CN114958785B - Laccase recombinant protein and application thereof - Google Patents

Laccase recombinant protein and application thereof Download PDF

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CN114958785B
CN114958785B CN202110200320.1A CN202110200320A CN114958785B CN 114958785 B CN114958785 B CN 114958785B CN 202110200320 A CN202110200320 A CN 202110200320A CN 114958785 B CN114958785 B CN 114958785B
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CN114958785A (en
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朱蕾蕾
杨建花
谭子瑊
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses laccase recombinant protein and application thereof. The invention provides laccase mutants, which are prepared according to the following steps: the amino acid sequences of LCC1, LCC2 and Mrl2 are subjected to sequence comparison together to obtain an amino acid comparison sequence matrix; taking a certain position between the 123 th and 141 th positions of the amino acid alignment sequence matrix as a recombination site 1, and taking a certain position between the 306 th and 345 th positions of the amino acid alignment sequence matrix as a recombination site 2; and recombining at least two of the three laccase proteins of the LCC1, the LCC2 and the Mrl through the recombination site 1 and/or the recombination site 2 to obtain a recombined laccase protein. The laccase mutant with improved enzyme activity and stability is obtained by a directional transformation method, and the efficiency of the laccase mutant in lignin degradation, organic synthesis and dye decolorization application is expected to be improved.

Description

Laccase recombinant protein and application thereof
Technical Field
The invention relates to the technical field of enzyme engineering, in particular to laccase recombinant protein and application thereof.
Background
Along with the rapid development of biotechnology, enzyme proteins are widely applied to various fields of foods, chemical industry, environment, energy sources and the like, and the sustainable development of industry and energy sources is promoted. However, natural enzymes are generally difficult to meet industrial demands, and their performance needs to be modified by directed evolution means, such as improvement of enzyme activity, stability, substrate specificity, stereoselectivity, and the like.
Directed evolution is a process of simulating natural evolution in a laboratory, wherein an important step is to build a high quality mutant library for downstream screening. At present, although mutation library construction methods such as error-prone PCR, gene recombination, CASTing, sequence saturation mutation and the like have been widely developed, the quality of the mutation library is low, and many nonsense mutations or recombinations exist, so that a large-capacity mutation library needs to be screened to obtain a target mutant. This limits the efficient progress of directed evolution to a large extent. It is therefore particularly important to construct a mutant library of smaller capacity but of higher quality.
The structural domain is one or more relatively independent compact three-dimensional entities formed by further folding due to the close association of adjacent super-secondary structures on the polypeptide chain. From a functional perspective, the active centers of the multi-domain enzyme proteins are often located between domains, and are mainly probably because the active centers with specific three-dimensional arrangement are easily constructed through the domains, and only one section of flexible peptide chain is often connected between each domain, so that the domains are easy to perform relative movement, which is beneficial to the combination of the active centers with substrates, the combination of the allosteric centers with modulators and the allosteric effects, and the catalysis of the enzyme on the reaction.
In multi-domain proteins, the domains of some proteins do not have a well-defined division of function or the domains coexist to function. Those skilled in the art generally recognize that the link between domains is not disrupted, i.e., replacement of any domain is likely to render the protein entirely nonfunctional. Laccase (EC 1.10.3.2) is a multifunctional oxidase, belongs to the family of blue multi-copper oxidases, can oxidize more than 250 substrates, and has wide industrial application prospects, such as lignin degradation, dye decolorization, organic pollutant degradation, biological detection, high-molecular synthesis, food industry and other fields. The laccase can effectively degrade lignin-rich crop straws, so that not only can the environmental problem be solved, but also various aromatic compounds derived from lignin can be obtained and used as precursors for chemical synthesis. Laccase uses molecular oxygen to oxidize various aromatic and non-aromatic compounds through a free radical-catalytic reaction mechanism, and the only byproduct is water, which is regarded as a green and environment-friendly biocatalyst. However, laccase from natural sources is relatively low in activity and is difficult to maintain stable in an industrial application environment, thereby limiting commercial production of laccase and use in large-scale lignin degradation, environmental remediation and the like. Therefore, how to produce cheap and efficient laccase (high activity and high stability) is getting more attention from a plurality of students at home and abroad. Most laccase has three domains, 4 copper ions participate in the reaction, 1T 1 copper is located in domain 3 and is responsible for obtaining electrons from the substrate; the 4 loops regions located near T1 copper in domain 2 and domain 3, respectively, are in the protein structure tableForming a light-sink on the surface for combining the substrates; a trinuclear central region consisting of 2T 3 copper and 1T 2 copper, located at the interface of domains 1 and 3, is responsible for the transfer of electrons from T1 copper to O 2 Water is produced. The three domains of laccase combine together to form an interfacial active center, so that the laccase obtains biochemical activity.
Disclosure of Invention
The applicant believes that the interaction of amino acids at the interface between domains has a great influence on the catalytic function of the enzyme, and therefore, the combination and reconstruction of the domains greatly change the catalytic performance, stability and the like of the enzyme protein. Therefore, the invention provides a concept of designing and reconstructing the protein by reconstructing the structural domain for the first time, and constructs a smart mutation library with high quality and small capacity, thereby achieving the purpose of rapidly reconstructing the target protein. And laccase is selected as a model enzyme protein to verify whether the method is effective, and finally laccase mutants with improved enzyme activity and stability are obtained.
In a first aspect, the invention claims a laccase mutant.
The laccase mutant disclosed by the invention can be prepared by the following steps: performing sequence comparison on laccase protein LCC1 from white rot fungi, laccase protein LCC2 from white rot fungi and laccase protein Mrl2 from basidiomycetes to obtain an amino acid comparison sequence matrix; taking a certain position between the 123 th and 141 th positions of the amino acid alignment sequence matrix as a recombination site 1, and taking a certain position between the 306 th and 345 th positions of the amino acid alignment sequence matrix as a recombination site 2; and recombining at least two of the three laccase proteins of the LCC1, the LCC2 and the Mrl through the recombination site 1 and/or the recombination site 2 to obtain a recombined laccase protein.
The amino acid alignment sequence matrix may specifically be a comparison result map directly output by software (the position information marked on the map is the position in the amino acid alignment sequence matrix, as shown in fig. 1) by using sequence alignment software (such as ClustalW, etc.) to align the amino acid sequences of the LCC1, LCC2 and Mrl.
Further, the recombination mode can be any one of the following modes:
(A1) Said LCC1 and said LCC2 recombine only at said recombination site 1;
(A2) Said LCC1 and said LCC2 recombine only at said recombination site 2;
(A3) Said LCC1 and said LCC2 recombine at said recombination site 1 and said recombination site 2 simultaneously;
(A4) Said LCC1 and said Mrl2 recombine at said recombination site 1;
(A5) Said LCC1 and said Mrl2 recombine only at said recombination site 2;
(A6) Said LCC1 and said Mrl2 recombine at said recombination site 1 and said recombination site 2 simultaneously;
(A7) Said LCC2 and said Mrl2 recombine at said recombination site 1;
(A8) Said LCC2 and said Mrl2 recombine only at said recombination site 2;
(A9) Said LCC2 and said Mrl2 recombine at said recombination site 1 and said recombination site 2 simultaneously;
(A10) The LCC1, LCC2 and Mrl2 recombine at the recombination site 1 and recombination site 2 simultaneously.
In the present invention, the (A1) specifically includes: 1) The part of the LCC1 positioned in front of the recombination site 1 in the amino acid alignment sequence matrix and the part of the LCC2 positioned behind the recombination site 1 in the amino acid alignment sequence matrix are recombined (L1L 2L2 is obtained); or, 2) the portion of the LCC1 located after the recombination site 1 in the amino acid alignment matrix and the portion of the LCC2 located before the recombination site 1 in the amino acid alignment matrix recombine (resulting in L2L 1).
In the present invention, the (A2) specifically is: 1) The part of the LCC1 positioned in front of the recombination site 2 in the amino acid alignment sequence matrix and the part of the LCC2 positioned behind the recombination site 2 in the amino acid alignment sequence matrix are recombined (L1L 1L2 is obtained); or, 2) the portion of the LCC1 located after the recombination site 2 in the amino acid alignment matrix and the portion of the LCC2 located before the recombination site 2 in the amino acid alignment matrix recombine (resulting in L2L 1).
In the present invention, the (A3) specifically includes: 1) The part of the LCC1 positioned in front of the recombination site 1 in the amino acid alignment sequence matrix, the part of the LCC2 positioned between the recombination site 1 and the recombination site 2 in the amino acid alignment sequence matrix and the part of the LCC1 positioned behind the recombination site 2 in the amino acid alignment sequence matrix are recombined (L1L 2L1 is obtained); or, 2) the portion of the LCC2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the LCC1 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the LCC2 located behind the recombination site 2 in the amino acid alignment matrix are recombined (resulting in L2L1L 2).
In the present invention, the (A4) specifically includes: 1) The portion of the LCC1 located in front of the recombination site 1 in the amino acid alignment matrix and the portion of the Mrl2 located behind the recombination site 1 in the amino acid alignment matrix recombine (resulting in L1 MM); or, 2) the portion of the LCC1 located after the recombination site 1 in the amino acid alignment matrix and the portion of the Mrl2 located before the recombination site 1 in the amino acid alignment matrix recombine (giving ML1L 1).
In the present invention, the (A5) specifically includes: 1) The portion of the LCC1 located in front of the recombination site 2 in the amino acid alignment matrix and the portion of the Mrl2 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in L1M); or, 2) the portion of the LCC1 located after the recombination site 2 in the amino acid alignment matrix and the portion of the Mrl2 located before the recombination site 2 in the amino acid alignment matrix recombine (resulting in MML 1).
In the present invention, the (A6) is: 1) The part of the LCC1 positioned in front of the recombination site 1 in the amino acid alignment sequence matrix, the part of the Mrl2 positioned between the recombination site 1 and the recombination site 2 in the amino acid alignment sequence matrix and the part of the LCC1 positioned behind the recombination site 2 in the amino acid alignment sequence matrix are recombined (L1 ML1 is obtained); or, 2) the portion of the Mrl2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the LCC1 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the Mrl2 located behind the recombination site 2 in the amino acid alignment matrix are recombined (resulting in ML 1M).
In the present invention, the (A7) is specifically: 1) The portion of the LCC2 located in front of the recombination site 1 in the amino acid alignment matrix and the portion of the Mrl2 located behind the recombination site 1 in the amino acid alignment matrix recombine (resulting in L2 MM); or, 2) the portion of the LCC2 located after the recombination site 1 in the amino acid alignment matrix and the portion of the Mrl2 located before the recombination site 1 in the amino acid alignment matrix recombine (giving ML2L 2).
In the present invention, the (A8) is specifically: 1) The portion of the LCC2 located in front of the recombination site 2 in the amino acid alignment matrix and the portion of the Mrl2 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in L2M); or, 2) the portion of the LCC2 located after the recombination site 2 in the amino acid alignment matrix and the portion of the Mrl2 located before the recombination site 2 in the amino acid alignment matrix recombine (resulting in MML 2).
In the present invention, the (A9) specifically includes: 1) The portion of the LCC2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the Mrl2 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the LCC2 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in L2ML 2); or, 2) the portion of the Mrl2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the LCC2 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the Mrl2 located behind the recombination site 2 in the amino acid alignment matrix are recombined (resulting in ML 2M).
In the present invention, the (a 10) specifically includes: 1) The part of the LCC1 positioned in front of the recombination site 1 in the amino acid alignment sequence matrix, the part of the LCC2 positioned between the recombination site 1 and the recombination site 2 in the amino acid alignment sequence matrix and the part of the Mrl2 positioned behind the recombination site 2 in the amino acid alignment sequence matrix are recombined (L1L 2M is obtained); or, 2) the portion of the LCC1 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the Mrl2 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the LCC2 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in L1ML 2); or, 3) the portion of the LCC2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the LCC1 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the Mrl2 located behind the recombination site 2 in the amino acid alignment matrix are recombined (resulting in L2L 1M); or, 4) the portion of the LCC2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the Mrl2 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the LCC1 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in L2ML 1); or, 5) the portion of the Mrl2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of the LCC1 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of the LCC2 located behind the recombination site 2 in the amino acid alignment matrix recombine (resulting in ML1L 2); or, 6) the portion of Mrl2 located in front of the recombination site 1 in the amino acid alignment matrix, the portion of LCC2 located between the recombination site 1 and the recombination site 2 in the amino acid alignment matrix, and the portion of LCC1 located behind the recombination site 2 in the amino acid alignment matrix are recombined (giving ML2L 1).
For the recombinant mutant substitutions, the following nomenclature was used: laccase is divided into three domains, and recombinant ABC represents that domain 1 is derived from A, domain 2 is derived from B, and domain 3 is derived from C; l1, L2, M represent domains derived from LCC1, LCC2, mrl2, respectively; recombination positions (i.e., positions in the amino acid alignment matrix).
In the invention, the amino acid sequence of LCC1 is specifically shown as SEQ ID No.1, wherein the 1 st to 123 th positions are domain I, the 124 th to 320 th positions are domain II, and the 321 th to 498 th positions are domain III. The amino acid sequence of LCC2 is shown in SEQ ID No.2, wherein the 1 st to 123 th positions are structural domain I, the 124 th to 320 th positions are structural domain II, and the 321 th to 499 th positions are structural domain III. The amino acid sequence of Mrl is specifically shown as SEQ ID No.3, wherein the 1 st to 123 th positions are structural domain I, the 124 th to 318 th positions are structural domain II, and the 319 th to 494 th positions are structural domain III.
In a specific embodiment of the present invention, the recombination site 1 is specifically between positions 123 and 124 of the amino acid alignment matrix; the recombination site 2 is specifically located between the 321 th and 322 th positions in the amino acid alignment sequence matrix.
Specifically, the laccase mutant is any one of the following proteins:
(B1) A protein (L1L 2L 2) with an amino acid sequence shown as SEQ ID No. 7;
(B2) A protein (L2L 2M) with an amino acid sequence shown in SEQ ID No. 8;
(B3) A protein (L2 ML 2) with an amino acid sequence shown in SEQ ID No. 9;
(B4) Protein (L1 MM) with amino acid sequence shown in SEQ ID No.10
(B5) A protein (L2 MM) with an amino acid sequence shown in SEQ ID No. 11;
(B6) A protein (L1L 2M) with an amino acid sequence shown in SEQ ID No. 12;
(B7) A protein (L1L 1M) with an amino acid sequence shown in SEQ ID No. 13;
(B8) A protein (ML 2L 2) with an amino acid sequence shown as SEQ ID No. 14;
(B9) A protein (MML 1) with an amino acid sequence shown as SEQ ID No. 15;
(B10) A protein having the same function as the amino acid sequence defined in any one of (B1) to (B9) by substitution and/or deletion and/or addition of one or more amino acid residues;
(B11) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the amino acid sequence defined in any one of (B1) to (B10) and having the same function;
(B12) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (B1) to (B11) with a protein tag.
Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above protein, the 95% or more homology may be at least 96%, 97%, 98% identity. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.
In a second aspect, the invention claims laccase protein variant related biological materials.
The laccase protein variant related biological material claimed by the invention can be specifically any one of the following materials:
(C1) A nucleic acid molecule encoding a laccase protein variant as described in the first aspect hereinbefore;
(C2) Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
Further, the nucleic acid molecule may be any one of the following:
(D1) A DNA molecule shown in SEQ ID No.16 (encoding nucleic acid for L1L 2);
(D2) A DNA molecule shown in SEQ ID No.17 (encoding nucleic acid for L2M);
(D3) A DNA molecule shown in SEQ ID No.18 (encoding nucleic acid for L2ML 2);
(D4) A DNA molecule shown in SEQ ID No.19 (encoding nucleic acid for L1 MM);
(D5) A DNA molecule shown in SEQ ID No.20 (encoding nucleic acid for L2 MM);
(D6) A DNA molecule shown in SEQ ID No.21 (encoding nucleic acid for L1L 2M);
(D7) A DNA molecule shown in SEQ ID No.22 (encoding nucleic acid for L1M);
(D8) A DNA molecule shown in SEQ ID No.23 (encoding nucleic acid for ML2L 2);
(D9) A DNA molecule shown in SEQ ID No.24 (encoding nucleic acid for M ML 1);
(D10) A DNA molecule which hybridizes under stringent conditions to a DNA molecule as defined in any one of (D1) to (D9) and which encodes said protein;
(D11) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined in any one of (D1) to (D10) and encoding said protein.
In the above nucleic acid molecule, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 And 1Hybridization in a mixed solution of mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na 3 PO 4 Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In the above nucleic acid molecules, homology refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of nucleotide sequences is searched for and calculated, and then the value (%) of identity can be obtained.
In the above nucleic acid molecules, the 95% or more homology may be at least 96%, 97%, 98% identity. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.
In a specific embodiment of the invention, the recombinant vector is in particular a pPICZ α -a vector comprising said nucleic acid molecule. The nucleic acid molecule is specifically constructed between the cleavage sites XhoI and XbaI of the pPICZ alpha-A vector.
In the invention, the recombinant bacterium is recombinant yeast, in particular recombinant Pichia pastoris.
In a third aspect, the invention claims the use of a laccase mutant as described in the first aspect hereinbefore or a biological material as described in the second aspect hereinbefore in any of the following:
(E1) Producing laccase or improving laccase yield;
(E2) Producing various organic compounds (such as sinapic acid, polyaniline, antibiotics);
(E3) For environmental pollutant treatment (e.g., fuel pollutant, pesticide, herbicide, etc.);
(E4) For textile, pulp and paper applications or as food additives;
(E5) As a biosensor, a biofuel cell or for medical diagnostic tools.
In a fourth aspect, the invention claims a method for improving laccase enzyme activity and/or stability.
The method for improving the enzyme activity and/or stability of laccase claimed by the invention can comprise the following steps: expressing the laccase variant of the first aspect in a recipient bacterium to obtain a recombinant bacterium; and (3) fermenting and culturing the recombinant bacteria to obtain laccase mutants with improved enzyme activity and/or stability from fermentation broth.
Further, expression of the laccase protein variant in the recipient bacterium is achieved by introducing the nucleic acid molecule as described in the second aspect above or the recombinant vector into the recipient bacterium.
Still further, the recipient is a yeast, such as pichia pastoris.
In the method, after the recombinant vector is introduced into the Pichia pastoris, the method further comprises the step of fermenting and culturing the recombinant yeast; the culture medium for fermentation culture can be BMM culture medium, the culture condition can be 20 ℃, and the culture is carried out for 5 days at 220r/min, and 1% of total volume of methanol is added into the fermentation system every day.
In a specific embodiment of the invention, the recombinant bacteria are inoculated in a BMG seed culture medium, cultured for 20 hours at 30 ℃ at 220r/min, then inoculated in a BMM fermentation culture medium at an inoculum size with an initial OD600 of 1.0, and cultured for 5 days at 20 ℃ at 220r/min, and 1% of the total volume of methanol is added every day.
The laccase mutant with improved enzyme activity and stability is obtained by a directional transformation method, and the efficiency of the laccase mutant in lignin degradation, organic synthesis and dye decolorization application is expected to be improved.
Drawings
FIG. 1 is a matrix of wild-type laccase amino acid alignment sequences.
FIG. 2 shows SDS-PAGE of wild-type laccase and recombinant variant supernatants.
FIG. 3 shows the thermostability of wild-type laccase and recombinant variant crude enzyme solutions.
FIG. 4 shows the organic solvent stability of wild-type laccase and recombinant variant crude enzyme solutions.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1, presentation of the method for the reconstruction of the Multi-Domain protein Domain
Through literature and database searches, the inventor of the invention finds that the active centers of the multi-domain proteins are all located between domains, and in this case, the interaction of amino acids on the interface between the domains can have great influence on the enzyme catalysis function, the stability thereof and the like. Furthermore, the functional division of domains within most multi-domain proteins is not clear. Therefore, the invention provides the concept of carrying out domain recombination on the multi-domain protein with similar structure (using TM-align (https:// zhanglab. Ccmb. Med. Umich. Edu/TM-align /) software to construct a high-quality mutation library. Laccase is an important industrial enzyme because laccase has wide application, and has multiple domains, and the functional division of each domain is ambiguous, so laccase is selected as the target enzyme. Because laccase LCC1 and LCC2 from white rot fungi have higher oxidation-reduction potential, have wider substrate spectrum, and LCC2 has higher stability, laccase Mrl from basidiomycetes has higher enzyme activity, and the invention aims to carry out domain recombination on the three laccases in order to obtain mutants with high activity, good stability and wide substrate spectrum. The three laccase are all three-structural-domain proteins, and the three laccase three-dimensional structures are extremely similar by utilizing TM-align (https:// zhanglab. Ccmb. Med. Umich. Edu/TM-align /) software to carry out on-line structural comparison, and the structure of the laccase three-dimensional structures is found to be compared pairwise, so that the three laccase three-dimensional structures are proved to be very similar, and the structural domain recombination is guessed to have less damage to the whole structure. Finally, LCC1, LCC2 and Mrl2 are selected as templates for domain reconstruction.
Wherein the amino acid sequence of LCC1 is shown as SEQ ID No.1, domain I is the first 123 amino acids from the nitrogen end, domain II is 197 amino acids between positions 124-320, and domain III is 178 amino acids between positions 321-498. The amino acid sequence of LCC2 is shown as SEQ ID No.2, domain I is the first 123 amino acids from the nitrogen end, domain II is 197 amino acids between positions 124-320, and domain III is 179 amino acids between positions 321-499. Mrl2 the amino acid sequence of which is shown in SEQ ID No.3, domain I is the first 123 amino acids from the nitrogen terminus, domain II is 195 amino acids between positions 124-318, and domain III is 176 amino acids between positions 319-494.
Performing sequence alignment on the amino acid sequences (SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3) of the LCC1, the LCC2 and the Mrl2 together to obtain an amino acid alignment sequence matrix (figure 1); taking the position between the 123 th and 124 th positions of the amino acid alignment sequence matrix as a recombination site 1, and taking the position between the 321 th and 322 th positions of the amino acid alignment sequence matrix as a recombination site 2; and recombining at least two of the three laccase proteins of the LCC1, the LCC2 and the Mrl through the recombination site 1 and/or the recombination site 2 to obtain a recombined laccase protein.
In the amino acid alignment sequence matrix, for LCC1, LCC2 and Mrl2 shown in SEQ ID No.1 to SEQ ID No.3, the amino acid sequences corresponding to domain I are all located upstream of the recombination site 1, the amino acid sequences corresponding to domain II are all located between the recombination site 1 and the recombination site 2, and the amino acid sequences corresponding to domain III are all located downstream of the recombination site 2.
For the recombinant mutant substitutions, the following nomenclature was used: laccase is divided into three domains, and recombinant ABC represents that domain I is derived from A, domain II is derived from B, and domain 3 is derived from II; l1, L2, M represent domains derived from LCC1, LCC2, mrl2, respectively.
EXAMPLE 2 Gene cloning of laccase
The laccase LCC1 gene (the sequence is shown as SEQ ID No. 4) and LCC2 gene (the sequence is shown as SEQ ID No. 5) which are derived from white rot fungi and laccase Mrl gene (the sequence is shown as SEQ ID No. 6) which are derived from basidiomycetes are artificially synthesized by the company limited Suzhou Gold intelligence biotechnology, then are connected to recombinant plasmids obtained after the enzyme cutting sites XhoI and XbaI of a pPICZ alpha-A vector (New England Biolabs company), are transformed into escherichia coli BL-21Gold (DE 3), positive clones are screened and cultured on LB plates containing bleomycin, plasmids are extracted, and the sequencing determines that the vectors are successfully constructed and are named as pPICZ alpha-A-LCC 1, pPICZ alpha-A-LCC 2 and pPICZ alpha-A-Mrl 2 respectively.
EXAMPLE 3 laccase recombinant variant construction
In order to improve the activity and stability of the laccase protein, the recombinant plasmids pPICZ alpha-A-lcc 1, pPICZ alpha-A-lcc 2 and pPICZ alpha-A-Mrl 2 constructed in example 2 are respectively used as templates for domain recombination, and the laccase has three domains and four fragments in total are added into the vector. In the homologous recombination method, the fewer fragments, the greater the success rate. Therefore, the three-fragment recombination method is designed in the experiment of the invention to improve the recombination success rate. A primer is designed on the vector, the vector is divided into two parts, one part forms a fragment (fragment 1) with the domain I, the other part forms a fragment (fragment 3) with the domain III, and the domain II is a fragment (fragment 2).
And amplifying the three fragments by using the three pairs of primers respectively, and recombining the three fragments to form a complete recombinant plasmid. Because the same fragment on three different genes is amplified by the same pair of primers, the binding position of the primers can be adjusted to a certain extent according to design requirements when the primers are designed. The corresponding upstream primer of fragment 1: 5'-CGTTGCTGGCGTTTTTCCATAG-3' and downstream primers: 5'-CCAGAAAGTACCAGCTTG-3'; the corresponding upstream primer of fragment 2: 5'-CAAGCTGGTACTTTCTGG-3' and downstream primers: 5'-GCSWARTCAACACCACC-3' (S is G or C, W is A or T, R is G or A); the corresponding upstream primer of fragment 3: 5'-GGTGGTGTTGAYTWSGC-3' (Y is T or C, W is A or T, S is G or C) and a downstream primer: 5'-CTATGGAAAAACGCCAGCAACG-3'), 24 recombinant plasmids are finally obtained, and the sequence is correct through sequencing verification.
The recombinant product was electrotransferred to E.coli cells, cultured overnight at 37℃to give recombinant bacteria, and the resulting monoclonal cells were inoculated into 3ml of LB liquid medium (25. Mu.g/ml bleomycin) with sterile toothpicks, respectively, and shake-cultured at 220rpm for 16 hours at 37℃to extract plasmids. Linearizing the plasmid by SacI, electrically converting the linearization product into pichia pastoris competent cells, and culturing at 30 ℃ for 72 hours to obtain recombinant strain monoclonal.
Example 4 expression of laccase proteins and recombinant variants in Pichia pastoris
The grown monoclonals were inoculated with 3ml of YPD medium (25. Mu.g/ml bleomycin) with sterile toothpicks, and after shaking at 220rpm for 20 hours at 30℃they were transferred to 10ml of BMG medium (final OD 0.2) (25. Mu.g/ml bleomycin) at 30℃and after shaking at 220rpm for 20 hours, they were transferred to 50ml of BMM medium (final OD 1.0) (25. Mu.g/ml bleomycin) at 20℃and shaking at 220rpm for 5 days, and 500. Mu.l of methanol was added to the fermentation system each day. After the completion of the culture, the supernatant (laccase expressed as secretion) was collected by centrifugation.
BMG medium: 13.4g/L YNB,10g/L glycerol, 0.0004g/L biotin, balance 100mM potassium phosphate buffer pH 6.0.
BMM medium: 13.4g/L YNB,0.0004g/L biotin, 10mM CuSO 4 8g/L L-alanine, balance 100mM potassium phosphate buffer pH 6.0.
Example 5 wild-type laccase and recombinant variant Property detection
Mu.l of the supernatant obtained in example 4 was mixed with 10. Mu.l of 5 Xloading dye (formula: 1.25ml of 1M Tris-HCl (pH 6.8), SDS 0.5g, bromophenol blue 25mg, glycerol 2.5ml, beta-mercaptoethanol 0.25 ml), treated at 100℃for 10min, and 10. Mu.l of the mixture was loaded, and the protein expression level and the molecular size were detected by SDS-PAGE.
Detecting the activity of the supernatant enzyme: 100. Mu.l of 100mM citrate-phosphate buffer, pH3.0, 60. Mu.l of supernatant, 40. Mu.l of ABTS (10 mM), 30℃were incubated at 30℃for 20min at 420nm for a single 10s detection.
Temperature stability test of recombinants and wild type: 100 μl of the supernatant was incubated at 60deg.C for 10min, and 60 μl was used to test the activity of the remaining enzyme, as described in the previous paragraph.
Organic solvent stability assay for recombinants and wild type: 100 μl of 50% ethanol (dissolved in 100mM citrate-phosphate buffer, pH 3.0), 60 μl of supernatant, 40 μl of ABTS (10 mM), and 30℃were incubated at 30deg.C for 20min at 30deg.C under light absorption of 420nm, and the relative residual enzyme activity was enzyme activity under ethanol/enzyme activity under pure buffer for 10 s.
As shown in FIG. 2, the target protein is about 75kDa in size. As shown in fig. 3, the ratio of the enzyme activity after heat treatment to the enzyme activity without heat treatment is the relative residual enzyme activity, and by comparing the relative residual enzyme activities after heat treatment of each recombinant, the thermal stability of the recombinants L1L2, L2M, L ML2, L1MM is found to be higher than that of the wild-type LCC2; l2MM, L1L2M are more thermostable than wild-type Mrl2; L1L1M is more stable than the closest wild-type LCC1. As shown in FIG. 4, ML2L2 has better ethanol tolerance than the most similar wild-type LCC2; L1L2M, L2L2M, L2ML2, L1L2L2, L1MM have better ethanol tolerance than Mrl2; MML1 is better resistant to ethanol than LCC1. By this method, only 24 recombinant mutants were selected, and 9 mutants with improved stability were obtained. The probability of obtaining dominant mutants by this recombination method (37.5%) is far higher than that of the general mutant library construction methods (epPCR, DNA shuffling, etc.).
Wherein the amino acid sequence of L1L2L2 is shown as SEQ ID No.7, and the coding gene is shown as SEQ ID No. 16. The amino acid sequence of L2L2M is shown as SEQ ID No.8, and the coding gene is shown as SEQ ID No. 17. The amino acid sequence of L2ML2 is shown as SEQ ID No.9, and the coding gene is shown as SEQ ID No. 18. The amino acid sequence of L1MM is shown as SEQ ID No.10, and the encoding gene is shown as SEQ ID No. 19. The amino acid sequence of L2MM is shown as SEQ ID No.11, and the encoding gene is shown as SEQ ID No. 20. The amino acid sequence of L1L2M is shown as SEQ ID No.12, and the coding gene is shown as SEQ ID No. 21. The amino acid sequence of L1L1M is shown as SEQ ID No.13, and the coding gene is shown as SEQ ID No. 22. The amino acid sequence of ML2L2 is shown as SEQ ID No.14, and the coding gene is shown as SEQ ID No. 23. The amino acid sequence of MML1 is shown as SEQ ID No.15, and the encoding gene is shown as SEQ ID No. 24.
EXAMPLE 6 purification of wild-type laccase and recombinant variant proteins
The supernatant collected in example 4 was subjected to protein purification by three steps of anion exchange chromatography, hydrophobic chromatography and molecular sieves. The supernatant was first filtered through a 0.22 μm pore size filter and concentrated to 50ml with QuixStand benchtop system, and the concentrated supernatant was further filtered through a 0.22 μm pore size filter, and then purified by a DEAE FF anion exchange chromatography column. 20 mmol.L by three column volumes -1 The column was washed with pH7.0 potassium phosphate buffer and 20 mmol.L -1 pH7.0 potassium phosphate buffer and 500 mmol.L -1 NaCl (dissolved in pH7.0, 20 mmol.L) -1 Potassium phosphate buffer) and the eluted protein was collected using a 96-well deep-well plate at 2 ml/well. Detecting ABTS enzyme activity of the eluate collected in each well, collecting the active liquid, further purifying with hydrophobic exchange chromatography (Hitrap Butyl FF), and purifying with 20mmol.L -1 pH7.0 potassium phosphate buffer and 2 mol.L -1 Ammonium sulfate (dissolved in pH7.0, 20 mmol.L) -1 Potassium phosphate buffer), the eluted active fraction was further purified by molecular sieve (Superdex 200) using 20 mmol.L at pH7.0 -1 Eluting with potassium phosphate buffer solution to obtain pure enzyme. The protein concentration of the purified enzyme protein was determined using the Braford method using Bovine Serum Albumin (BSA) as a standard. And freeze-drying the purified enzyme solutionThe powder was stored at-80℃until further use.
Example 7 kinetic parameter determination of wild-type laccase and recombinant variant pure enzyme
The pure enzyme obtained in example 6 with the same concentration is added into a reaction system, and substrates with different concentrations are taken for absorbance value detection, so that the initial rate of each reaction is determined. The specific detection method comprises the following steps:
1. with ddH 2 O the purified enzyme protein was diluted to a concentration of 2.5 ng/. Mu.l.
2. ABTS solutions of different concentrations (final concentrations of 0-2mM, respectively) were added as substrates to the above reaction system. 150. Mu.l of 100mM citrate-phosphate buffer, pH3.0, 10. Mu.l of enzyme solution, 40. Mu.l of ABTS (0-10 mM), 30℃were incubated at 30℃for 20min under light absorption at 420 nm.
3. According to the detection result, the initial speed of the enzymatic reaction under different substrate concentrations is calculated, the Km value and the Vmax are calculated, the kcat value is further calculated, and the calculation result is shown in the table 1. The kcat values of both recombinant variants L2L2M, L1MM, L2MM were higher than those of wild type Mrl and LCC2, indicating that the catalytic efficiencies of both recombinants L2L2M, L1MM, L2MM were higher than those of the wild type.
TABLE 1 kinetic parameters of wild-type laccase and recombinant variants
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> laccase recombinant protein and application thereof
<130> GNCLN210328
<160> 24
<170> PatentIn version 3.5
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ggtgataact tccaaatcaa cgttattgat aacttgtcta acgaaaccat gttgaagtct 180
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gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccaa tggttgttta tgatccatct gatccacatg ctgacttgta cgatgttgat 420
gacgaaacca ccattattac cttgtctgat tggtatcata ctgctgcttc attgggtgct 480
gcattcccaa ttggttctga ttctaccttg attaacggtt tgggtagatt tgctggtggt 540
gattctactg atttggctgt tattaccgtt gaacaaggta agagatacag aatgagatta 600
ttgtctttgt cttgtgatcc aaactacgtt ttctctattg atggtcataa catgaccatt 660
attgaagctg atgctgttaa ccatgaacca ttgaccgttg attctattca aatctacgct 720
ggtcaaagat actctttcgt tttgaccgct gatcaagata ttgataacta cttcattaga 780
gctttgccat ctgctggtac tacatctttc gatggtggta ttaactctgc tattttgaga 840
tactctggtg cttctgaagt tgatccaact accactgaaa ctacctctgt tttgcctttg 900
gatgaagcta atttggttcc attggattct ccagctgctc caggtgatcc aaatattggt 960
ggtgttgatt acgctttgaa cttggatttc aacttcgatg gtacaaactt cttcattaac 1020
gatgtttctt tcgtttctcc aaccgttcca gttttgttgc aaattttgtc tggtactacc 1080
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gaaatttctt tcccaattac tgctactaat gcaccaggtg ctcctcatcc atttcacttg 1200
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ccagttagaa gagatgttgt taacaccggt actgctggtg ataacgttac cattagattc 1320
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ggtttcgcta tcgttttgtc tgaagatacc gcagatgttt ctaataccac tactccatct 1440
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<213> Artificial sequence
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ggtgatagat tccaattgaa cgttattgat aacttgaccg atcataccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtccagct 240
ttcatcaatc aatgtccaat ttcttctggt cattctttct tgtacgattt ccaagttcca 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccat tcgttgttta tgatccaaac gatccagctg cagacttgta cgatgttgat 420
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gcttttccat tgggtgctga tgctactttg attaacggta agggtagatc tccatctact 540
accaccgctg atttgacagt tatttctgtt accccaggta agagatacag attcagattg 600
gtttctttgt cttgtgatcc aaaccatacc ttctctattg atggtcataa catgaccatt 660
attgaaaccg attctattaa caccgctcca ttggttgttg attctattca aattttcgct 720
gctcaaagat actctttcgt tttggaagct aatcaagctg ttgataacta ctggattaga 780
gctaatccat ctttcggtaa cgttggtttc actggtggta ttaactctgc tattttgaga 840
tacgatggtg ctgctgctat tgaacctact actactcaaa ctacctctac cgaaccattg 900
aacgaagtta acttgcatcc attggttgat actgttgttc caggttctcc agttgctggt 960
ggtgttgatt tggctattaa catggctttc aacttcaacg gtacaaactt cttcattaac 1020
ggtgcttcat tcactccacc aaccgttcca gttttgttgc aaattatttc tggtgctcaa 1080
aacgctcaag atttgttgcc atctggttct gtttattctt tgccatctaa cgctgatatt 1140
gaaatttctt tccctgctac tgctgctgct cctggtgctc ctcatccatt tcacttgcat 1200
ggtcatgctt ttgctgttgt tagatctgct ggttctaccg tttacaacta cgataaccca 1260
attttcagag atgttgtttc tactggtaca ccagcagccg gtgataacgt taccattaga 1320
ttcagaactg ataaccctgg tccatggttc ttgcattgtc atattgattt ccatttggaa 1380
gctggtttcg cagttgtttt cgctgaagat attccagatg ttgcttctgc taatccagtt 1440
cctcaagcct ggtctgattt gtgtccaact tacgatgcta gagatccatc tgatcaatga 1500
<210> 6
<211> 1485
<212> DNA
<213> Artificial sequence
<400> 6
tctatcggtc caatcgctga cttggttatc tctaaccaag acgtttctcc agacggtttc 60
actagatctg ctgttgttgc tggtggtgac actatcggtc cattgatcgt tggtaacaag 120
aacgacaact tgcaaatcaa cgttgttaac aacttggacg acgacactat gttgcaatct 180
acttctatcc actggcacgg tttcttccaa caatctacta actgggctga cggtactgct 240
ttcgttaacc aatgtccaat cgctaagggt aactctttct tgtacgactt cgacgctact 300
gaccaagctg gtactttctg gtaccactct cacttgtcta ctcaatactg tgacggtttg 360
agaggtccaa tcgttatcta cgacccagac gacccacacg cttctttgta cgacgttgac 420
gacgaatcta ctgttatcac tttggctgac tggtaccaca ctaaggctaa ggaaatcact 480
ttcggtactc cagactctac tttgatcaac ggtttgggta gatggtctca aggtaacgaa 540
actgacttgt ctgttatcac tgttacttct ggtcaaagat acagaatgag attgatcaac 600
actgcttgtg acgctgctta cactttctct atcgacaacc acactatgac tgttatcgaa 660
gctgacgctg ttaacatcga accaatcgaa gttgactctt tgactatcta cgctggtcaa 720
agatactctt tcgttttgaa cgctgaccaa gctgttggta actactggat cagagctaac 780
ccaaacatcg gtactatggg ttacactaac ggtatcaact ctgctatctt gagatacgac 840
actgctgaag aagaagaacc agacgttttg gacatcactt ctactaactc tttgtctgaa 900
gctgacttgg ttccattgga aaacccaggt gctccaggtg acccagttgt tggtggtgtt 960
gactacgctt tgcacttgga cttcgctttc acttctgctg ctactttcac tgttaacgac 1020
gctactttcg ttccaccaac tgttccagtt ttgttgcaaa tcttgtctgg tgctcaaact 1080
gctgacactt tgttgccatc tggttctgtt gttgctttgc catctaactc tactatcgaa 1140
ttgtctatga ctggtggttt gttgggtttg gaacacccaa tccacttgca cggtcacaac 1200
ttcgacgttg ttagagttgc tggttctact gaatacaact acgaaaaccc aatcagaaga 1260
gacgttgtta acgctggttc tacttctgac aacgttacta tcagattcac tactgacaac 1320
ccaggtccat ggatcttgca ctgtcacatc gactggcact tggaagctgg tttcgctatc 1380
gttttcgctg aagctactga cgaatgggtt gacactatcg acccatctga cgcttgggaa 1440
aacttgtgtc caacttacga cgctttgtct gacgacgact tgtaa 1485
<210> 7
<211> 499
<212> PRT
<213> Artificial sequence
<400> 7
Ala Ile Gly Pro Val Thr Asp Leu Thr Ile Ser Asn Ala Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ala Ala Val Leu Ala Asn Gly Val Phe Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Asn Phe Gln Ile Asn Val
35 40 45
Ile Asp Asn Leu Ser Asn Glu Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Ala Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Thr Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Thr Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Phe Val Val Tyr Asp
115 120 125
Pro Asn Asp Pro Ala Ala Asp Leu Tyr Asp Val Asp Asn Asp Asp Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Val Ala Ala Lys Leu Gly Pro
145 150 155 160
Ala Phe Pro Leu Gly Ala Asp Ala Thr Leu Ile Asn Gly Lys Gly Arg
165 170 175
Ser Pro Ser Thr Thr Thr Ala Asp Leu Thr Val Ile Ser Val Thr Pro
180 185 190
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Leu Ser Cys Asp Pro Asn
195 200 205
His Thr Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu Thr Asp
210 215 220
Ser Ile Asn Thr Ala Pro Leu Val Val Asp Ser Ile Gln Ile Phe Ala
225 230 235 240
Ala Gln Arg Tyr Ser Phe Val Leu Glu Ala Asn Gln Ala Val Asp Asn
245 250 255
Tyr Trp Ile Arg Ala Asn Pro Ser Phe Gly Asn Val Gly Phe Thr Gly
260 265 270
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Gly Ala Ala Ala Ile Glu
275 280 285
Pro Thr Thr Thr Gln Thr Thr Ser Thr Glu Pro Leu Asn Glu Val Asn
290 295 300
Leu His Pro Leu Val Asp Thr Val Val Pro Gly Ser Pro Val Ala Gly
305 310 315 320
Gly Val Asp Leu Ala Ile Asn Met Ala Phe Asn Phe Asn Gly Thr Asn
325 330 335
Phe Phe Ile Asn Gly Ala Ser Phe Thr Pro Pro Thr Val Pro Val Leu
340 345 350
Leu Gln Ile Ile Ser Gly Ala Gln Asn Ala Gln Asp Leu Leu Pro Ser
355 360 365
Gly Ser Val Tyr Ser Leu Pro Ser Asn Ala Asp Ile Glu Ile Ser Phe
370 375 380
Pro Ala Thr Ala Ala Ala Pro Gly Ala Pro His Pro Phe His Leu His
385 390 395 400
Gly His Ala Phe Ala Val Val Arg Ser Ala Gly Ser Thr Val Tyr Asn
405 410 415
Tyr Asp Asn Pro Ile Phe Arg Asp Val Val Ser Thr Gly Thr Pro Ala
420 425 430
Ala Gly Asp Asn Val Thr Ile Arg Phe Arg Thr Asp Asn Pro Gly Pro
435 440 445
Trp Phe Leu His Cys His Ile Asp Phe His Leu Glu Ala Gly Phe Ala
450 455 460
Val Val Phe Ala Glu Asp Ile Pro Asp Val Ala Ser Ala Asn Pro Val
465 470 475 480
Pro Gln Ala Trp Ser Asp Leu Cys Pro Thr Tyr Asp Ala Arg Asp Pro
485 490 495
Ser Asp Gln
<210> 8
<211> 496
<212> PRT
<213> Artificial sequence
<400> 8
Gly Ile Gly Pro Val Ala Asp Leu Thr Ile Thr Asn Ala Ala Val Ser
1 5 10 15
Pro Asp Gly Phe Ser Arg Gln Ala Val Val Val Asn Gly Gly Thr Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Met Gly Asp Arg Phe Gln Leu Asn Val
35 40 45
Ile Asp Asn Leu Thr Asp His Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala
65 70 75 80
Phe Ile Asn Gln Cys Pro Ile Ser Ser Gly His Ser Phe Leu Tyr Asp
85 90 95
Phe Gln Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Phe Val Val Tyr Asp
115 120 125
Pro Asn Asp Pro Ala Ala Asp Leu Tyr Asp Val Asp Asn Asp Asp Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Val Ala Ala Lys Leu Gly Pro
145 150 155 160
Ala Phe Pro Leu Gly Ala Asp Ala Thr Leu Ile Asn Gly Lys Gly Arg
165 170 175
Ser Pro Ser Thr Thr Thr Ala Asp Leu Thr Val Ile Ser Val Thr Pro
180 185 190
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Leu Ser Cys Asp Pro Asn
195 200 205
His Thr Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu Thr Asp
210 215 220
Ser Ile Asn Thr Ala Pro Leu Val Val Asp Ser Ile Gln Ile Phe Ala
225 230 235 240
Ala Gln Arg Tyr Ser Phe Val Leu Glu Ala Asn Gln Ala Val Asp Asn
245 250 255
Tyr Trp Ile Arg Ala Asn Pro Ser Phe Gly Asn Val Gly Phe Thr Gly
260 265 270
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Gly Ala Ala Ala Ile Glu
275 280 285
Pro Thr Thr Thr Gln Thr Thr Ser Thr Glu Pro Leu Asn Glu Val Asn
290 295 300
Leu His Pro Leu Val Asp Thr Val Val Pro Gly Ser Pro Val Ala Gly
305 310 315 320
Gly Val Asp Tyr Ala Leu His Leu Asp Phe Ala Phe Thr Ser Ala Ala
325 330 335
Thr Phe Thr Val Asn Asp Ala Thr Phe Val Pro Pro Thr Val Pro Val
340 345 350
Leu Leu Gln Ile Leu Ser Gly Ala Gln Thr Ala Asp Thr Leu Leu Pro
355 360 365
Ser Gly Ser Val Val Ala Leu Pro Ser Asn Ser Thr Ile Glu Leu Ser
370 375 380
Met Thr Gly Gly Leu Leu Gly Leu Glu His Pro Ile His Leu His Gly
385 390 395 400
His Asn Phe Asp Val Val Arg Val Ala Gly Ser Thr Glu Tyr Asn Tyr
405 410 415
Glu Asn Pro Ile Arg Arg Asp Val Val Asn Ala Gly Ser Thr Ser Asp
420 425 430
Asn Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Ile Leu
435 440 445
His Cys His Ile Asp Trp His Leu Glu Ala Gly Phe Ala Ile Val Phe
450 455 460
Ala Glu Ala Thr Asp Glu Trp Val Asp Thr Ile Asp Pro Ser Asp Ala
465 470 475 480
Trp Glu Asn Leu Cys Pro Thr Tyr Asp Ala Leu Ser Asp Asp Asp Leu
485 490 495
<210> 9
<211> 497
<212> PRT
<213> Artificial sequence
<400> 9
Gly Ile Gly Pro Val Ala Asp Leu Thr Ile Thr Asn Ala Ala Val Ser
1 5 10 15
Pro Asp Gly Phe Ser Arg Gln Ala Val Val Val Asn Gly Gly Thr Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Met Gly Asp Arg Phe Gln Leu Asn Val
35 40 45
Ile Asp Asn Leu Thr Asp His Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala
65 70 75 80
Phe Ile Asn Gln Cys Pro Ile Ser Ser Gly His Ser Phe Leu Tyr Asp
85 90 95
Phe Gln Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Ile Val Ile Tyr Asp
115 120 125
Pro Asp Asp Pro His Ala Ser Leu Tyr Asp Val Asp Asp Glu Ser Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Thr Lys Ala Lys Glu Ile Thr
145 150 155 160
Phe Gly Thr Pro Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg Trp Ser
165 170 175
Gln Gly Asn Glu Thr Asp Leu Ser Val Ile Thr Val Thr Ser Gly Gln
180 185 190
Arg Tyr Arg Met Arg Leu Ile Asn Thr Ala Cys Asp Ala Ala Tyr Thr
195 200 205
Phe Ser Ile Asp Asn His Thr Met Thr Val Ile Glu Ala Asp Ala Val
210 215 220
Asn Ile Glu Pro Ile Glu Val Asp Ser Leu Thr Ile Tyr Ala Gly Gln
225 230 235 240
Arg Tyr Ser Phe Val Leu Asn Ala Asp Gln Ala Val Gly Asn Tyr Trp
245 250 255
Ile Arg Ala Asn Pro Asn Ile Gly Thr Met Gly Tyr Thr Asn Gly Ile
260 265 270
Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Glu Glu Glu Glu Pro Asp
275 280 285
Val Leu Asp Ile Thr Ser Thr Asn Ser Leu Ser Glu Ala Asp Leu Val
290 295 300
Pro Leu Glu Asn Pro Gly Ala Pro Gly Asp Pro Val Val Gly Gly Val
305 310 315 320
Asp Leu Ala Ile Asn Met Ala Phe Asn Phe Asn Gly Thr Asn Phe Phe
325 330 335
Ile Asn Gly Ala Ser Phe Thr Pro Pro Thr Val Pro Val Leu Leu Gln
340 345 350
Ile Ile Ser Gly Ala Gln Asn Ala Gln Asp Leu Leu Pro Ser Gly Ser
355 360 365
Val Tyr Ser Leu Pro Ser Asn Ala Asp Ile Glu Ile Ser Phe Pro Ala
370 375 380
Thr Ala Ala Ala Pro Gly Ala Pro His Pro Phe His Leu His Gly His
385 390 395 400
Ala Phe Ala Val Val Arg Ser Ala Gly Ser Thr Val Tyr Asn Tyr Asp
405 410 415
Asn Pro Ile Phe Arg Asp Val Val Ser Thr Gly Thr Pro Ala Ala Gly
420 425 430
Asp Asn Val Thr Ile Arg Phe Arg Thr Asp Asn Pro Gly Pro Trp Phe
435 440 445
Leu His Cys His Ile Asp Phe His Leu Glu Ala Gly Phe Ala Val Val
450 455 460
Phe Ala Glu Asp Ile Pro Asp Val Ala Ser Ala Asn Pro Val Pro Gln
465 470 475 480
Ala Trp Ser Asp Leu Cys Pro Thr Tyr Asp Ala Arg Asp Pro Ser Asp
485 490 495
Gln
<210> 10
<211> 494
<212> PRT
<213> Artificial sequence
<400> 10
Ala Ile Gly Pro Val Thr Asp Leu Thr Ile Ser Asn Ala Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ala Ala Val Leu Ala Asn Gly Val Phe Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Asn Phe Gln Ile Asn Val
35 40 45
Ile Asp Asn Leu Ser Asn Glu Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Ala Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Thr Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Thr Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Ile Val Ile Tyr Asp
115 120 125
Pro Asp Asp Pro His Ala Ser Leu Tyr Asp Val Asp Asp Glu Ser Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Thr Lys Ala Lys Glu Ile Thr
145 150 155 160
Phe Gly Thr Pro Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg Trp Ser
165 170 175
Gln Gly Asn Glu Thr Asp Leu Ser Val Ile Thr Val Thr Ser Gly Gln
180 185 190
Arg Tyr Arg Met Arg Leu Ile Asn Thr Ala Cys Asp Ala Ala Tyr Thr
195 200 205
Phe Ser Ile Asp Asn His Thr Met Thr Val Ile Glu Ala Asp Ala Val
210 215 220
Asn Ile Glu Pro Ile Glu Val Asp Ser Leu Thr Ile Tyr Ala Gly Gln
225 230 235 240
Arg Tyr Ser Phe Val Leu Asn Ala Asp Gln Ala Val Gly Asn Tyr Trp
245 250 255
Ile Arg Ala Asn Pro Asn Ile Gly Thr Met Gly Tyr Thr Asn Gly Ile
260 265 270
Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Glu Glu Glu Glu Pro Asp
275 280 285
Val Leu Asp Ile Thr Ser Thr Asn Ser Leu Ser Glu Ala Asp Leu Val
290 295 300
Pro Leu Glu Asn Pro Gly Ala Pro Gly Asp Pro Val Val Gly Gly Val
305 310 315 320
Asp Tyr Ala Leu His Leu Asp Phe Ala Phe Thr Ser Ala Ala Thr Phe
325 330 335
Thr Val Asn Asp Ala Thr Phe Val Pro Pro Thr Val Pro Val Leu Leu
340 345 350
Gln Ile Leu Ser Gly Ala Gln Thr Ala Asp Thr Leu Leu Pro Ser Gly
355 360 365
Ser Val Val Ala Leu Pro Ser Asn Ser Thr Ile Glu Leu Ser Met Thr
370 375 380
Gly Gly Leu Leu Gly Leu Glu His Pro Ile His Leu His Gly His Asn
385 390 395 400
Phe Asp Val Val Arg Val Ala Gly Ser Thr Glu Tyr Asn Tyr Glu Asn
405 410 415
Pro Ile Arg Arg Asp Val Val Asn Ala Gly Ser Thr Ser Asp Asn Val
420 425 430
Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Ile Leu His Cys
435 440 445
His Ile Asp Trp His Leu Glu Ala Gly Phe Ala Ile Val Phe Ala Glu
450 455 460
Ala Thr Asp Glu Trp Val Asp Thr Ile Asp Pro Ser Asp Ala Trp Glu
465 470 475 480
Asn Leu Cys Pro Thr Tyr Asp Ala Leu Ser Asp Asp Asp Leu
485 490
<210> 11
<211> 494
<212> PRT
<213> Artificial sequence
<400> 11
Gly Ile Gly Pro Val Ala Asp Leu Thr Ile Thr Asn Ala Ala Val Ser
1 5 10 15
Pro Asp Gly Phe Ser Arg Gln Ala Val Val Val Asn Gly Gly Thr Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Met Gly Asp Arg Phe Gln Leu Asn Val
35 40 45
Ile Asp Asn Leu Thr Asp His Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Pro Ala
65 70 75 80
Phe Ile Asn Gln Cys Pro Ile Ser Ser Gly His Ser Phe Leu Tyr Asp
85 90 95
Phe Gln Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Ile Val Ile Tyr Asp
115 120 125
Pro Asp Asp Pro His Ala Ser Leu Tyr Asp Val Asp Asp Glu Ser Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Thr Lys Ala Lys Glu Ile Thr
145 150 155 160
Phe Gly Thr Pro Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg Trp Ser
165 170 175
Gln Gly Asn Glu Thr Asp Leu Ser Val Ile Thr Val Thr Ser Gly Gln
180 185 190
Arg Tyr Arg Met Arg Leu Ile Asn Thr Ala Cys Asp Ala Ala Tyr Thr
195 200 205
Phe Ser Ile Asp Asn His Thr Met Thr Val Ile Glu Ala Asp Ala Val
210 215 220
Asn Ile Glu Pro Ile Glu Val Asp Ser Leu Thr Ile Tyr Ala Gly Gln
225 230 235 240
Arg Tyr Ser Phe Val Leu Asn Ala Asp Gln Ala Val Gly Asn Tyr Trp
245 250 255
Ile Arg Ala Asn Pro Asn Ile Gly Thr Met Gly Tyr Thr Asn Gly Ile
260 265 270
Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Glu Glu Glu Glu Pro Asp
275 280 285
Val Leu Asp Ile Thr Ser Thr Asn Ser Leu Ser Glu Ala Asp Leu Val
290 295 300
Pro Leu Glu Asn Pro Gly Ala Pro Gly Asp Pro Val Val Gly Gly Val
305 310 315 320
Asp Tyr Ala Leu His Leu Asp Phe Ala Phe Thr Ser Ala Ala Thr Phe
325 330 335
Thr Val Asn Asp Ala Thr Phe Val Pro Pro Thr Val Pro Val Leu Leu
340 345 350
Gln Ile Leu Ser Gly Ala Gln Thr Ala Asp Thr Leu Leu Pro Ser Gly
355 360 365
Ser Val Val Ala Leu Pro Ser Asn Ser Thr Ile Glu Leu Ser Met Thr
370 375 380
Gly Gly Leu Leu Gly Leu Glu His Pro Ile His Leu His Gly His Asn
385 390 395 400
Phe Asp Val Val Arg Val Ala Gly Ser Thr Glu Tyr Asn Tyr Glu Asn
405 410 415
Pro Ile Arg Arg Asp Val Val Asn Ala Gly Ser Thr Ser Asp Asn Val
420 425 430
Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Ile Leu His Cys
435 440 445
His Ile Asp Trp His Leu Glu Ala Gly Phe Ala Ile Val Phe Ala Glu
450 455 460
Ala Thr Asp Glu Trp Val Asp Thr Ile Asp Pro Ser Asp Ala Trp Glu
465 470 475 480
Asn Leu Cys Pro Thr Tyr Asp Ala Leu Ser Asp Asp Asp Leu
485 490
<210> 12
<211> 496
<212> PRT
<213> Artificial sequence
<400> 12
Ala Ile Gly Pro Val Thr Asp Leu Thr Ile Ser Asn Ala Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ala Ala Val Leu Ala Asn Gly Val Phe Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Asn Phe Gln Ile Asn Val
35 40 45
Ile Asp Asn Leu Ser Asn Glu Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Ala Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Thr Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Thr Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Phe Val Val Tyr Asp
115 120 125
Pro Asn Asp Pro Ala Ala Asp Leu Tyr Asp Val Asp Asn Asp Asp Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Val Ala Ala Lys Leu Gly Pro
145 150 155 160
Ala Phe Pro Leu Gly Ala Asp Ala Thr Leu Ile Asn Gly Lys Gly Arg
165 170 175
Ser Pro Ser Thr Thr Thr Ala Asp Leu Thr Val Ile Ser Val Thr Pro
180 185 190
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Leu Ser Cys Asp Pro Asn
195 200 205
His Thr Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu Thr Asp
210 215 220
Ser Ile Asn Thr Ala Pro Leu Val Val Asp Ser Ile Gln Ile Phe Ala
225 230 235 240
Ala Gln Arg Tyr Ser Phe Val Leu Glu Ala Asn Gln Ala Val Asp Asn
245 250 255
Tyr Trp Ile Arg Ala Asn Pro Ser Phe Gly Asn Val Gly Phe Thr Gly
260 265 270
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Gly Ala Ala Ala Ile Glu
275 280 285
Pro Thr Thr Thr Gln Thr Thr Ser Thr Glu Pro Leu Asn Glu Val Asn
290 295 300
Leu His Pro Leu Val Asp Thr Val Val Pro Gly Ser Pro Val Ala Gly
305 310 315 320
Gly Val Asp Tyr Ala Leu His Leu Asp Phe Ala Phe Thr Ser Ala Ala
325 330 335
Thr Phe Thr Val Asn Asp Ala Thr Phe Val Pro Pro Thr Val Pro Val
340 345 350
Leu Leu Gln Ile Leu Ser Gly Ala Gln Thr Ala Asp Thr Leu Leu Pro
355 360 365
Ser Gly Ser Val Val Ala Leu Pro Ser Asn Ser Thr Ile Glu Leu Ser
370 375 380
Met Thr Gly Gly Leu Leu Gly Leu Glu His Pro Ile His Leu His Gly
385 390 395 400
His Asn Phe Asp Val Val Arg Val Ala Gly Ser Thr Glu Tyr Asn Tyr
405 410 415
Glu Asn Pro Ile Arg Arg Asp Val Val Asn Ala Gly Ser Thr Ser Asp
420 425 430
Asn Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Ile Leu
435 440 445
His Cys His Ile Asp Trp His Leu Glu Ala Gly Phe Ala Ile Val Phe
450 455 460
Ala Glu Ala Thr Asp Glu Trp Val Asp Thr Ile Asp Pro Ser Asp Ala
465 470 475 480
Trp Glu Asn Leu Cys Pro Thr Tyr Asp Ala Leu Ser Asp Asp Asp Leu
485 490 495
<210> 13
<211> 496
<212> PRT
<213> Artificial sequence
<400> 13
Ala Ile Gly Pro Val Thr Asp Leu Thr Ile Ser Asn Ala Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ala Ala Val Leu Ala Asn Gly Val Phe Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Asn Phe Gln Ile Asn Val
35 40 45
Ile Asp Asn Leu Ser Asn Glu Thr Met Leu Lys Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Lys Gly Thr Asn Trp Ala Asp Gly Ala Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Thr Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Thr Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Met Val Val Tyr Asp
115 120 125
Pro Ser Asp Pro His Ala Asp Leu Tyr Asp Val Asp Asp Glu Thr Thr
130 135 140
Ile Ile Thr Leu Ser Asp Trp Tyr His Thr Ala Ala Ser Leu Gly Ala
145 150 155 160
Ala Phe Pro Ile Gly Ser Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg
165 170 175
Phe Ala Gly Gly Asp Ser Thr Asp Leu Ala Val Ile Thr Val Glu Gln
180 185 190
Gly Lys Arg Tyr Arg Met Arg Leu Leu Ser Leu Ser Cys Asp Pro Asn
195 200 205
Tyr Val Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu Ala Asp
210 215 220
Ala Val Asn His Glu Pro Leu Thr Val Asp Ser Ile Gln Ile Tyr Ala
225 230 235 240
Gly Gln Arg Tyr Ser Phe Val Leu Thr Ala Asp Gln Asp Ile Asp Asn
245 250 255
Tyr Phe Ile Arg Ala Leu Pro Ser Ala Gly Thr Thr Ser Phe Asp Gly
260 265 270
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Ser Gly Ala Ser Glu Val Asp
275 280 285
Pro Thr Thr Thr Glu Thr Thr Ser Val Leu Pro Leu Asp Glu Ala Asn
290 295 300
Leu Val Pro Leu Asp Ser Pro Ala Ala Pro Gly Asp Pro Asn Ile Gly
305 310 315 320
Gly Val Asp Tyr Ala Leu His Leu Asp Phe Ala Phe Thr Ser Ala Ala
325 330 335
Thr Phe Thr Val Asn Asp Ala Thr Phe Val Pro Pro Thr Val Pro Val
340 345 350
Leu Leu Gln Ile Leu Ser Gly Ala Gln Thr Ala Asp Thr Leu Leu Pro
355 360 365
Ser Gly Ser Val Val Ala Leu Pro Ser Asn Ser Thr Ile Glu Leu Ser
370 375 380
Met Thr Gly Gly Leu Leu Gly Leu Glu His Pro Ile His Leu His Gly
385 390 395 400
His Asn Phe Asp Val Val Arg Val Ala Gly Ser Thr Glu Tyr Asn Tyr
405 410 415
Glu Asn Pro Ile Arg Arg Asp Val Val Asn Ala Gly Ser Thr Ser Asp
420 425 430
Asn Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Ile Leu
435 440 445
His Cys His Ile Asp Trp His Leu Glu Ala Gly Phe Ala Ile Val Phe
450 455 460
Ala Glu Ala Thr Asp Glu Trp Val Asp Thr Ile Asp Pro Ser Asp Ala
465 470 475 480
Trp Glu Asn Leu Cys Pro Thr Tyr Asp Ala Leu Ser Asp Asp Asp Leu
485 490 495
<210> 14
<211> 499
<212> PRT
<213> Artificial sequence
<400> 14
Ser Ile Gly Pro Ile Ala Asp Leu Val Ile Ser Asn Gln Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ser Ala Val Val Ala Gly Gly Asp Thr Ile
20 25 30
Gly Pro Leu Ile Val Gly Asn Lys Asn Asp Asn Leu Gln Ile Asn Val
35 40 45
Val Asn Asn Leu Asp Asp Asp Thr Met Leu Gln Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Gln Ser Thr Asn Trp Ala Asp Gly Thr Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Lys Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Asp Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Phe Val Val Tyr Asp
115 120 125
Pro Asn Asp Pro Ala Ala Asp Leu Tyr Asp Val Asp Asn Asp Asp Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Val Ala Ala Lys Leu Gly Pro
145 150 155 160
Ala Phe Pro Leu Gly Ala Asp Ala Thr Leu Ile Asn Gly Lys Gly Arg
165 170 175
Ser Pro Ser Thr Thr Thr Ala Asp Leu Thr Val Ile Ser Val Thr Pro
180 185 190
Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Leu Ser Cys Asp Pro Asn
195 200 205
His Thr Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu Thr Asp
210 215 220
Ser Ile Asn Thr Ala Pro Leu Val Val Asp Ser Ile Gln Ile Phe Ala
225 230 235 240
Ala Gln Arg Tyr Ser Phe Val Leu Glu Ala Asn Gln Ala Val Asp Asn
245 250 255
Tyr Trp Ile Arg Ala Asn Pro Ser Phe Gly Asn Val Gly Phe Thr Gly
260 265 270
Gly Ile Asn Ser Ala Ile Leu Arg Tyr Asp Gly Ala Ala Ala Ile Glu
275 280 285
Pro Thr Thr Thr Gln Thr Thr Ser Thr Glu Pro Leu Asn Glu Val Asn
290 295 300
Leu His Pro Leu Val Asp Thr Val Val Pro Gly Ser Pro Val Ala Gly
305 310 315 320
Gly Val Asp Leu Ala Ile Asn Met Ala Phe Asn Phe Asn Gly Thr Asn
325 330 335
Phe Phe Ile Asn Gly Ala Ser Phe Thr Pro Pro Thr Val Pro Val Leu
340 345 350
Leu Gln Ile Ile Ser Gly Ala Gln Asn Ala Gln Asp Leu Leu Pro Ser
355 360 365
Gly Ser Val Tyr Ser Leu Pro Ser Asn Ala Asp Ile Glu Ile Ser Phe
370 375 380
Pro Ala Thr Ala Ala Ala Pro Gly Ala Pro His Pro Phe His Leu His
385 390 395 400
Gly His Ala Phe Ala Val Val Arg Ser Ala Gly Ser Thr Val Tyr Asn
405 410 415
Tyr Asp Asn Pro Ile Phe Arg Asp Val Val Ser Thr Gly Thr Pro Ala
420 425 430
Ala Gly Asp Asn Val Thr Ile Arg Phe Arg Thr Asp Asn Pro Gly Pro
435 440 445
Trp Phe Leu His Cys His Ile Asp Phe His Leu Glu Ala Gly Phe Ala
450 455 460
Val Val Phe Ala Glu Asp Ile Pro Asp Val Ala Ser Ala Asn Pro Val
465 470 475 480
Pro Gln Ala Trp Ser Asp Leu Cys Pro Thr Tyr Asp Ala Arg Asp Pro
485 490 495
Ser Asp Gln
<210> 15
<211> 496
<212> PRT
<213> Artificial sequence
<400> 15
Ser Ile Gly Pro Ile Ala Asp Leu Val Ile Ser Asn Gln Asp Val Ser
1 5 10 15
Pro Asp Gly Phe Thr Arg Ser Ala Val Val Ala Gly Gly Asp Thr Ile
20 25 30
Gly Pro Leu Ile Val Gly Asn Lys Asn Asp Asn Leu Gln Ile Asn Val
35 40 45
Val Asn Asn Leu Asp Asp Asp Thr Met Leu Gln Ser Thr Ser Ile His
50 55 60
Trp His Gly Phe Phe Gln Gln Ser Thr Asn Trp Ala Asp Gly Thr Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Lys Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Asp Ala Thr Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Ile Val Ile Tyr Asp
115 120 125
Pro Asp Asp Pro His Ala Ser Leu Tyr Asp Val Asp Asp Glu Ser Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Thr Lys Ala Lys Glu Ile Thr
145 150 155 160
Phe Gly Thr Pro Asp Ser Thr Leu Ile Asn Gly Leu Gly Arg Trp Ser
165 170 175
Gln Gly Asn Glu Thr Asp Leu Ser Val Ile Thr Val Thr Ser Gly Gln
180 185 190
Arg Tyr Arg Met Arg Leu Ile Asn Thr Ala Cys Asp Ala Ala Tyr Thr
195 200 205
Phe Ser Ile Asp Asn His Thr Met Thr Val Ile Glu Ala Asp Ala Val
210 215 220
Asn Ile Glu Pro Ile Glu Val Asp Ser Leu Thr Ile Tyr Ala Gly Gln
225 230 235 240
Arg Tyr Ser Phe Val Leu Asn Ala Asp Gln Ala Val Gly Asn Tyr Trp
245 250 255
Ile Arg Ala Asn Pro Asn Ile Gly Thr Met Gly Tyr Thr Asn Gly Ile
260 265 270
Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Glu Glu Glu Glu Pro Asp
275 280 285
Val Leu Asp Ile Thr Ser Thr Asn Ser Leu Ser Glu Ala Asp Leu Val
290 295 300
Pro Leu Glu Asn Pro Gly Ala Pro Gly Asp Pro Val Val Gly Gly Val
305 310 315 320
Asp Tyr Ala Leu Asn Leu Asp Phe Asn Phe Asp Gly Thr Asn Phe Phe
325 330 335
Ile Asn Asp Val Ser Phe Val Ser Pro Thr Val Pro Val Leu Leu Gln
340 345 350
Ile Leu Ser Gly Thr Thr Ser Ala Ala Asp Leu Leu Pro Ser Gly Ser
355 360 365
Leu Phe Ala Leu Pro Ser Asp Ser Thr Ile Glu Ile Ser Phe Pro Ile
370 375 380
Thr Ala Thr Asn Ala Pro Gly Ala Pro His Pro Phe His Leu His Gly
385 390 395 400
His Thr Phe Ser Ile Val Arg Thr Ala Gly Ser Thr Asp Thr Asn Phe
405 410 415
Val Asn Pro Val Arg Arg Asp Val Val Asn Thr Gly Thr Ala Gly Asp
420 425 430
Asn Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Phe Leu
435 440 445
His Cys His Ile Asp Phe His Leu Glu Ala Gly Phe Ala Ile Val Leu
450 455 460
Ser Glu Asp Thr Ala Asp Val Ser Asn Thr Thr Thr Pro Ser Thr Ala
465 470 475 480
Trp Glu Asp Leu Cys Pro Thr Tyr Asn Ala Leu Asp Ser Ser Asp Leu
485 490 495
<210> 16
<211> 1500
<212> DNA
<213> Artificial sequence
<400> 16
gcaattggtc cagttaccga tttgaccatt tctaacgctg atgtttctcc agatggtttt 60
actagagctg ctgttttggc taatggtgtt ttcccaggtc cattgattac tggtaacaag 120
ggtgataact tccaaatcaa cgttattgat aacttgtcta acgaaaccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtgctgct 240
tttgttaatc aatgtccaat tgctaccggt aactctttct tgtacgattt cactgctact 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccat tcgttgttta tgatccaaac gatccagctg cagacttgta cgatgttgat 420
aacgatgata ccgttattac cttggctgat tggtatcatg ttgctgctaa attgggtcca 480
gcttttccat tgggtgctga tgctactttg attaacggta agggtagatc tccatctact 540
accaccgctg atttgacagt tatttctgtt accccaggta agagatacag attcagattg 600
gtttctttgt cttgtgatcc aaaccatacc ttctctattg atggtcataa catgaccatt 660
attgaaaccg attctattaa caccgctcca ttggttgttg attctattca aattttcgct 720
gctcaaagat actctttcgt tttggaagct aatcaagctg ttgataacta ctggattaga 780
gctaatccat ctttcggtaa cgttggtttc actggtggta ttaactctgc tattttgaga 840
tacgatggtg ctgctgctat tgaacctact actactcaaa ctacctctac cgaaccattg 900
aacgaagtta acttgcatcc attggttgat actgttgttc caggttctcc agttgctggt 960
ggtgttgatt tggctattaa catggctttc aacttcaacg gtacaaactt cttcattaac 1020
ggtgcttcat tcactccacc aaccgttcca gttttgttgc aaattatttc tggtgctcaa 1080
aacgctcaag atttgttgcc atctggttct gtttattctt tgccatctaa cgctgatatt 1140
gaaatttctt tccctgctac tgctgctgct cctggtgctc ctcatccatt tcacttgcat 1200
ggtcatgctt ttgctgttgt tagatctgct ggttctaccg tttacaacta cgataaccca 1260
attttcagag atgttgtttc tactggtaca ccagcagccg gtgataacgt taccattaga 1320
ttcagaactg ataaccctgg tccatggttc ttgcattgtc atattgattt ccatttggaa 1380
gctggtttcg cagttgtttt cgctgaagat attccagatg ttgcttctgc taatccagtt 1440
cctcaagcct ggtctgattt gtgtccaact tacgatgcta gagatccatc tgatcaatga 1500
<210> 17
<211> 1491
<212> DNA
<213> Artificial sequence
<400> 17
ggtattggtc cagttgctga tttgactatt accaacgctg ctgtttctcc agatggtttc 60
tcaagacaag ctgttgttgt taatggtggt actccaggtc cattgattac tggtaacatg 120
ggtgatagat tccaattgaa cgttattgat aacttgaccg atcataccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtccagct 240
ttcatcaatc aatgtccaat ttcttctggt cattctttct tgtacgattt ccaagttcca 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccat tcgttgttta tgatccaaac gatccagctg cagacttgta cgatgttgat 420
aacgatgata ccgttattac cttggctgat tggtatcatg ttgctgctaa attgggtcca 480
gcttttccat tgggtgctga tgctactttg attaacggta agggtagatc tccatctact 540
accaccgctg atttgacagt tatttctgtt accccaggta agagatacag attcagattg 600
gtttctttgt cttgtgatcc aaaccatacc ttctctattg atggtcataa catgaccatt 660
attgaaaccg attctattaa caccgctcca ttggttgttg attctattca aattttcgct 720
gctcaaagat actctttcgt tttggaagct aatcaagctg ttgataacta ctggattaga 780
gctaatccat ctttcggtaa cgttggtttc actggtggta ttaactctgc tattttgaga 840
tacgatggtg ctgctgctat tgaacctact actactcaaa ctacctctac cgaaccattg 900
aacgaagtta acttgcatcc attggttgat actgttgttc caggttctcc agttgctggt 960
ggtgttgact acgctttgca cttggacttc gctttcactt ctgctgctac tttcactgtt 1020
aacgacgcta ctttcgttcc accaactgtt ccagttttgt tgcaaatctt gtctggtgct 1080
caaactgctg acactttgtt gccatctggt tctgttgttg ctttgccatc taactctact 1140
atcgaattgt ctatgactgg tggtttgttg ggtttggaac acccaatcca cttgcacggt 1200
cacaacttcg acgttgttag agttgctggt tctactgaat acaactacga aaacccaatc 1260
agaagagacg ttgttaacgc tggttctact tctgacaacg ttactatcag attcactact 1320
gacaacccag gtccatggat cttgcactgt cacatcgact ggcacttgga agctggtttc 1380
gctatcgttt tcgctgaagc tactgacgaa tgggttgaca ctatcgaccc atctgacgct 1440
tgggaaaact tgtgtccaac ttacgacgct ttgtctgacg acgacttgta a 1491
<210> 18
<211> 1494
<212> DNA
<213> Artificial sequence
<400> 18
ggtattggtc cagttgctga tttgactatt accaacgctg ctgtttctcc agatggtttc 60
tcaagacaag ctgttgttgt taatggtggt actccaggtc cattgattac tggtaacatg 120
ggtgatagat tccaattgaa cgttattgat aacttgaccg atcataccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtccagct 240
ttcatcaatc aatgtccaat ttcttctggt cattctttct tgtacgattt ccaagttcca 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccaa tcgttatcta cgacccagac gacccacacg cttctttgta cgacgttgac 420
gacgaatcta ctgttatcac tttggctgac tggtaccaca ctaaggctaa ggaaatcact 480
ttcggtactc cagactctac tttgatcaac ggtttgggta gatggtctca aggtaacgaa 540
actgacttgt ctgttatcac tgttacttct ggtcaaagat acagaatgag attgatcaac 600
actgcttgtg acgctgctta cactttctct atcgacaacc acactatgac tgttatcgaa 660
gctgacgctg ttaacatcga accaatcgaa gttgactctt tgactatcta cgctggtcaa 720
agatactctt tcgttttgaa cgctgaccaa gctgttggta actactggat cagagctaac 780
ccaaacatcg gtactatggg ttacactaac ggtatcaact ctgctatctt gagatacgac 840
actgctgaag aagaagaacc agacgttttg gacatcactt ctactaactc tttgtctgaa 900
gctgacttgg ttccattgga aaacccaggt gctccaggtg acccagttgt tggtggtgtt 960
gatttggcta ttaacatggc tttcaacttc aacggtacaa acttcttcat taacggtgct 1020
tcattcactc caccaaccgt tccagttttg ttgcaaatta tttctggtgc tcaaaacgct 1080
caagatttgt tgccatctgg ttctgtttat tctttgccat ctaacgctga tattgaaatt 1140
tctttccctg ctactgctgc tgctcctggt gctcctcatc catttcactt gcatggtcat 1200
gcttttgctg ttgttagatc tgctggttct accgtttaca actacgataa cccaattttc 1260
agagatgttg tttctactgg tacaccagca gccggtgata acgttaccat tagattcaga 1320
actgataacc ctggtccatg gttcttgcat tgtcatattg atttccattt ggaagctggt 1380
ttcgcagttg ttttcgctga agatattcca gatgttgctt ctgctaatcc agttcctcaa 1440
gcctggtctg atttgtgtcc aacttacgat gctagagatc catctgatca atga 1494
<210> 19
<211> 1485
<212> DNA
<213> Artificial sequence
<400> 19
gcaattggtc cagttaccga tttgaccatt tctaacgctg atgtttctcc agatggtttt 60
actagagctg ctgttttggc taatggtgtt ttcccaggtc cattgattac tggtaacaag 120
ggtgataact tccaaatcaa cgttattgat aacttgtcta acgaaaccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtgctgct 240
tttgttaatc aatgtccaat tgctaccggt aactctttct tgtacgattt cactgctact 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccaa tcgttatcta cgacccagac gacccacacg cttctttgta cgacgttgac 420
gacgaatcta ctgttatcac tttggctgac tggtaccaca ctaaggctaa ggaaatcact 480
ttcggtactc cagactctac tttgatcaac ggtttgggta gatggtctca aggtaacgaa 540
actgacttgt ctgttatcac tgttacttct ggtcaaagat acagaatgag attgatcaac 600
actgcttgtg acgctgctta cactttctct atcgacaacc acactatgac tgttatcgaa 660
gctgacgctg ttaacatcga accaatcgaa gttgactctt tgactatcta cgctggtcaa 720
agatactctt tcgttttgaa cgctgaccaa gctgttggta actactggat cagagctaac 780
ccaaacatcg gtactatggg ttacactaac ggtatcaact ctgctatctt gagatacgac 840
actgctgaag aagaagaacc agacgttttg gacatcactt ctactaactc tttgtctgaa 900
gctgacttgg ttccattgga aaacccaggt gctccaggtg acccagttgt tggtggtgtt 960
gactacgctt tgcacttgga cttcgctttc acttctgctg ctactttcac tgttaacgac 1020
gctactttcg ttccaccaac tgttccagtt ttgttgcaaa tcttgtctgg tgctcaaact 1080
gctgacactt tgttgccatc tggttctgtt gttgctttgc catctaactc tactatcgaa 1140
ttgtctatga ctggtggttt gttgggtttg gaacacccaa tccacttgca cggtcacaac 1200
ttcgacgttg ttagagttgc tggttctact gaatacaact acgaaaaccc aatcagaaga 1260
gacgttgtta acgctggttc tacttctgac aacgttacta tcagattcac tactgacaac 1320
ccaggtccat ggatcttgca ctgtcacatc gactggcact tggaagctgg tttcgctatc 1380
gttttcgctg aagctactga cgaatgggtt gacactatcg acccatctga cgcttgggaa 1440
aacttgtgtc caacttacga cgctttgtct gacgacgact tgtaa 1485
<210> 20
<211> 1485
<212> DNA
<213> Artificial sequence
<400> 20
ggtattggtc cagttgctga tttgactatt accaacgctg ctgtttctcc agatggtttc 60
tcaagacaag ctgttgttgt taatggtggt actccaggtc cattgattac tggtaacatg 120
ggtgatagat tccaattgaa cgttattgat aacttgaccg atcataccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtccagct 240
ttcatcaatc aatgtccaat ttcttctggt cattctttct tgtacgattt ccaagttcca 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccaa tcgttatcta cgacccagac gacccacacg cttctttgta cgacgttgac 420
gacgaatcta ctgttatcac tttggctgac tggtaccaca ctaaggctaa ggaaatcact 480
ttcggtactc cagactctac tttgatcaac ggtttgggta gatggtctca aggtaacgaa 540
actgacttgt ctgttatcac tgttacttct ggtcaaagat acagaatgag attgatcaac 600
actgcttgtg acgctgctta cactttctct atcgacaacc acactatgac tgttatcgaa 660
gctgacgctg ttaacatcga accaatcgaa gttgactctt tgactatcta cgctggtcaa 720
agatactctt tcgttttgaa cgctgaccaa gctgttggta actactggat cagagctaac 780
ccaaacatcg gtactatggg ttacactaac ggtatcaact ctgctatctt gagatacgac 840
actgctgaag aagaagaacc agacgttttg gacatcactt ctactaactc tttgtctgaa 900
gctgacttgg ttccattgga aaacccaggt gctccaggtg acccagttgt tggtggtgtt 960
gactacgctt tgcacttgga cttcgctttc acttctgctg ctactttcac tgttaacgac 1020
gctactttcg ttccaccaac tgttccagtt ttgttgcaaa tcttgtctgg tgctcaaact 1080
gctgacactt tgttgccatc tggttctgtt gttgctttgc catctaactc tactatcgaa 1140
ttgtctatga ctggtggttt gttgggtttg gaacacccaa tccacttgca cggtcacaac 1200
ttcgacgttg ttagagttgc tggttctact gaatacaact acgaaaaccc aatcagaaga 1260
gacgttgtta acgctggttc tacttctgac aacgttacta tcagattcac tactgacaac 1320
ccaggtccat ggatcttgca ctgtcacatc gactggcact tggaagctgg tttcgctatc 1380
gttttcgctg aagctactga cgaatgggtt gacactatcg acccatctga cgcttgggaa 1440
aacttgtgtc caacttacga cgctttgtct gacgacgact tgtaa 1485
<210> 21
<211> 1491
<212> DNA
<213> Artificial sequence
<400> 21
gcaattggtc cagttaccga tttgaccatt tctaacgctg atgtttctcc agatggtttt 60
actagagctg ctgttttggc taatggtgtt ttcccaggtc cattgattac tggtaacaag 120
ggtgataact tccaaatcaa cgttattgat aacttgtcta acgaaaccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtgctgct 240
tttgttaatc aatgtccaat tgctaccggt aactctttct tgtacgattt cactgctact 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccat tcgttgttta tgatccaaac gatccagctg cagacttgta cgatgttgat 420
aacgatgata ccgttattac cttggctgat tggtatcatg ttgctgctaa attgggtcca 480
gcttttccat tgggtgctga tgctactttg attaacggta agggtagatc tccatctact 540
accaccgctg atttgacagt tatttctgtt accccaggta agagatacag attcagattg 600
gtttctttgt cttgtgatcc aaaccatacc ttctctattg atggtcataa catgaccatt 660
attgaaaccg attctattaa caccgctcca ttggttgttg attctattca aattttcgct 720
gctcaaagat actctttcgt tttggaagct aatcaagctg ttgataacta ctggattaga 780
gctaatccat ctttcggtaa cgttggtttc actggtggta ttaactctgc tattttgaga 840
tacgatggtg ctgctgctat tgaacctact actactcaaa ctacctctac cgaaccattg 900
aacgaagtta acttgcatcc attggttgat actgttgttc caggttctcc agttgctggt 960
ggtgttgact acgctttgca cttggacttc gctttcactt ctgctgctac tttcactgtt 1020
aacgacgcta ctttcgttcc accaactgtt ccagttttgt tgcaaatctt gtctggtgct 1080
caaactgctg acactttgtt gccatctggt tctgttgttg ctttgccatc taactctact 1140
atcgaattgt ctatgactgg tggtttgttg ggtttggaac acccaatcca cttgcacggt 1200
cacaacttcg acgttgttag agttgctggt tctactgaat acaactacga aaacccaatc 1260
agaagagacg ttgttaacgc tggttctact tctgacaacg ttactatcag attcactact 1320
gacaacccag gtccatggat cttgcactgt cacatcgact ggcacttgga agctggtttc 1380
gctatcgttt tcgctgaagc tactgacgaa tgggttgaca ctatcgaccc atctgacgct 1440
tgggaaaact tgtgtccaac ttacgacgct ttgtctgacg acgacttgta a 1491
<210> 22
<211> 1491
<212> DNA
<213> Artificial sequence
<400> 22
gcaattggtc cagttaccga tttgaccatt tctaacgctg atgtttctcc agatggtttt 60
actagagctg ctgttttggc taatggtgtt ttcccaggtc cattgattac tggtaacaag 120
ggtgataact tccaaatcaa cgttattgat aacttgtcta acgaaaccat gttgaagtct 180
acctctattc attggcatgg tttcttccaa aaaggtacta attgggctga tggtgctgct 240
tttgttaatc aatgtccaat tgctaccggt aactctttct tgtacgattt cactgctact 300
gatcaagctg gtactttctg gtatcattct catttgtcta cccaatactg tgatggtttg 360
agaggtccaa tggttgttta tgatccatct gatccacatg ctgacttgta cgatgttgat 420
gacgaaacca ccattattac cttgtctgat tggtatcata ctgctgcttc attgggtgct 480
gcattcccaa ttggttctga ttctaccttg attaacggtt tgggtagatt tgctggtggt 540
gattctactg atttggctgt tattaccgtt gaacaaggta agagatacag aatgagatta 600
ttgtctttgt cttgtgatcc aaactacgtt ttctctattg atggtcataa catgaccatt 660
attgaagctg atgctgttaa ccatgaacca ttgaccgttg attctattca aatctacgct 720
ggtcaaagat actctttcgt tttgaccgct gatcaagata ttgataacta cttcattaga 780
gctttgccat ctgctggtac tacatctttc gatggtggta ttaactctgc tattttgaga 840
tactctggtg cttctgaagt tgatccaact accactgaaa ctacctctgt tttgcctttg 900
gatgaagcta atttggttcc attggattct ccagctgctc caggtgatcc aaatattggt 960
ggtgttgact acgctttgca cttggacttc gctttcactt ctgctgctac tttcactgtt 1020
aacgacgcta ctttcgttcc accaactgtt ccagttttgt tgcaaatctt gtctggtgct 1080
caaactgctg acactttgtt gccatctggt tctgttgttg ctttgccatc taactctact 1140
atcgaattgt ctatgactgg tggtttgttg ggtttggaac acccaatcca cttgcacggt 1200
cacaacttcg acgttgttag agttgctggt tctactgaat acaactacga aaacccaatc 1260
agaagagacg ttgttaacgc tggttctact tctgacaacg ttactatcag attcactact 1320
gacaacccag gtccatggat cttgcactgt cacatcgact ggcacttgga agctggtttc 1380
gctatcgttt tcgctgaagc tactgacgaa tgggttgaca ctatcgaccc atctgacgct 1440
tgggaaaact tgtgtccaac ttacgacgct ttgtctgacg acgacttgta a 1491
<210> 23
<211> 1500
<212> DNA
<213> Artificial sequence
<400> 23
tctatcggtc caatcgctga cttggttatc tctaaccaag acgtttctcc agacggtttc 60
actagatctg ctgttgttgc tggtggtgac actatcggtc cattgatcgt tggtaacaag 120
aacgacaact tgcaaatcaa cgttgttaac aacttggacg acgacactat gttgcaatct 180
acttctatcc actggcacgg tttcttccaa caatctacta actgggctga cggtactgct 240
ttcgttaacc aatgtccaat cgctaagggt aactctttct tgtacgactt cgacgctact 300
gaccaagctg gtactttctg gtaccactct cacttgtcta ctcaatactg tgacggtttg 360
agaggtccat tcgttgttta tgatccaaac gatccagctg cagacttgta cgatgttgat 420
aacgatgata ccgttattac cttggctgat tggtatcatg ttgctgctaa attgggtcca 480
gcttttccat tgggtgctga tgctactttg attaacggta agggtagatc tccatctact 540
accaccgctg atttgacagt tatttctgtt accccaggta agagatacag attcagattg 600
gtttctttgt cttgtgatcc aaaccatacc ttctctattg atggtcataa catgaccatt 660
attgaaaccg attctattaa caccgctcca ttggttgttg attctattca aattttcgct 720
gctcaaagat actctttcgt tttggaagct aatcaagctg ttgataacta ctggattaga 780
gctaatccat ctttcggtaa cgttggtttc actggtggta ttaactctgc tattttgaga 840
tacgatggtg ctgctgctat tgaacctact actactcaaa ctacctctac cgaaccattg 900
aacgaagtta acttgcatcc attggttgat actgttgttc caggttctcc agttgctggt 960
ggtgttgatt tggctattaa catggctttc aacttcaacg gtacaaactt cttcattaac 1020
ggtgcttcat tcactccacc aaccgttcca gttttgttgc aaattatttc tggtgctcaa 1080
aacgctcaag atttgttgcc atctggttct gtttattctt tgccatctaa cgctgatatt 1140
gaaatttctt tccctgctac tgctgctgct cctggtgctc ctcatccatt tcacttgcat 1200
ggtcatgctt ttgctgttgt tagatctgct ggttctaccg tttacaacta cgataaccca 1260
attttcagag atgttgtttc tactggtaca ccagcagccg gtgataacgt taccattaga 1320
ttcagaactg ataaccctgg tccatggttc ttgcattgtc atattgattt ccatttggaa 1380
gctggtttcg cagttgtttt cgctgaagat attccagatg ttgcttctgc taatccagtt 1440
cctcaagcct ggtctgattt gtgtccaact tacgatgcta gagatccatc tgatcaatga 1500
<210> 24
<211> 1491
<212> DNA
<213> Artificial sequence
<400> 24
tctatcggtc caatcgctga cttggttatc tctaaccaag acgtttctcc agacggtttc 60
actagatctg ctgttgttgc tggtggtgac actatcggtc cattgatcgt tggtaacaag 120
aacgacaact tgcaaatcaa cgttgttaac aacttggacg acgacactat gttgcaatct 180
acttctatcc actggcacgg tttcttccaa caatctacta actgggctga cggtactgct 240
ttcgttaacc aatgtccaat cgctaagggt aactctttct tgtacgactt cgacgctact 300
gaccaagctg gtactttctg gtaccactct cacttgtcta ctcaatactg tgacggtttg 360
agaggtccaa tcgttatcta cgacccagac gacccacacg cttctttgta cgacgttgac 420
gacgaatcta ctgttatcac tttggctgac tggtaccaca ctaaggctaa ggaaatcact 480
ttcggtactc cagactctac tttgatcaac ggtttgggta gatggtctca aggtaacgaa 540
actgacttgt ctgttatcac tgttacttct ggtcaaagat acagaatgag attgatcaac 600
actgcttgtg acgctgctta cactttctct atcgacaacc acactatgac tgttatcgaa 660
gctgacgctg ttaacatcga accaatcgaa gttgactctt tgactatcta cgctggtcaa 720
agatactctt tcgttttgaa cgctgaccaa gctgttggta actactggat cagagctaac 780
ccaaacatcg gtactatggg ttacactaac ggtatcaact ctgctatctt gagatacgac 840
actgctgaag aagaagaacc agacgttttg gacatcactt ctactaactc tttgtctgaa 900
gctgacttgg ttccattgga aaacccaggt gctccaggtg acccagttgt tggtggtgtt 960
gattacgctt tgaacttgga tttcaacttc gatggtacaa acttcttcat taacgatgtt 1020
tctttcgttt ctccaaccgt tccagttttg ttgcaaattt tgtctggtac tacctctgct 1080
gctgatttgt tgccatctgg ttctttgttt gctttgccat cagattctac cattgaaatt 1140
tctttcccaa ttactgctac taatgcacca ggtgctcctc atccatttca cttgcatggt 1200
cataccttct ctatcgttag aactgctggt tctactgata ccaacttcgt taacccagtt 1260
agaagagatg ttgttaacac cggtactgct ggtgataacg ttaccattag attcactact 1320
gataaccctg gtccatggtt cttgcattgt catattgatt tccatttgga agctggtttc 1380
gctatcgttt tgtctgaaga taccgcagat gtttctaata ccactactcc atctactgct 1440
tgggaagatt tgtgtccaac ctacaacgct ttggattctt ctgatttgtg a 1491

Claims (8)

1. Laccase mutants, which are any of the following proteins:
(B1) A protein with an amino acid sequence shown as SEQ ID No. 7;
(B2) A protein with an amino acid sequence shown as SEQ ID No. 8;
(B3) A protein with an amino acid sequence shown as SEQ ID No. 9;
(B4) A protein with an amino acid sequence shown as SEQ ID No. 10;
(B5) A protein with an amino acid sequence shown as SEQ ID No. 11;
(B6) The amino acid sequence of the protein is shown as SEQ ID No. 14.
2. Laccase mutant related biological material is any one of the following:
(C1) A nucleic acid molecule encoding the laccase mutant of claim 1;
(C2) Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing said nucleic acid molecules.
3. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule is any one of the following:
(D1) A DNA molecule shown in SEQ ID No. 16;
(D2) A DNA molecule shown in SEQ ID No. 17;
(D3) A DNA molecule shown in SEQ ID No. 18;
(D4) A DNA molecule shown in SEQ ID No. 19;
(D5) A DNA molecule shown in SEQ ID No. 20;
(D6) A DNA molecule shown in SEQ ID No. 23.
4. Use of the laccase mutant of claim 1 or the biomaterial of claim 2 or 3 in any one of the following:
(E1) Producing laccase;
(E2) As laccase.
5. A method for improving laccase enzyme activity and/or stability, comprising the steps of: expressing the laccase mutant of claim 1 in a recipient bacterium to obtain a recombinant bacterium; and (3) fermenting and culturing the recombinant bacteria to obtain laccase mutants with improved enzyme activity and/or stability from fermentation broth.
6. The method according to claim 5, wherein: expression of the laccase mutant in the recipient bacterium is achieved by introducing the nucleic acid molecule of claim 2 or 3 into the recipient bacterium.
7. The method according to claim 6, wherein: the recipient bacteria are yeasts.
8. The method according to claim 7, wherein: the yeast is Pichia pastoris.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
KR20140011062A (en) * 2012-07-17 2014-01-28 한국화학연구원 Dye compounds-degradable laccase and methods of preparation thereof
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CN108192878A (en) * 2018-02-09 2018-06-22 福建农林大学 The endogenous laccase protein Ma-Lac1 of Monochamus alternatus, encoding gene and its application

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KR20140011062A (en) * 2012-07-17 2014-01-28 한국화학연구원 Dye compounds-degradable laccase and methods of preparation thereof
CN107475219A (en) * 2017-09-29 2017-12-15 天津科技大学 Three kinds recombinate carbohydrase and preparation method and application
CN108192878A (en) * 2018-02-09 2018-06-22 福建农林大学 The endogenous laccase protein Ma-Lac1 of Monochamus alternatus, encoding gene and its application

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