodiments having combinations of all or some of the features described. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Relative increase of volumetric activity. Graph showing the relative increase of volumetric activity in parallel cultures in  E. coli  of wild-type (non-mutated) versus variant laccases according to SEQ ID NO: 1 and SEQ ID NO: 2. The abbreviation “SEQ” followed by a number refers to the SEQ ID NO: of the respective number; SEQ1 refers to SEQ ID NO: 1. SEQ1 149T refers to the polypeptide according to SEQ ID NO: 1, wherein the amino acid corresponding to position 149 is replaced by a T (Thr or threonine). 
         FIG. 2 : Relative increase of volumetric activity. Graph showing the relative increase of volumetric activity in parallel cultures in  E. coli  of wild-type (non-mutated) versus variant laccases according to SEQ ID NO: 5. The abbreviation “SEQ” followed by a number refers to the SEQ ID NO: of the respective number; SEQ5 refers to SEQ ID NO: 5. SEQ5 149T refers to the polypeptide according to SEQ ID NO: 5, wherein the amino acid corresponding to position 149 is replaced by a T (Thr or threonine). 
         FIG. 3 : Relative increase of volumetric activity. Graph showing the relative increase of volumetric activity in parallel cultures in  E. coli  of wild-type (non-mutated) versus variant laccases according to SEQ ID NO: 15. The abbreviation “SEQ” followed by a number refers to the SEQ ID NO: of the respective number; SEQ15 refers to SEQ ID NO: 15. SEQ 15 149T refers to the polypeptide according to SEQ ID NO: 15, wherein the amino acid corresponding to position 149 is replaced by a T (Thr or threonine). 
         FIG. 4 : Relative increase of volumetric activity. Graph showing the relative increase of volumetric activity in parallel cultures in  Pichia  pastoris of wild-type (non-mutated) versus variant laccases according to SEQ ID NO: 1 and SEQ ID NO: 2. The abbreviation “SEQ” followed by a number refers to the SEQ ID NO: of the respective number; SEQ1 refers to SEQ ID NO: 1. SEQ1 149T refers to the polypeptide according to SEQ ID NO: 1, wherein the amino acid corresponding to position 149 is replaced by a threonine residue (Thr or T). 
     
    
    
     DETAILED DESCRIPTION 
     This disclosure is based on the observation that a single amino acid substitution in different laccases improves the yield of that laccase by at least 50% when expressed in prokaryotes as well as in eukaryotes. It was also found that the variant laccase remains active. 
     The term “amino acid substitution” is used herein the same way as it is commonly used, i.e., the term refers to a replacement of one or more amino acids in a protein with another amino acid. Artificial amino acid substitutions may also be referred to as mutations. 
     The term “small amino acid” as used herein is intended to cover a group of amino acids that are usually considered as small or tiny amino acids. In a preferred embodiment, this group is limited to amino acids threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine. 
     SEQ ID NO: 1 is a CotA laccase from  Bacillus subtilis  newly disclosed herein, whereas SEQ ID NO: 2 is a CotA laccase that has been previously disclosed in WO 2013/038062. It was found that laccase variants that have a small amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine at an amino acid position corresponding to position 149 in SEQ ID NO: 1 provided a higher yield when expressed in a heterologous expression system. 
     Laccase variants with an amino acid residue selected from the group consisting of threonine, proline, asparagine and aspartic acid at an amino acid position corresponding to position 149 in SEQ ID NO: 1 are preferred in the context of this disclosure. 
     Variants carrying a threonine residue at an amino acid position corresponding to position 149 in SEQ ID NO: 1 are most preferred since they resulted in the highest yield of recombinantly expressed laccase. 
     SEQ ID NO: 3 and SEQ ID NO: 4 disclose  B. subtilis  spore coat proteins with laccase activity (CotA laccase), that carry a 149T mutation. In fact, SEQ ID NO: 3 is a variant from SEQ ID NO: 1 wherein an isoleucine residue at position 149 has been replaced by a threonine residue. SEQ ID NO: 4 is a variant from SEQ ID NO: 2 wherein an isoleucine residue at position 149 has been replaced by a threonine residue. 
     A homology search for proteins homologous to SEQ ID NO: 1 was performed using SEQ ID NO: 1 as the query sequence in the “Standard protein BLAST” software, available on the World Wide Web at blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&amp;PAGE_TYPE=BlastSearch&amp;LINK_LOC=blasthome. More information on the software and database versions is available at the National Center for Biotechnology Information at National library of Medicine at the National institute of Health internet site ncbi.nlm.nih.gov. Therein, a number of molecular biology tools including BLAST (Basic Logical Alignment Search Tool) is to be found. BLAST makes use of the following databases: all non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples from WGS projects. The search as reported herein was performed online on 19 Feb. 2014 and employed BLASTP version 2.2.29+. 
     The search revealed 44 sequences with at least 75% sequence identity to SEQ ID NO: 1 (Table 1). These sequences are herein provided as SEQ ID NOS: 51 to 94. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequences obtained from a BLAST search disclosing 44 sequences with at least 75% identity to SEQ ID NO: 1 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
                   
                   
                 AA at pos 
               
               
                 ID 
                 BLAST 
                   
                   
                 Overall 
                 AA # corr. 
                 corr. to 
               
               
                 NO: 
                 No: 
                 Description 
                 Accession No: 
                 identity (1)   
                 to pos 149 (2)   
                 AA (3)   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 1 
                 CotA laccase from  B.   subtilis  (query sequence) 
                   
                 100%  
                 149 
                 Ile 
               
               
                 51 
                 2 
                 laccase [ Bacillus   subtilis ] 
                 AGZ16504.1 
                 98% 
                 149 
                 Ile 
               
               
                 52 
                 3 
                 spore copper-dependent laccase (outer coat) 
                 YP_003865004.1 
                 98% 
                 149 
                 Ile 
               
               
                   
                   
                 [ Bacillus   subtilis  subsp.  spizizenii  str. W23] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|WP_003219376.1|copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   subtilis ] &gt;gb|EFG93543.1|spore copper-dependent 
                   
                   
                   
                   
               
               
                   
                   
                 laccase [ Bacillus   subtilis  subsp.  spizizenii  ATCC 
                   
                   
                   
                   
               
               
                   
                   
                 6633] &gt;gb|ADM36695.1|spore copper-dependent 
                   
                   
                   
                   
               
               
                   
                   
                 laccase (outer coat) [ Bacillus   subtilis  subsp. 
                   
                   
                   
                   
               
               
                   
                   
                   spizizenii  str. W23] 
                   
                   
                   
                   
               
               
                 53 
                 4 
                 spore copper-dependent laccase [ Bacillus   subtilis ] 
                 WP_004397739.1 
                 96% 
                 149 
                 Ile 
               
               
                   
                   
                 &gt;gb|ELS60660.1|spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  subsp.  inaquosorum  KCTC 13429] 
                   
                   
                   
                   
               
               
                 54 
                 5 
                 copper oxidase [ Bacillus   subtilis ] 
                 WP_019713492.1 
                 96% 
                 149 
                 Ile 
               
               
                 55 
                 6 
                 laccase [ Bacillus   vallismortis ] 
                 AGR50961.1 
                 95% 
                 149 
                 Ile 
               
               
                 56 
                 7 
                 spore coat protein A [ Bacillus   subtilis  XF-1] 
                 YP_007425830.1 
                 96% 
                 151 
                 Ile 
               
               
                   
                   
                 &gt;ref|WP_015382982.1|spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus ] &gt;gb|AGE62493.1|spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  XF-1] &gt;gb|ERI42893.1|copper 
                   
                   
                   
                   
               
               
                   
                   
                 oxidase [ Bacillus  sp. EGD-AK10] 
                   
                   
                   
                   
               
               
                 57 
                 8 
                 spore copper-dependent laccase [ Bacillus   subtilis   
                 YP_004206641.1 
                 96% 
                 149 
                 Ile 
               
               
                   
                   
                 BSn5] &gt;ref|YP_005559844.1|spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  subsp.  natto  BEST195] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|YP_007210655.1|Spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  subsp.  subtilis  str. BSP1] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|WP_014479048.1|copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   subtilis ] &gt;dbj|BAI84141.1|spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  subsp.  natto  BEST195] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|ADV95614.1|spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  BSn5] &gt;gb|ADZ57279.1|laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus  sp. LS02] &gt;gb|ADZ57280.1|laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus  sp. LS03] &gt;gb|ADZ57283.1|laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus  sp. WN01] &gt;gb|ADZ57284.1|laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis ] &gt;gb|AGA20638.1|Spore coat 
                   
                   
                   
                   
               
               
                   
                   
                 protein A [ Bacillus   subtilis  subsp.  subtilis  str. 
                   
                   
                   
                   
               
               
                   
                   
                 BSP1] 
                   
                   
                   
                   
               
               
                 58 
                 9 
                 CotA [ Bacillus  sp. JS] &gt;ref|WP_014663045.1| 
                 YP_006230497.1 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 copper oxidase [ Bacillus  sp. JS] &gt;gb|AFI27241.1| 
                   
                   
                   
                   
               
               
                   
                   
                 CotA [ Bacillus  sp. JS] 
                   
                   
                   
                   
               
               
                 59 
                 10 
                 copper oxidase [ Bacillus   subtilis  QH-1] 
                 EXF51833.1 
                 95% 
                 149 
                 Ile 
               
               
                 60 
                 11 
                 copper oxidase [ Bacillus   subtilis ] &gt;gb|EHA29133.1| 
                 WP_003234000.1 
                 95% 
                 151 
                 Ile 
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   subtilis   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  subtilis  str. SC-8] 
                   
                   
                   
                   
               
               
                 61 
                 12 
                 outer spore coat copper-dependent laccase [ Bacillus   
                 YP_006628799.1 
                 95% 
                 151 
                 Ile 
               
               
                   
                   
                   subtilis  QB928] &gt;ref|WP_014906195.1|copper 
                   
                   
                   
                   
               
               
                   
                   
                 oxidase [ Bacillus   subtilis ] &gt;dbj|BAA22774.1|spore 
                   
                   
                   
                   
               
               
                   
                   
                 coat protein A [ Bacillus   subtilis ] &gt;gb|AFQ56549.1| 
                   
                   
                   
                   
               
               
                   
                   
                 Outer spore coat copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   subtilis  QB928] 
                   
                   
                   
                   
               
               
                 62 
                 13 
                 spore coat protein A [ Bacillus   subtilis  subsp.  subtilis   
                 NP_388511.1 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 str. 168] 
                   
                   
                   
                   
               
               
                 63 
                 14 
                 spore coat protein A [ Bacillus   subtilis  subsp.  subtilis   
                 YP_007661398.1 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 str. BAB-1] &gt;ref|WP_015482891.1|spore coat 
                   
                   
                   
                   
               
               
                   
                   
                 protein A [ Bacillus   subtilis ] &gt;gb|AGI27890.1|spore 
                   
                   
                   
                   
               
               
                   
                   
                 coat protein A [ Bacillus   subtilis  subsp.  subtilis  str. 
                   
                   
                   
                   
               
               
                   
                   
                 BAB-1] 
                   
                   
                   
                   
               
               
                 64 
                 15 
                 Chain A, Mutations In The Neighbourhood Of 
                 4AKQ_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: E498d Mutant 
                   
                   
                   
                   
               
               
                 65 
                 16 
                 Chain A, Mutations In The Neighbourhood Of 
                 4A68_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: D116n Mutant 
                   
                   
                   
                   
               
               
                 66 
                 17 
                 Chain A, Mutations In The Neighbourhood Of 
                 4A66_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: D116a Mutant 
                   
                   
                   
                   
               
               
                 67 
                 18 
                 spore coat protein [ Bacillus   subtilis ] 
                 ACS44284.1 
                 95% 
                 149 
                 Ile 
               
               
                 68 
                 19 
                 spore coat protein [ Bacillus   subtilis ] 
                 AGK12417.1 
                 95% 
                 149 
                 Ile 
               
               
                 69 
                 20 
                 Chain A, Crystal Structure Of The Reconstituted 
                 2X87_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota 
                   
                   
                   
                   
               
               
                 70 
                 21 
                 laccase [ Bacillus  sp. ZW2531-1] 
                 AFN66123.1 
                 95% 
                 149 
                 Ile 
               
               
                 71 
                 22 
                 Chain A, Mutations In The Neighbourhood Of 
                 4A67_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: D116e Mutant 
                   
                   
                   
                   
               
               
                 72 
                 23 
                 Chain A, Proximal Mutations At The Type 1 Cu 
                 2WSD_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Site Of Cota-Laccase: I494a Mutant 
                   
                   
                   
                   
               
               
                 73 
                 24 
                 Chain A, Mutations In The Neighbourhood Of 
                 4AKP_A 
                 95% 
                 149 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: e498t Mutant 
                   
                   
                   
                   
               
               
                 74 
                 25 
                 laccase [ Bacillus  sp. HR03] 
                 ACM46021.1 
                 94% 
                 149 
                 Ile 
               
               
                 75 = 
                 26 
                 copper oxidase [ Bacillus   vallismortis ] 
                 WP_010329056.1 
                 94% 
                 149 
                 Ile 
               
               
                 15 
                   
                   
                   
                   
                   
                   
               
               
                 76 
                 27 
                 laccase [ Bacillus   subtilis ] 
                 AEK80414.1 
                 92% 
                 149 
                 Ile 
               
               
                 77 
                 28 
                 copper oxidase [ Bacillus   mojavensis ] 
                 WP_010333230.1 
                 91% 
                 149 
                 Ile 
               
               
                 78 
                 29 
                 Chain A, Mutations In The Neighbourhood Of 
                 4AKO_A 
                 94% 
                 145 
                 Ile 
               
               
                   
                   
                 Cota-Laccase Trinuclear Site: E498l Mutant 
                   
                   
                   
                   
               
               
                 79 
                 30 
                 CotA [ Bacillus   subtilis ] 
                 AAB62305.1 
                 89% 
                 149 
                 Ile 
               
               
                 80 
                 31 
                 spore copper-dependent laccase [ Bacillus   
                 YP_003972023.1 
                 81% 
                 149 
                 Ile 
               
               
                   
                   
                   atrophaeus  1942] &gt;ref|WP_003328493.1|copper 
                   
                   
                   
                   
               
               
                   
                   
                 oxidase [ Bacillus   atrophaeus ] &gt;gb|ADP31092.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase (outer coat) 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   atrophaeus  1942] &gt;gb|EIM09308.1|spore 
                   
                   
                   
                   
               
               
                   
                   
                 copper-dependent laccase [ Bacillus   atrophaeus  C89] 
                   
                   
                   
                   
               
               
                 81 
                 32 
                 Spore coat protein A [ Bacillus   atrophaeus ] 
                 WP_010787813.1 
                 81% 
                 149 
                 Ile 
               
               
                   
                   
                 &gt;gb|EOB38473.1|Spore coat protein A [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   atrophaeus  UCMB-5137] 
                   
                   
                   
                   
               
               
                 82 
                 33 
                 copper oxidase [ Bacillus  sp. 5B6] &gt;gb|EIF12180.1| 
                 WP_007609818.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                 CotA [ Bacillus  sp. 5B6] 
                   
                   
                   
                   
               
               
                 83 
                 34 
                 outer spore coat copper-dependent laccase [ Bacillus   
                 YP_007496315.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  UCMB5036] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|YP_008411651.1|outer spore coat copper- 
                   
                   
                   
                   
               
               
                   
                   
                 dependent laccase [ Bacillus   amyloliquefaciens   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  plantarum  UCMB5033] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|YP_008420054.1|outer spore coat copper- 
                   
                   
                   
                   
               
               
                   
                   
                 dependent laccase [ Bacillus   amyloliquefaciens   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  plantarum  UCMB5113] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|WP_015416957.1|outer spore coat copper- 
                   
                   
                   
                   
               
               
                   
                   
                 dependent laccase [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;emb|CCP20645.1|outer spore coat copper- 
                   
                   
                   
                   
               
               
                   
                   
                 dependent laccase [ Bacillus   amyloliquefaciens   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  plantarum  UCMB5036] &gt;emb|CDG28620.1| 
                   
                   
                   
                   
               
               
                   
                   
                 outer spore coat copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  UCMB5033] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;emb|CDG24919.1|outer spore coat copper- 
                   
                   
                   
                   
               
               
                   
                   
                 dependent laccase [ Bacillus   amyloliquefaciens   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  plantarum  UCMB5113] 
                   
                   
                   
                   
               
               
                 84 
                 35 
                 spore coat protein CotA [ Bacillus   amyloliquefaciens   
                 YP_005419918.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                 subsp.  plantarum  YAU B9601-Y2] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|YP_006327430.1|spore coat protein A 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   amyloliquefaciens  Y2] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|WP_014417082.1|copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens ] &gt;gb|ADZ57285.1 |laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus  sp. LC02] &gt;emb|CCG48602.1|spore coat 
                   
                   
                   
                   
               
               
                   
                   
                 protein CotA [ Bacillus   amyloliquefaciens  subsp. 
                   
                   
                   
                   
               
               
                   
                   
                   plantarum  YAU B9601-Y2] &gt;gb|AFJ60705.1|spore 
                   
                   
                   
                   
               
               
                   
                   
                 coat protein A [ Bacillus   amyloliquefaciens  Y2] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;dbj|BAM49543.1|spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   subtilis  BEST7613] &gt;dbj|BAM56813.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   subtilis   
                   
                   
                   
                   
               
               
                   
                   
                 BEST7003] 
                   
                   
                   
                   
               
               
                 85 
                 36 
                 bilirubin oxidase [ Bacillus   amyloliquefaciens  subsp. 
                 YP_008625231.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                   plantarum  NAU-B3] &gt;ref|WP_022552695.1| 
                   
                   
                   
                   
               
               
                   
                   
                 bilirubin oxidase [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;emb|CDH94370.1| bilirubin oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  NAU-B3] 
                   
                   
                   
                   
               
               
                 86 
                 37 
                 spore coat protein A [ Bacillus   amyloliquefaciens   
                 YP_007185316.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                 subsp.  plantarum  AS43.3] &gt;ref|WP_015239305.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore coat protein A [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|AFZ89646.1| spore coat protein A [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  AS43.3] 
                   
                   
                   
                   
               
               
                 87 = 5 
                 38 
                 CotA [ Bacillus   amyloliquefaciens  subsp.  plantarum   
                 YP_001420286.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                 str. FZB42] &gt;ref|YP_008725930.1| cotA [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  CC178] &gt;ref|WP_012116986.1| 
                   
                   
                   
                   
               
               
                   
                   
                 copper oxidase [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|ABS73055.1| CotA [ Bacillus   amyloliquefaciens   
                   
                   
                   
                   
               
               
                   
                   
                 subsp.  plantarum  str. FZB42] &gt;gb|AGZ55352.1| 
                   
                   
                   
                   
               
               
                   
                   
                 CotA [ Bacillus   amyloliquefaciens  CC178] 
                   
                   
                   
                   
               
               
                 88 
                 39 
                 laccase [ Bacillus  sp. LC03] 
                 ADZ57286.1 
                 76% 
                 149 
                 Leu 
               
               
                 89 
                 40 
                 copper oxidase [ Bacillus  sp. 916] &gt;gb|EJD67619.1| 
                 WP_007408880.1 
                 77% 
                 149 
                 Leu 
               
               
                   
                   
                 CotA [ Bacillus  sp. 916] 
                   
                   
                   
                   
               
               
                 90 
                 41 
                 copper oxidase [ Bacillus   amyloliquefaciens ] 
                 WP_021495201.1 
                 76% 
                 149 
                 Leu 
               
               
                   
                   
                 &gt;gb|ERH51073.1| copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  EGD-AQ14] 
                   
                   
                   
                   
               
               
                 91 
                 42 
                 bilirubin oxidase [ Bacillus   amyloliquefaciens  subsp. 
                 YP_005129370.1 
                 76% 
                 149 
                 Leu 
               
               
                   
                   
                   plantarum  CAU B946] &gt;ref|YP_007446652.1| 
                   
                   
                   
                   
               
               
                   
                   
                 bilirubin oxidase [ Bacillus   amyloliquefaciens  IT-45] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;ref|YP_008949033.1| copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  LFB112] &gt;ref|WP_003155789.1| 
                   
                   
                   
                   
               
               
                   
                   
                 copper oxidase [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|ADZ57278.1| laccase [ Bacillus  sp. LS01] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|ADZ57282.1| laccase [ Bacillus  sp. LS05] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;emb|CCF04175.1| bilirubin oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  CAU B946] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|EKE46469.1| bilirubin oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  subsp.  plantarum  M27] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|AGF28771.1| bilirubin oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  IT-45] &gt;gb|ERK81509.1| copper 
                   
                   
                   
                   
               
               
                   
                   
                 oxidase [ Bacillus   amyloliquefaciens  UASWS BA1] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|AHC41184.1| copper oxidase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  LFB112] 
                   
                   
                   
                   
               
               
                 92 
                 43 
                 copper oxidase [ Bacillus   amyloliquefaciens  subsp. 
                 AHK48246.1 
                 76% 
                 149 
                 Leu 
               
               
                   
                   
                   plantarum  TrigoCor1448] 
                   
                   
                   
                   
               
               
                 93 
                 44 
                 spore copper-dependent laccase [ Bacillus   
                 YP_003919218.1 
                 76% 
                 149 
                 Leu 
               
               
                   
                   
                   amyloliquefaciens  DSM 7] &gt;ref|YP_005540261.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  TA208] &gt;ref|YP_005544441.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  LL3] &gt;ref|YP_005548603.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  XH7] &gt;ref|WP_013351262.1| 
                   
                   
                   
                   
               
               
                   
                   
                 copper oxidase [ Bacillus   amyloliquefaciens ] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;emb|CBI41748.1| spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   amyloliquefaciens  DSM 7] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|AEB22768.1| spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   amyloliquefaciens  TA208] 
                   
                   
                   
                   
               
               
                   
                   
                 &gt;gb|AEB62213.1| spore copper-dependent laccase 
                   
                   
                   
                   
               
               
                   
                   
                 [ Bacillus   amyloliquefaciens  LL3] &gt;gb|AEK87755.1| 
                   
                   
                   
                   
               
               
                   
                   
                 spore copper-dependent laccase [ Bacillus   
                   
                   
                   
                   
               
               
                   
                   
                   amyloliquefaciens  XH7] 
                   
                   
                   
                   
               
               
                 94 
                 45 
                 copper oxidase [ Bacillus   siamensis ] 
                 WP_016937040.1 
                 75% 
                 149 
                 Leu 
               
               
                   
               
               
                   (1) Overall identity of selected sequence with SEQ ID NO: 1, the query sequence. 
               
               
                   (2) Position number of the selected sequence that corresponds with position 149 in SEQ ID NO: 1. 
               
               
                   (3) Amino acid at a position of the selected sequence that corresponds with position 149 in SEQ ID NO: 1. 
               
            
           
         
       
     
     Analysis of the homologous proteins revealed that all proteins with at least 75% sequence identity to SEQ ID NO: 1, belong to the species of  Bacillus . All sequences with at least 75% sequence identity to SEQ ID NO: 1 were copper-dependent oxidases (laccases) and most of them were annotated as spore coat proteins. Thus, it was concluded that sequences with this extent (at least 75%) of identity to SEQ ID NO: 1 represent a functionally and structurally highly related group of proteins that are likely to have similar structural traits and folding pathways. 
     Several amino acid substitutions were performed in a variety of laccases at a position corresponding to position 149 in SEQ ID NO: 1, finding that the yield of soluble recombinant laccase protein could be improved when the original amino acid (Ile or Leu) occurring at that position was replaced with a small or tiny amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine. 
     In other words, the disclosure relates to a spore coat polypeptide with laccase activity wherein the polypeptide comprises an amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine, at an amino acid position corresponding to position 149 in SEQ ID NO: 1. 
     Although marginal differences in yield were observed between the variants carrying the different amino acids, variants carrying a threonine residue at position 149 showed the highest yield. In a preferred embodiment, the disclosure, therefore, relates to a laccase with a threonine residue at a position corresponding to amino acid 149 of SEQ ID NO: 1. 
     In a further preferred embodiment, the polypeptide according to the disclosure is a polypeptide mutated as described above wherein the wild-type sequence is encoded by the genome of a  Bacillus  species, such as  Bacillus subtilis.    
     None of the 45 laccases from Table 1 (44 sequences from the search plus SEQ ID NO: 1 used as the query sequence) has an amino acid selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine at a position corresponding to position 149 of SEQ ID NO: 1. Thus, it may be concluded that a laccase with at least 75% sequence identity to SEQ ID NO: 1 comprising an amino acid selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine at a position corresponding to position 149 of SEQ ID NO: 1 has not yet been described in the prior art. 
     It is remarkable that the amino acid corresponding to position 149 in SEQ ID NO: 1 is rather well conserved within the group of 44 sequences of Table 1. An isoleucine residue occurs at that position in 31 out of 44 cases (70%), whereas thirteen sequences appear to have a leucine residue at that position (30%). 
     It was further observed that the search identified two different groups of sequences. The first group comprises 26 sequences with between 94% and 100% identity with SEQ ID NO: 1. Those sequences were almost all annotated as  Bacillus subtilis  CotA spore coat proteins, apart from two  Bacillus vallismortis  CotA (SEQ ID NO: 55 and SEQ ID NO: 75). 
     A second group of 15 sequences was also identified with an identity of between 75% and 81% with the sequence of SEQ ID NO: 1. 
     It was found that 41 out of 44 sequences from the search (93%) belonged to either one of these two groups. 
     Introduction of a specific mutation in a recombinant gene is among the routine skills of a molecular biologist. Specific guidance may be obtained from  Methods in Molecular Biology , Vol. 182, “In vitro mutagenesis protocols,” eds. Jeff Braman, Humana Press 2002. There are commercially available kits for performing site-directed mutagenesis (for example, QUIKCHANGE® II XL Site-Directed Mutagenesis kit, Agilent Technologies cat. No. 200521). 
     Variants of representatives of laccases were prepared from each of the above-described two groups. This includes laccases with an amino acid sequence according to SEQ ID NO: 1 and SEQ ID NO: 2 as representatives of group 1 (94%400% identity). The sequences of these variants are shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively, wherein the isoleucine residue at position 149 of SEQ ID NO: 1 and SEQ ID NO: 2 was replaced by a threonine. When expressed in  E. coli , both variants showed an increased yield of active enzyme of 240% and 260%, respectively ( FIG. 1 ). In other words, the volumetric activity of both variants was increased to at least 240%. 
     As a control experiment, it was determined whether this improved volumetric activity may be attributable to an increased specific activity of the enzyme. This appeared not to be the case. The increase in the amount of mutated enzyme (149T) in the soluble fraction of cell lysate was proportional to the increase in volumetric activity, so it has to be concluded that more variant enzyme may be recovered, thereby completely accounting for the increase in volumetric activity. Hence, the yield of the laccase enzyme is increased rather than its specific activity. 
     A 149T variant was also prepared from a representative of laccases from the second group (75%-81% identity). The naturally occurring Leu at position 149 in SEQ ID NO: 5 (77% identity to SEQ ID NO: 1) was replaced by a threonine in order to obtain the variant according to SEQ ID NO: 6. 
     When expressed in  E. coli , the 149T variant showed an increased yield of active enzyme of 250%. In other words, the 149T variant had an increased volumetric activity of at least a factor 2.5 ( FIG. 2 ). 
     The naturally occurring amino acid at position 149 could also be replaced with other amino acids. Variants of representative laccases were prepared from each of the two groups identified above: SEQ ID NO: 5 and SEQ ID NO: 15. The naturally occurring Leu in SEQ ID NO: 5 and the naturally occurring Ile at position 149 of SEQ ID NO: 15 were replaced with either a threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine or a valine residue. The variants of SEQ ID NO: 5 are represented by SEQ ID NOS: 6-14, respectively, whereas the variants of SEQ ID NO: 15 are represented by SEQ ID NOS: 16-24 (Tables 2 and 3). 
     Sequences of SEQ ID NO: 1 to SEQ ID NO: 24 are shown in Table 5. 
     Each of these variants exhibited an improved yield of at least 50% when expressed in a heterologous expression system ( FIGS. 2 and 3 ). 
     The variants according to SEQ ID NO: 3 and SEQ ID NO: 4 were also expressed in  Pichia pastoris . In accordance with the data obtained in a prokaryotic expression system ( E. coli , see above) the eukaryotic expression also showed an increased yield. The yield was improved to at least 200% when the expression of the variant sequences was compared with their wild-type, SEQ ID NO: 1 and SEQ ID NO: 2, respectively ( FIG. 4 ). 
     Accordingly, the disclosure relates to a polypeptide with laccase activity comprising an amino acid sequence that is at least 75% identical to the amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises an amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine, at an amino acid position corresponding to position 149 in SEQ ID NO: 1. 
     In a preferred embodiment, the disclosure relates to a polypeptide with laccase activity consisting of an amino acid sequence that is at least 75% identical to the amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises a small amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine at an amino acid position corresponding to position 149 in SEQ ID NO: 1. 
     In a further preferred embodiment, the disclosure relates to a polypeptide with laccase activity comprising or consisting of an amino acid sequence that is at least 75% identical to the amino acid sequence according to SEQ ID NO: 1, wherein the polypeptide comprises a threonine residue at a position corresponding to position 149 in SEQ ID NO: 1. 
     This variant laccase is also referred to herein as amino acid variant 149Thr or 149T. In a further preferred embodiment, the polypeptide is isolated. 
     The above finding that spore coat proteins occur in two distinct groups allows defining the disclosure in yet another way, such as the structural relationship between the polypeptide according to the disclosure and the reference polypeptides according to the sequences herein. Hence, the disclosure relates to a polypeptide comprising an amino acid sequence that is at least 94% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 51-75 as representatives of group 1, as well as a polypeptide comprising an amino acid sequence that is at least 94% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 80-94 as representatives of group 2, wherein the polypeptide comprises an amino acid residue selected from the group consisting of threonine, proline, alanine, glycine, serine, cysteine, aspartic acid, asparagine and valine, at an amino acid position corresponding to position 149 in SEQ ID NO: 1. 
     The term “at least 94%” is used herein to include at least 95%, such as 96%, 97%, 98%, 99% or even 100%. 
     The term “amino acid variant,” “laccase variant” or “sequence variant” or equivalent has a meaning well recognized in the art and is accordingly used herein to indicate an amino acid sequence that has at least one amino acid difference as compared to another amino acid sequence, such as the amino acid sequence from which it was derived. 
     The term “at least 75%” is used herein to include at least 76%, such as at least 77%, 78%, 79%, 80% or more such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%. 
     The term “laccase activity” is used herein to mean the property of a polypeptide to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction. Laccase activity may be determined by standard oxidation assays known in the art including, such as, for example, by measurement of oxidation of syringaldazine, according to Sigma online protocol, or according to Cantarella et al., 2003. [7]   
     An example of determining relative laccase activity is presented in Example 4. Any substrate suitable for the enzyme in question may be used in the activity measurements. A non-limiting example of a substrate suitable for use in assessing the enzymatic activity of laccase variants is ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Laccases are able to oxidize this substrate. 
     As used herein, the term “increased (or improved) laccase-specific activity” refers to a laccase activity higher than that of a corresponding non-mutated laccase enzyme under the same conditions. 
     The term “increased yield” or equivalent means that the yield of the active enzyme from the same culture volume obtained in a standard purification or recovery protocol is improved by at least 50% or a factor 1.5. The increase may be even more, such as a factor 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. 
     Recovery of a laccase variant produced by a host cell may be performed by any technique known to those skilled in the art. Possible techniques include, but are not limited to, secretion of the protein into the expression medium, and purification of the protein from cellular biomass. The production method may further comprise a step of purifying the laccase variant obtained. For thermostable laccases, non-limiting examples of such methods include heating of the disintegrated cells and removing coagulated thermo-labile proteins from the solution. For secreted proteins, non-limiting examples of such methods include ion exchange chromatography, and ultra-filtration of the expression medium. It is important that the purification method of choice is such that the purified protein retains its activity, preferably its laccase activity. 
     The laccase variants according to this disclosure may be used in a wide range of different industrial processes and applications, such as cellulose recovery from lignocellulosic biomass, decreasing refining energy in wood refining and pulp preparation, in pulp delignification, textile dye bleaching, wastewater detoxification, xenobiotic detoxification, and detergent manufacturing. 
     Amino acid variations as described herein may be introduced into any of the amino acid sequences disclosed herein, or other homologous sequences, by standard methods known in the art, such as site-directed mutagenesis. In this way, the yield of the laccases from a heterologous expression system may be improved. 
     Kits for performing site-directed mutagenesis are commercially available in the art (e.g., QUIKCHANGE® II XL Site-Directed Mutagenesis kit by Agilent Technologies). Further suitable methods for introducing the above mutations into a recombinant gene are disclosed, e.g., in  Methods in Molecular Biology,  2002. [8]   
     Thus, some embodiments of this disclosure relate to laccase variants or mutants that comprise a small amino acid residue, preferably a threonine residue (Thr) in a position that corresponds to the position 149 of the amino acid sequence depicted in SEQ ID NO: 1, and have an increased yield as compared to that of a corresponding non-mutated control when expressed in a heterologous expression system. 
     The term “heterologous expression system” or equivalent means a system for expressing a DNA sequence from one host organism in a recipient organism from a different species or genus than the host organism. The most prevalent recipients, known as heterologous expression systems, are usually chosen because they are easy to transfer DNA into or because they allow for a simpler assessment of the protein&#39;s function. Heterologous expression systems are also preferably used because they allow the upscaling of the production of a protein encoded by the DNA sequence in an industrial process. Preferred recipient organisms for use as heterologous expression systems include bacterial, fungal and yeast organisms, such as, for example,  Escherichia coli, Bacillus, Corynebacterium, Pseudomonas, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, filamentus fungi  and many more systems well known in the art. 
     As used herein, the degree of identity between two or more amino acid sequences is equivalent to a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions divided by the total number of positions×100), excluding gaps, which need to be introduced for optimal alignment of the two sequences, and overhangs. The comparison of sequences and determination of percent identity between two or more sequences can be accomplished using standard methods known in the art. For example, a freeware conventionally used for this purpose is “Align” tool at NCBI recourse on the World Wide Web at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&amp;BLAST_SPEC=blast2seq&amp;LINK_LOC=align2seq. 
     In a preferred embodiment, the alignment of two sequences is to be performed over the full length of the polypeptides. 
     The present laccase polypeptides or proteins may be fused to additional sequences, by attaching or inserting, including, but not limited to, affinity tags, facilitating protein purification (S-tag, maltose binding domain, chitin binding domain), domains or sequences assisting folding (such as thioredoxin domain, SUMO protein), sequences affecting protein localization (periplasmic localization signals, etc.), proteins bearing additional function, such as green fluorescent protein (GFP), or sequences representing another enzymatic activity. Other suitable fusion partners for the present laccases are known to those skilled in the art. 
     This disclosure also relates to polynucleotides encoding any of the laccase variants disclosed herein. Means and methods for cloning and isolating such polynucleotides are well known in the art. 
     Furthermore, this disclosure relates to a vector comprising a polynucleotide according to the disclosure, optionally operably linked to one or more control sequences. Suitable control sequences are readily available in the art and include, but are not limited to, promoter, leader, polyadenylation, and signal sequences. 
     Laccase variants according to various embodiments of this disclosure may be obtained by standard recombinant methods known in the art. Briefly, such a method may comprise the steps of i) culturing a desired recombinant host cell under conditions suitable for the production of a present laccase polypeptide variant, and ii) recovering the polypeptide variant obtained. The polypeptide may then optionally be further purified. 
     A large number of vector-host systems known in the art may be used for recombinant production of laccase variants. Possible vectors include, but are not limited to, plasmids or modified viruses that are maintained in the host cell as autonomous DNA molecule or integrated in genomic DNA. The vector system must be compatible with the host cell used as is well known in the art. Non-limiting examples of suitable host cells include bacteria (e.g.,  E. coli, bacilli ), yeast (e.g.,  Pichia Pastoris, Saccharomyces Cerevisae ), fungi (e.g.,  filamentous fungi ), and insect cells (e.g., Sf9). 
     A polypeptide according to the disclosure may be advantageously used in an application selected from the group consisting of pulp delignification, degrading or decreasing the structural integrity of lignocellulosic material, textile dye bleaching, wastewater detoxification, xenobiotic detoxification, production of a sugar from a lignocellulosic material and recovering cellulose from a biomass. 
     In yet other terms, the disclosure relates to a method for improving the yield of a polypeptide with laccase activity in a heterologous expression system comprising the step of altering the amino acid at a position corresponding to position 149 in SEQ ID NO: 1 to a threonine residue. 
     EXAMPLES 
     Example 1 
     Construction of Laccases with Improved Properties 
     Mutations as described herein were introduced into various recombinant genes by standard site-directed mutagenesis essentially as described in WO 2013/038062. In more detail, in order to introduce mutation 149T into the gene of SEQ ID NO: 1, two separate PCRs were carried out: 
     
       
         
           
               
               
            
               
                   
                 (1) with primers Primer1  
               
               
                   
                 (SEQ ID NO: 25) 
               
               
                   
                 GAAATTAATACGACTCACTATAGG 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 Primer2 (Seq1) 
               
               
                   
                 (SEQ ID NO: 26) 
               
               
                   
                 AGCACCGCGTTGCTGATTCGGATAATGATA, 
               
               
                   
                   
               
               
                   
                 (2) with Primer3 (Seq1) 
               
               
                   
                 (SEQ ID NO: 27) 
               
               
                   
                 CGAATCAGCAACGCGGTGCTAccTTGTGGTATC 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 Primer4 
               
               
                   
                 (SEQ ID NO: 28) 
               
               
                   
                 GGTTATGCTAGTTATTGCTCAGCGGTG. 
               
            
           
         
       
     
     In both reactions, recombinant gene without the mutation was used as the template. Primers 1 and 4 bind inside the vector sequence and are not specific to the recombinant gene. Primers 2 and 3 bind inside the recombinant gene and their binding sites overlap. Primer 3 binding site contains the mutation site. Primer 3 represents the mutated (desired) sequence, which is not 100% matching the template (lower case type font in the primer sequence indicate the mis-matched nucleotides); however, the primer has enough affinity and specificity to the binding site to produce the desired PCR product. Purified PCR products from reactions (1) and (2) were combined and used as template for PCR reaction with Primer1 and Primer4. The product of this reaction, containing the mutant sequence of the gene, was cloned in a plasmid vector for expression in  E. coli.    
     Similarly, for introducing a 149T mutation into other genes (corresponding to SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 15), the same Primer1 and Primer4 were used, whereas Primer2 and Primer3 were specific for each gene. 
     In the polypeptide comprising the sequence according to SEQ ID NO: 2, there is an isoleucine at position 149, the position corresponding to amino acid 149 in SEQ ID NO: 1. For introducing the 149T mutation into the polypeptide comprising the sequence according to SEQ ID NO: 2, the following primers3 and 2 were used: 
     
       
         
           
               
               
            
               
                   
                 Primer3 
               
               
                   
                 (SEQ ID NO: 29) 
               
               
                   
                 CGAATCAGCAGCGCGGCGCTAccTTGTGGTATC; 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 Primer2 
               
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
                 AGCGCCGCGCTGCTGATTCGGATAATGATA. 
               
            
           
         
       
     
     In the polypeptide comprising the sequence according to SEQ ID NO: 5, there is a leucine at position 149, the position corresponding to amino acid 149 in SEQ ID NO: 1. For introducing several mutations at position 149 into the polypeptide comprising the sequence according to SEQ ID NO: 5, the following primer2 was used in combination with the primers 3 listed in Table 2: 
     
       
         
           
               
               
            
               
                   
                 Primer2 
               
               
                   
                 (SEQ ID NO: 31) 
               
               
                   
                 GCCCCGCGCTGTTTGTTTGGATAA. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Primers used to introduce various amino 
               
               
                 acid substitutions into SEQ ID NO: 5 
               
            
           
           
               
               
            
               
                 Specific primer3 used 
                 Variant obtained 
               
            
           
           
               
               
               
            
               
                 to introduce variations 
                 AA change 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                 introduced at 
                 SEQ 
               
               
                 Primer3 sequence 
                 ID NO: 
                 position 149 
                 ID NO: 
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 32 
                 Thr 
                  6 
               
               
                 GCTacGTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 33 
                 Pro 
                  7 
               
               
                 GCTCcGTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 34 
                 Ala 
                  8 
               
               
                 GCTgcGTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 35 
                 Gly 
                  9 
               
               
                 GCTggtTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 36 
                 Ser 
                 10 
               
               
                 GCTtctTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 37 
                 Cys 
                 11 
               
               
                 GCTtgtTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 38 
                 Asp 
                 12 
               
               
                 GCTgatTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 39 
                 Asn 
                 13 
               
               
                 GCTaacTTATGGTATCAC 
                   
                   
                   
               
               
                   
               
               
                 TCCAAACAAACAGCGCGGG 
                 40 
                 Val 
                 14 
               
               
                 GCTgTGTTATGGTATCAC 
               
               
                   
               
            
           
         
       
     
     In the polypeptide comprising the sequence according to SEQ ID NO: 15, there is an isoleucine at position 149, the position corresponding to amino acid 149 in SEQ ID NO: 1. For introducing a 149T mutation into the polypeptide comprising the sequence according to SEQ ID NO: 15, the following primer2 was used in combination with the various primers3 in Table 3: 
     
       
         
           
               
               
            
               
                   
                 Primer2: 
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 AGCACCGCGCTGCTGATTCGGATAATGATA. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primers used to introduce various amino 
               
               
                 acid substitutions into SEQ ID NO: 15 
               
            
           
           
               
               
            
               
                 Specific primer3 used 
                 Variant obtained 
               
            
           
           
               
               
               
            
               
                 to introduce mutation 
                 AA change 
                   
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                 introduced at 
                 SEQ 
               
               
                 Primer3 sequence 
                 ID NO: 
                 position 149 
                 ID NO: 
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTAc 
                 42 
                 Thr 
                 16 
               
               
                 TTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTcc 
                 43 
                 Pro 
                 17 
               
               
                 gTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTgc 
                 44 
                 Ala 
                 18 
               
               
                 gTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTgg 
                 45 
                 Gly 
                 19 
               
               
                 TTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTtc 
                 46 
                 Ser 
                 20 
               
               
                 TTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTtg 
                 47 
                 Cys 
                 21 
               
               
                 TTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTga 
                 48 
                 Asp 
                 22 
               
               
                 TTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTAa 
                 49 
                 Asn 
                 23 
               
               
                 cTTGTGGTATCACGATCACGC 
                   
                   
                   
               
               
                   
               
               
                 CGAATCAGCAGCGCGGTGCTgT 
                 50 
                 Val 
                 24 
               
               
                 gTTGTGGTATCACGATCACGC 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Heterologous Expression of Variant and Non-Mutated Laccases 
     Variant laccases were expressed in  E. coli  and  Pichia pastoris.    
     For expression in  Pichia Pastoris , recombinant genes were cloned into a commercial  Pichia Pastoris  expression vector pPICZ-A, available from Invitrogen (Life Technologies). This vector provides secreted protein expression under the control of methanol-inducible AOX1 promoter upon integration of the construct into genomic DNA of the yeast cell. 
     Linearized plasmid DNA was introduced into yeast cells by electroporation, and clones with integrated recombinant gene were selected on agar medium plates with Zeocin (25 μg/ml). Ten colonies from each construct were tested in small liquid cultures (3 ml) with a 72-hour cultivation in humidified shaker at 28° C. according to the plasmid manufacturer manual (World Wide Web at tools.lifetechnologies.com/content/sfs/manuals/ppiczalpha_man.pdf). The medium recommended by the manufacturer was supplemented with 1 mM CuCl, as laccase protein contains copper as a cofactor. Activity in the medium was measured by ABTS oxidation (see Example 4), and the two best-producing clones were selected for each gene. Parallel cultures of the selected clones were grown in flask scale according to the plasmid manufacturer manual (see above) at 28° C. for 105 hours. Cells were removed by centrifugation and medium containing the recombinant protein was collected. These preparations were used for comparison of volumetric activities of variant and non-mutated genes. 
     For recombinant expression in  E. coli , recombinant genes were cloned into pET-28 commercial expression vector under the control of T7 bacteriophage promoter. Protein production was carried out in  E. coli  BL21(DE3) strain according to the plasmid manufacturer protocol on the World Wide Web at richsingiser.com/4402/Novagen %20pET %20system %20manual.pdf. The medium recommended by the manufacturer was supplemented with 1 mM CuCl, as laccase protein contains copper as a cofactor. The incubation temperature for protein production was 30° C., which was found optimal for maximum yield of the active protein. Cells were lysed using lysis buffer (50 mM Tris-HCl pH 7.4, 1% TRITON®-X100, 1 mM CuCl) and heated at 70° C. for 20 minutes. Coagulated cell debris was removed by centrifugation. The recombinant laccase, being a thermostable protein, remained in soluble fraction. Enzymatic activity was detectable only in soluble fraction. Analysis of soluble and insoluble fractions by gel-electrophoresis reveals that over 90% of the recombinant protein is present in insoluble inactive form as inclusion bodies (in accordance with literature data). 
     Example 3 
     Measurement of Yield 
     The relative yields of mutated and non-mutated soluble laccases were determined by densitometry of protein bands after denaturing polyacrylamide gel electrophoresis. To this end, samples of soluble proteins after thermal treatment (see Example 2) obtained from parallel cultures of mutated and non-mutated clones, were analyzed by gel-electrophoresis under denaturing conditions (a standard method well known in the art of molecular biology). After staining the gel with Coomassie Brilliant Blue, the gel was scanned to obtain a bitmap image, and intensity of the band corresponding to recombinant laccase was quantified by ImageJ software (a public freeware developed at National Institute of Health and online available on the World Wide Web at imagej.nih.gov/ij/). 
     Example 4 
     Relative Activity Measurement of Laccase 
     As stated above, the term “laccase activity” is used herein to mean the capability to act as a laccase enzyme, which may be expressed as the maximal initial rate of the specific oxidation reaction. Relative activity was measured by oxidation of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Reaction course was monitored by change in absorbance at 405 nM (green color development). The appropriate reaction time was determined to provide initial rates of oxidation when color development is linear with time. Substrate (ABTS) concentration was 5 mM to provide maximum initial rates (substrate saturation conditions). 
     Typically, reactions were carried out in 96-well flat bottom plates, each well containing 2 μl of enzyme preparation in 200 μl of 100 mM Succinic acid pH 5. The reaction was initiated by simultaneous addition of the substrate (22 μl of 50 mM ABTS) in each well. After the reaction time has elapsed, absorbance at 405 nm of the reaction mixtures was determined by a plate reader (Multiscan Go, Thermo Scientific). In order to determine relative activity of mutated laccase, the absorbance of the reference laccase sample was taken for 100%, and relative activity was determined as fraction of this absorbance. 
     Example 5 
     Identification of the Amino Acid Position Corresponding to Position 149 
     In order to identify the amino acid position that corresponds to position 149 in SEQ ID NO: 1 in a given sequence X, the sequence X is aligned with the sequence of SEQ ID NO: 1 using standard software available in the art, in this case the “Align” tool at NCBI recourse on the World Wide Web at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&amp;BLAST_SPEC=blast2seq&amp;LINK_LOC=align2seq. 
     As an example, sequences 51-94 were aligned with SEQ ID NO: 1. Only a fragment of that alignment is shown in Table 4, i.e., the fragment corresponding to amino acids 125-152 of SEQ ID NO: 1. It is immediately evident that this particular region is highly conserved or highly homologous between the various sequences provided in the table, leading to a high degree of identity in all examined sequences. For example, the isoleucine (I) residue at position 149 of SEQ ID NO: 1 corresponds to an isoleucine residue at position 149 in SEQ ID NO: 51, to an isoleucine residue at position 151 in SEQ ID NO: 56, and to a leucine residue (L) at position 149 in SEQ ID NO: 85. The position of the first and last amino acid of each fragment is shown in Table 4. The amino acid corresponding to position 149 in SEQ ID NO: 1 in each fragment is indicated in bold. 
     Sequences of SEQ ID NO: 1 to SEQ ID NO: 24 are shown in Table 5. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Alignment of SEQ ID NO: 1 with SEQ ID NOS: 51-94, 
               
               
                 fragments 125-152 of the alignment are shown. 
               
               
                 The amino acid at the position corresponding to 
               
               
                 amino acid 149 in SEQ ID NO: 1 is shown in bold. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 First 
                 Sequence corresponding to 
                 Last 
                 SEQ 
                 Fragment of 
               
               
                 Accession No: 
                 AA 
                 AA 125-152 of SEQ ID NO: 1. 
                 AA 
                 ID NO: 
                 SEQ ID NO: 
               
               
                   
               
               
                 SEQ ID NO: 1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                  95 
                  1 
               
               
                   
               
               
                 AGZ16504.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                  96 
                 51 
               
               
                   
               
               
                 YP_003865004.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                  97 
                 52 
               
               
                   
               
               
                 WP_004397739.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                  98 
                 53 
               
               
                   
               
               
                 WP_019713492.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                  99 
                 54 
               
               
                   
               
               
                 AGR50961.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 100 
                 55 
               
               
                   
               
               
                 YP_007425830.1 
                 127 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 154 
                 101 
                 56 
               
               
                   
               
               
                 YP_004206641.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 102 
                 57 
               
               
                   
               
               
                 YP_006230497.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 103 
                 58 
               
               
                   
               
               
                 EXF51833.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 104 
                 59 
               
               
                   
               
               
                 WP_003234000.1 
                 127 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 154 
                 105 
                 60 
               
               
                   
               
               
                 YP_006628799.1 
                 127 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 154 
                 106 
                 61 
               
               
                   
               
               
                 NP_388511.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 107 
                 62 
               
               
                   
               
               
                 YP_007661398.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 108 
                 63 
               
               
                   
               
               
                 4AKQ_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 109 
                 64 
               
               
                   
               
               
                 4A68_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 110 
                 65 
               
               
                   
               
               
                 4A66_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 111 
                 66 
               
               
                   
               
               
                 AC544284.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 112 
                 67 
               
               
                   
               
               
                 AGK12417.1 
                 125 
                 KDLEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 113 
                 68 
               
               
                   
               
               
                 2X87_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 114 
                 69 
               
               
                   
               
               
                 AFN66123.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 115 
                 70 
               
               
                   
               
               
                 4A67_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 116 
                 71 
               
               
                   
               
               
                 2WSD_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 117 
                 72 
               
               
                   
               
               
                 4AKP_A 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 118 
                 73 
               
               
                   
               
               
                 ACM46021.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 119 
                 74 
               
               
                   
               
               
                 WP_010329056.1 
                 125 
                 KDFEQTGPYFKREIYHYPNQQRGA I LWY 
                 152 
                 120 
                 75 
               
               
                   
               
               
                 AEK80414.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 121 
                 76 
               
               
                   
               
               
                 WP_010333230.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 122 
                 77 
               
               
                   
               
               
                 4AKO_A 
                 121 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 148 
                 123 
                 78 
               
               
                   
               
               
                 AAB62305.1 
                 125 
                 KDFEQTGPYFKREVYHYPNQQRGA I LWY 
                 152 
                 124 
                 79 
               
               
                   
               
               
                 YP_003972023.1 
                 125 
                 KGFQETGPYFSREIYHYPNQQRGA I LWY 
                 152 
                 125 
                 80 
               
               
                   
               
               
                 WP_010787813.1 
                 125 
                 KGFQETGPYFSREIYHYPNQQRGA I LWY 
                 152 
                 126 
                 81 
               
               
                   
               
               
                 WP_007609818.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 127 
                 82 
               
               
                   
               
               
                 YP_007496315.1 
                 125 
                 KDFKETGPYFEREVYHYPNKQRGA L LWY 
                 152 
                 128 
                 83 
               
               
                   
               
               
                 YP_005419918.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 129 
                 84 
               
               
                   
               
               
                 YP_008625231.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 130 
                 85 
               
               
                   
               
               
                 YP_007185316.1 
                 125 
                 KDFKETGPYFEREVYHYPNKQRGA L LWY 
                 152 
                 131 
                 86 
               
               
                   
               
               
                 YP_001420286.1 
                 125 
                 KDFKETGPYFEREVYHYPNKQRGA L LWY 
                 152 
                 132 
                 87 
               
               
                   
               
               
                 ADZ57286.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 133 
                 88 
               
               
                   
               
               
                 WP_007408880.1 
                 125 
                 KDFKETGPYFEREVYHYPNKQRGA L LWY 
                 152 
                 134 
                 89 
               
               
                   
               
               
                 WP_021495201.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 135 
                 90 
               
               
                   
               
               
                 YP_005129370.1 
                 125 
                 KDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 136 
                 91 
               
               
                   
               
               
                 AHK48246.1 
                 125 
                 KDFKETGPYFEREVYHYPNKQRGA L LWY 
                 152 
                 137 
                 92 
               
               
                   
               
               
                 YP_003919218.1 
                 125 
                 RDFKEKGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 138 
                 93 
               
               
                   
               
               
                 WP_016937040.1 
                 125 
                 RDFKETGPYFEKEVYHYPNKQRGA L LWY 
                 152 
                 139 
                 94 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Sequences of SEQ ID NOS: 1-24. 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID NO: 
                 Name 
                 Organism 
                 Sequence 
               
               
                   
               
               
                  1 
                 Cot1 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPDTLKPVQQTTEKTYYEVTMEECAHQLHRDLPPTRLW 
               
               
                   
                   
                 
                   subtilis 
                 
                 GYNGLFPGPTIEVKRNENVYVKWMNNLPSEHFLPIDHTIHHSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRG 
               
               
                   
                   
                   
                 AILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINEDGSLFYPSGPENPSPSLPKPSIVPAFCGDTILVNGKVWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNGGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGESIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIQNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKAGTTEIWSIVNPTQGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAVTFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                  2 
                 Cot2 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLW 
               
               
                   
                   
                 
                   subtilis 
                 
                 GYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRG 
               
               
                   
                   
                   
                 AILWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSEEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINEDGSLFYPSGPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNGGEFIQIGSDGGLLPRSVKLTSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSAGCGGDVNPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIQNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKAGTTEIWSIINPTRGTHPIHLHLVSFRVIDRRPFDIAHY 
               
               
                   
                   
                   
                 QESGALSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHKSDPNSSSVDKLHRTRAPPPPPLRSGC 
               
               
                   
               
               
                  3 
                 Cot1 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPDTLKPVQQTTEKTYYEVTMEECAHQLHRDLPPTRLW 
               
               
                   
                 149T 
                 
                   subtilis 
                 
                 GYNGLFPGPTIEVKRNENVYVKWMNNLPSERFLPIDHTIHHSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRG 
               
               
                   
                   
                   
                 ATLWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINEDGSLFYPSGPENPSPSLPKPSIVPAFCGDTILVNGKVWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNGGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGESIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIQNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKAGTTEIWSIVNPTQGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAVTFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                  4 
                 Cot2 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPDTLKPVQQSKEKTYYEVTMEECTHQLHRDLPPTRLW 
               
               
                   
                 149T 
                 
                   subtilis 
                 
                 GYNGLFPGPTIEVKRNENVYVKWMNNLPSTHFLPIDHTIHHSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPDDSDGYPEAWFSKDFEQTGPYFKREVYHYPNQQRG 
               
               
                   
                   
                   
                 ATLWYHDHAMALTRLNVYAGLVGAYIIHDPKEKRLKLPSEEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINEDGSLFYPSGPENPSPSLPNPSIVPAFCGETILVNGKVWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNGGEFIQIGSDGGLLPRSVKLTSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSAGCGGDVNPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIQNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKAGTTEIWSIINPTRGTHPIHLHLVSFRVIDRRPFDIAHY 
               
               
                   
                   
                   
                 QESGALSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHKSDPNSSSVDKLHRTRAPPPPPLRSGC 
               
               
                   
               
               
                  5 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                   
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ALLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                  6 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149T 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ATLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                  7 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149P 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 APLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                  8 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149A 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 AALWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                  9 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149G 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 AGLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 10 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149S 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ASLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 11 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149C 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ACLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 12 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149D 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ADLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 13 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149N 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 ANLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 14 
                 seq 61 
                 
                   Bacillus 
                 
                 MALEKFADELPIIETLQPQKKSDGSTYYEVTMKECFHKLHRDLPPTRLW 
               
               
                   
                 149V 
                 
                   amylo- 
                 
                 GYNGLFPGPTIDVNKDENVYIKWMNDLPDKHFLPVDHTIHHSESGHQEP 
               
               
                   
                   
                 
                   liquefaciens 
                 
                 DVKTVVHLHGGATPPDSDGYPEAWFTKDFKETGPYFEREVYHYPNKQRG 
               
               
                   
                   
                   
                 AVLWYHDHAMAITRLNVYAGLAGMYIIRERKEKQLKLPSGEYDVPLMIL 
               
               
                   
                   
                   
                 DRTLNDDGSLSYPSGPANPSETLPDPSIVPFFCGNTILVNGKAWPYMEV 
               
               
                   
                   
                   
                 EPRTYRFRILNASNTRTFSLSLNNGGKFIQIGSDGGLLPRSVKTQSISL 
               
               
                   
                   
                   
                 APAERYDVLIDFSAFDGEHIILTNGTGCGGDADPDTDANVMQFRVTKPL 
               
               
                   
                   
                   
                 KGADTSRKPKYLSTMPNVTSKRIHNIRMLKLTNTQDKYGRPVLTLNNKR 
               
               
                   
                   
                   
                 WHDPVTEAPKLGTTEIWSIINPMGGTHPIHLHLVSFQILDRRPFDLERY 
               
               
                   
                   
                   
                 NKFGDIVYTGPAVPPPPSEKGWKDTVQAHSGEVIRIAATFAPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDVTDKQ 
               
               
                   
               
               
                 15 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                   
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 AILWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 16 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149T 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 ATLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 17 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149P 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 APLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 18 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149A 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 AALWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 19 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149G 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 AGLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 20 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149S 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 ASLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 21 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149C 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 ACLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 22 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149D 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 ADLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 23 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149N 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHRSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 ANLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
               
                 24 
                 Seq 49 
                 
                   Bacillus 
                 
                 MTLEKFVDALPIPETLKPVQQTKEKTYYEVTMEECAHKLHRDLPPTRLW 
               
               
                   
                 149V 
                 
                   vallismortis 
                 
                 GYNCQFPGPTIEVNRNENVYVKWMNHLSSTHFLPVDHTIHHSDSQHEEP 
               
               
                   
                   
                   
                 EVKTVVHLHGGVTPEDSDGYPEAWFTKDFEQTGPYFKREIYHYPNQQRG 
               
               
                   
                   
                   
                 AVLWYHDHAMALTRLNVYAGLIGAYLIHDPKEKRLKLPSGEYDVPLLIT 
               
               
                   
                   
                   
                 DRTINGDGSLFYPNGPENPSPSLPNPSIVPAFCGETILVNGKAWPYLEV 
               
               
                   
                   
                   
                 EPRKYRFRVINASNTRTYNLSLDNDGEFIQIGSDGGLLPRSVKLNSFSL 
               
               
                   
                   
                   
                 APAERYDIIIDFTAYEGQSIILANSEGCGGDANPETDANIMQFRVTKPL 
               
               
                   
                   
                   
                 AQKDESRKPKYLASYPSVQNERIHNIRTLKLAGTQDEYGRPVLLLNNKR 
               
               
                   
                   
                   
                 WHDPVTETPKAGTTEIWSIINPTRGTHPIHLHLVSFRVLDRRPFDIARY 
               
               
                   
                   
                   
                 QERGELSYTGPAVPPPPSEKGWKDTIQAHAGEVLRIAATFGPYSGRYVW 
               
               
                   
                   
                   
                 HCHILEHEDYDMMRPMDITDPHK 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     
         
         1. Martins L. O., C. M. Soares, M. M. Pereira, M. Teixeira, T. Costa, and G. H. Jones, et al. Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the  Bacillus subtilis  endospore coat.  J. Biol. Chem.  2002, 277:18849-59. 
         2. Bento I., L. O. Martins, G. Gato Lopes, M. Arménia Carrondo, and P. F. Lindley. Dioxygen reduction by multi-copper oxidases: a structural perspective.  Dalton Trans  2005, 21:3507-13. 
         3. Brissos V., L. Pereira, F. D. Munteanu, A. Cavaco-Paulo, and L. O. Martins. Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes.  Biotechnol. J.  2009, 4:558-63. 
         4. Suzuki T., K. Endo, M. Ito, H. Tsujibo, K. Miyamoto, and Y. Inamori. A thermostable laccase from  Streptomyces lavendulae  REN-7: purification, characterization, nucleotide sequence and expression.  Biosci. Biotechnol. Biochem.  2003, 67:2167-75. 
         5. Kumar et al., “Combined sequence and structure analysis of the fungal laccase family,”  Biotechnol. Bioeng.  2003, 83:386-394. 
         6. Morozova et al., “Blue laccases,”  Biochemistry  (Moscow), 2007, 72:1136-1150. 
         7. Cantarella et al., “Determination of laccase activity in mixed solvents: Comparison between two chromogens in a spectrophotometric assay,”  Biotechnology and Bioengineering,  2003, V. 82(4):395-398. 
         8 . Methods in Molecular Biology,  2002, vol. 182, “In vitro mutagenesis protocols,” eds. Jeff Braman, Humana Press.