CN113832120B - Formaldehyde conversion mutant protein and application thereof - Google Patents

Formaldehyde conversion mutant protein and application thereof Download PDF

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CN113832120B
CN113832120B CN202111161482.5A CN202111161482A CN113832120B CN 113832120 B CN113832120 B CN 113832120B CN 202111161482 A CN202111161482 A CN 202111161482A CN 113832120 B CN113832120 B CN 113832120B
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CN113832120A (en
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朱蕾蕾
李天真
唐梓静
谭子瑊
马延和
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses formaldehyde-converting muteins and their use. The mutein of the invention can catalyze formaldehyde to prepare 1, 3-dihydroxyacetone, and lactic acid can be further synthesized by the 1, 3-dihydroxyacetone. The obtained mutant greatly improves the catalytic efficiency of formaldehyde to 1, 3-dihydroxyacetone, and can be carried out under mild conditions. Has better application prospect.

Description

Formaldehyde conversion mutant protein and application thereof
The application relates to a Chinese patent application 202010983978.X filed on 9 months and 17 days in 2020, and the name of the application is formaldehyde conversion mutant protein and application thereof.
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to formaldehyde-converted mutant protein and application thereof.
Background
1,3-Dihydroxyacetone (1, 3-dihydroxyacetone) is the simplest three-carbon ketose existing in nature and has wider application. Can be used as raw material of cosmetics, has good skin protection effect, and can be used for synthesizing polyester compounds. In addition, 1,3-dihydroxyacetone is also an important metabolic intermediate, which can be further converted into high value-added chemicals and fuel molecules such as ethanol, butanol, lactic acid, succinic acid and the like by a microbial fermentation method, and other saccharides with higher value such as erythrulose, sorbose and the like can be synthesized. So the 1,3-dihydroxyacetone is an important chemical synthesis intermediate and is widely applied to industries such as cosmetic manufacture, food development, medicine, chemical synthesis and the like.
At present, the production method of the 1, 3-dihydroxyacetone mainly comprises a microbial method and a chemical method, the microbial method mainly utilizes a microbial fermentation method to convert glycerol into the 1, 3-dihydroxyacetone, but the microbial method has low production capacity, needs to strictly control the condition of microbial culture, has strict requirements on the production environment of the microorganism, and has complex purification process of the later-stage 1, 3-dihydroxyacetone, high cost and environmental pollution. The chemical method mainly uses a metal catalyst to oxidize glycerol to generate 1, 3-dihydroxyacetone, but the chemical method uses the metal catalyst to pollute the environment and is not beneficial to the environmental protection.
In recent years, a carbon compound has been attracting attention as a green energy substance having a great application prospect, and the carbon compound can be used for synthesizing basic organic chemical raw materials, fuels and other high-added-value chemicals. Because of the characteristics of low cost and easy availability, the carbon compound becomes the compound which has the most development prospect for preparing high-value compounds by replacing petroleum, and has important scientific significance and development value in the fields of medicine, food and chemical industry. Meanwhile, the utilization and conversion of the carbon compound can also obviously reduce the negative influence on the ecological environment caused by people in the use process of fossil fuel and synthetic materials. The formaldehyde can be converted from other carbon compounds, further is converted into a bioavailable intermediate substance, has the characteristics of wide sources, low price and the like, and has important application prospect in synthesizing high-value compounds by taking formaldehyde as a precursor.
Disclosure of Invention
In order to solve the above problems, an object of the present invention is to provide a formaldehyde conversion mutant protein, which is a protein having activity of catalyzing formaldehyde to synthesize 1, 3-dihydroxyacetone, and which is characterized by thermal stability, wherein the mutant BFD1 of a benzoic acid decarboxylase derived from Pseudomonas putida is subjected to error-prone PCR, and a target gene is subjected to random mutation.
A second object of the present invention is to provide the use of the above formaldehyde-converting mutein.
The invention adopts the following technical scheme to realize the purposes:
Firstly, the invention provides a formaldehyde conversion mutein which can catalyze formaldehyde to synthesize 1, 3-dihydroxyacetone, wherein the amino acid sequence of the formaldehyde conversion mutein is that at least one amino acid residue at S26, L43, F66, R86, T87, G109, A204, H281, A322, F397, M460, W463, V467, V473 and S525 corresponding to SEQ ID NO. 1 is mutated; or the amino acid sequence of the formaldehyde-converting mutein has a mutation site in the mutated amino acid sequence and has an amino acid sequence having a homology of 80% or more, preferably 90% or more, 95% or more, or 98% or more with the mutated amino acid sequence.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein comprises at least one of the following mutation sites: serine S corresponding to position 26 of SEQ ID NO. 1 is mutated to phenylalanine F, leucine L at position 43 is mutated to glutamine Q, phenylalanine F at position 66 is mutated to leucine L, arginine R at position 86 is mutated to cysteine C, threonine T at position 87 is mutated to alanine A, glycine G at position 109 is mutated to serine S, alanine A at position 204 is mutated to valine V, histidine H at position 281 is mutated to tyrosine Y, alanine A at position 322 is mutated to threonine T, phenylalanine F at position 397 is mutated to either leucine L or serine S, methionine M at position 460 is mutated to threonine T, tryptophan W at position 463 is mutated to arginine R, valine V at position 467 is mutated to alanine A, valine V at position 473 is mutated to alanine A, and serine S at position 525 is mutated to alanine A. Specifically, the formaldehyde-converting mutein comprises a substitution corresponding to SEQ ID NO. 1, at least one of the following sites or a combination of two or more different sites: S26F, L43Q, F, L, R86C, T A, G109S, A34204V, H281Y, A322T, F397L/F397S, M460T, W463R, V467A, V473A, S525A.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises at least: histidine H at position 281 corresponding to SEQ ID NO. 1 is mutated, for example to tyrosine Y. Further, the formaldehyde-converting mutein may also include mutations at least one other site, for example, a combination of any one or more of the sites S26F, L43Q, F66L, T87A, G S, A204V, A322T, F L/F397S, M460T, W463 467A, V473A, S a.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises at least: serine S at position 26 corresponding to SEQ ID NO. 1 is mutated, for example to phenylalanine F. Further, the formaldehyde-converting mutein may also comprise mutations at least one other site, for example a combination of any one or more of the sites L43Q, F L, T87A, G109S, A204V, H281Y, A32322T, F397L/F397S, M460T, W463R, V467A, V473A, S525A.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises at least: a phenylalanine F mutation at position 397 corresponding to SEQ ID NO. 1, e.g., to either leucine L or serine S. Further, the formaldehyde-converting mutein may also comprise mutations at least one other site, for example a combination of any one or more of the sites S26F, L43Q, F66L, T87A, G109S, A V, H Y, A322T, M463T, W463R, V467A, V473A, S525A.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises at least: tryptophan W at position 463, corresponding to SEQ ID NO. 1, is mutated, for example, to arginine R. Further, the formaldehyde-converting mutein may also comprise mutations at least one other site, for example a combination of any one or more of the sites S26F, L, Q, F, L, T, 87, A, G, S, A, V, H, Y, A, 322, T, F, 397L/F397S, M, 460T, V, A, V, 473, A, S, 525A.
In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises: histidine H at position 281 corresponding to SEQ ID NO. 1 is tyrosine Y and serine S at position 26 is mutated to phenylalanine F. Further, the formaldehyde-converting mutein may also comprise mutations at least one other site, for example W463R, L43Q, G109S, F397S/F397L, M460T, S525A, V I, V467A, F66L, A204V, A T, a combination of any one or more of the sites. In one embodiment, the amino acid sequence of the formaldehyde-converting mutein described above comprises mutations at positions S26F, H281Y and F397S corresponding to SEQ ID NO. 1; in yet another embodiment, the amino acid sequence of the formaldehyde converting mutein comprises mutations corresponding to the S26F, H281Y and W463R sites of SEQ ID NO. 1.
As an exemplary embodiment of the present invention, the amino acid sequence of the above formaldehyde-converting mutein is specifically any one of the following 1) to 18):
1) The 281 th histidine of SEQ ID NO.1 is mutated into tyrosine, other amino acid residues are kept unchanged, and the obtained amino acid sequence (the nucleotide sequence of the amino acid sequence is SEQ ID NO. 3);
2) The 86 th arginine of SEQ ID NO.1 is mutated into cysteine, and other amino acid residues are kept unchanged, so that the amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 4) is obtained;
3) Mutation of 281 th histidine of SEQ ID NO. 1 to tyrosine, mutation of 26 th serine to phenylalanine, and other amino acid residues unchanged, the obtained amino acid sequence (the nucleotide sequence is SEQ ID NO. 5);
4) The 281 th histidine of SEQ ID NO. 1 is mutated into tyrosine, the 397 th phenylalanine is mutated into leucine, and other amino acid residues are kept unchanged, so that the obtained amino acid sequence (the nucleotide sequence of the amino acid sequence is SEQ ID NO. 6);
5) The 281 th histidine of SEQ ID NO. 1 is mutated into tyrosine, the 473 th valine is mutated into alanine, and other amino acid residues are kept unchanged, so as to obtain an amino acid sequence (the nucleotide sequence of the amino acid sequence is SEQ ID NO. 7);
6) Mutation of 281 th histidine of SEQ ID NO.1 to tyrosine, mutation of 26 th serine to phenylalanine, mutation of 43 rd leucine to glutamine, and remaining other amino acid residues unchanged, to obtain an amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 8);
7) Mutation of 281 th histidine of SEQ ID NO.1 to tyrosine, mutation of 26 th serine to phenylalanine, mutation of 66 th phenylalanine to leucine, and remaining other amino acid residues unchanged, to obtain an amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 9);
8) Mutation of 281 th histidine of SEQ ID NO. 1 to tyrosine, mutation of 26 th serine to phenylalanine, mutation of 109 th glycine to serine, and remaining other amino acid residues unchanged, to obtain an amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 10);
9) Mutation of 281 th histidine to tyrosine, mutation of 26 th serine to phenylalanine, mutation of 204 th alanine to valine, and other amino acid residues remained unchanged, thus obtaining an amino acid sequence (the nucleotide sequence is SEQ ID NO: 11);
10 281 th histidine of SEQ ID NO. 1 into tyrosine, 26 th serine into phenylalanine, 397 th phenylalanine into serine, and other amino acid residues are kept unchanged, thus obtaining an amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 12);
11 Mutation of histidine 281 to tyrosine, serine 26 to phenylalanine, tryptophan 463 to arginine, and other amino acid residues unchanged in SEQ ID NO. 1 to obtain an amino acid sequence (the nucleotide sequence is SEQ ID NO. 13);
12 Mutation of histidine 281 to tyrosine, serine 26 to phenylalanine, valine 467 to alanine, and other amino acid residues to obtain an amino acid sequence (the nucleotide sequence is SEQ ID NO: 14);
13 Mutation of the 281 th histidine of SEQ ID NO. 1 to tyrosine, mutation of the 87 th threonine to alanine, mutation of the 322 rd alanine to threonine, and remaining other amino acid residues unchanged, the resulting amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 15);
14 281 th histidine of SEQ ID NO.1 into tyrosine, 26 th serine into phenylalanine, 463 th tryptophan into arginine, 109 th glycine into serine, and other amino acid residues unchanged, the obtained amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 16);
15 281 th histidine of SEQ ID NO. 1 into tyrosine, 26 th serine into phenylalanine, 397 th phenylalanine into serine, 109 th glycine into serine, and other amino acid residues unchanged, the resulting amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 17);
16 281 th histidine of SEQ ID NO. 1 into tyrosine, 26 th serine into phenylalanine, 460 th methionine into threonine, 525 th serine into alanine, other amino acid residues are kept unchanged, and the obtained amino acid sequence (the nucleotide sequence of which is SEQ ID NO. 18);
17 281 st histidine of SEQ ID NO. 1 to tyrosine, 26 th serine to phenylalanine, 397 th phenylalanine to serine, 109 th glycine to serine, 322 rd alanine to threonine, other amino acid residues remaining unchanged, the resulting amino acid sequence (nucleotide sequence of which is SEQ ID NO. 19);
18 281 th histidine of SEQ ID NO. 1 into tyrosine, 26 th serine into phenylalanine, 463 th tryptophan into arginine, 109 th glycine into serine, 397 th phenylalanine into serine, and the other amino acid residues unchanged, the resulting amino acid sequence (nucleotide sequence of which is SEQ ID NO. 20);
The invention also provides a polynucleotide which codes for the formaldehyde-converting mutein.
The invention also provides a recombinant vector, an expression cassette, a transgenic cell line and/or a recombinant bacterium containing the formaldehyde conversion mutant protein and/or the polynucleotide.
The invention also provides the use of the formaldehyde-converting mutein as described above as a catalyst, preferably for catalyzing the preparation of 1, 3-dihydroxyacetone and/or lactic acid, for example for catalyzing the preparation of 1, 3-dihydroxyacetone and/or lactic acid from formaldehyde.
The invention also provides the use of recombinant vectors, expression cassettes, transgenic cell lines and/or recombinant bacteria containing the polynucleotides as catalysts, preferably for the catalytic preparation of 1, 3-dihydroxyacetone and/or lactic acid, for example for the catalytic preparation of 1, 3-dihydroxyacetone and/or lactic acid from formaldehyde.
When the formaldehyde conversion mutein of the present invention catalyzes formaldehyde to prepare a compound of interest, it comprises: contacting formaldehyde conversion mutant protein with formaldehyde to catalyze the formaldehyde conversion mutant protein to generate 1, 3-dihydroxyacetone; further, an alkali metal hydroxide and/or an alkaline earth metal hydroxide may be optionally added to catalyze the formation of lactic acid from 1, 3-dihydroxyacetone.
The recombinant vector, transgenic cell line or recombinant strain containing the formaldehyde-converting mutein and/or the polynucleotide in the invention comprises the following components in catalyzing formaldehyde to prepare a target compound: contacting a recombinant vector, transgenic cell line or recombinant strain comprising said formaldehyde converting mutein and/or said polynucleotide with formaldehyde to catalyze it to 1, 3-dihydroxyacetone; further, an alkali metal hydroxide and/or an alkaline earth metal hydroxide may be optionally added to catalyze the formation of lactic acid from 1, 3-dihydroxyacetone.
Advantageous effects
The formaldehyde-converted mutant protein obtained by molecular modification of the formaldehyde-converted protein greatly improves the efficiency of producing 1, 3-dihydroxyacetone by formaldehyde condensation and provides a raw material for synthesis of lactic acid.
Drawings
Fig. 1: the product 1, 3-dihydroxyacetone was checked by HPLC in example 7.
Fig. 2: HPLC in example 8 detected the end product lactic acid produced by alkali metal hydroxide catalysis.
Fig. 3: the final product lactic acid produced by alkaline earth metal hydroxide catalysis was checked by HPLC in example 8.
Fig. 4: formaldehyde conversion mutein structure diagram.
Terminology and definitions
In the context of the present invention, "formaldehyde-converting protein" in the present invention refers to a protein capable of catalyzing the synthesis of 1, 3-dihydroxyacetone from formaldehyde, so long as it has a function of catalyzing the conversion of formaldehyde into 1, 3-dihydroxyacetone, and the amino acid sequence and source thereof are not particularly limited. By way of example only, but not limitation, it may be, for example, a benzoic acid decarboxylase (benzoylformate decarboxylases, BFD) derived from pseudomonas putida (pseudomonas putida) and a benzaldehyde lyase (benzaldehyde lyase, BAL) derived from pseudomonas fluorescens (Pseudomonas fluorescens biovar I). "Formaldehyde-converting mutein" means that the "formaldehyde-converting protein" is subjected to amino acid mutation to give a protein having the above-mentioned functions.
In the present invention, amino acids are represented by single letter or three letter codes, having the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).
In the present invention, "homology" has the meaning conventional in the art, referring to "identity" between two nucleic acid or amino acid sequences, the percentage of which represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences to be compared obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length.
In the present invention, the terms "mutant" and "variant" and "mutein" are used interchangeably and "modified" or "mutant" are used interchangeably and these expressions refer to the formaldehyde-converting mutein of SEQ ID NO. 1 as a starting sequence, or derived from such a protein, comprising alterations at one or more positions, i.e. substitutions, insertions and/or deletions, relative to the amino acid of the unmodified or engineered protein, and still retain its activity. Muteins can be obtained by a variety of techniques known in the art. In particular, exemplary techniques for modifying a DNA sequence encoding a wild-type protein include, but are not limited to, directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides.
The term "substitution" with respect to an amino acid position or residue means that the amino acid at a particular position has been replaced with another amino acid. Substitutions may be conservative or non-conservative.
The mutations are described in terms of their mutation at a specific residue, the position of which is determined by an amino acid sequence SEQ ID NO.1 alignment or reference sequence SEQ ID NO.1 as starting protein. In the context of the present invention, it also relates to any variant carrying these same mutations at functionally equivalent residues.
The term "corresponding to" as used herein has a meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponding to" means that two sequences are aligned by homology or sequence identity, and that one sequence corresponds to a specified position in the other sequence. In the present invention, "corresponding to SEQ ID NO:1 "indicates that the position of the mutation site is determined by comparison with SEQ ID NO. 1. Thus, for example, in the case of "amino acid residue corresponding to position 40 of the amino acid sequence shown in SEQ ID NO. 1", if a 6 XHis tag is added to one end of any of the amino acid sequences shown in SEQ ID NO. 1, then position 40 of the resulting mutant corresponding to the amino acid sequence shown in SEQ ID NO. 1 may be position 46 of the mutant.
It will be appreciated by those skilled in the art that "corresponding to SEQ ID NO:1 "only indicates that SEQ ID NO. 1 is used as a reference for determining the position of the mutation site, but does not represent that the mutein of the present invention can be obtained only by modifying the amino acid sequence shown in SEQ ID NO. 1. In one embodiment, the man skilled in the art can obtain the formaldehyde-converting muteins of the invention starting from the amino acid sequence of any formaldehyde-converting enzyme known in the art, provided that the resulting formaldehyde-converting mutein is mutated at the site in question according to the invention in comparison with SEQ ID NO:1 and retains the catalytic activity of the formaldehyde-converting enzyme, i.e.that it is not necessary within the scope of the invention that all but the mutated site corresponds exactly to SEQ ID NO: 1. In one embodiment, the formaldehyde-converting muteins of the invention are obtained by carrying out the mutation or substitution of the invention starting from the protein shown in SEQ ID NO. 1. In one embodiment, the inventive mutation or substitution is performed with the sequence having homology of SEQ ID NO. 1 as a starting sequence to obtain the formaldehyde conversion mutein of the present invention, the sequence having homology of SEQ ID NO. 1 is identical to the S26, L43, F66, R86, T87, G109, A204, H281, A322, F397, M460, W463, V467, V473, S525 sites of SEQ ID NO. 1, so that the inventive mutation or substitution may occur, but other sites may be identical or different. In one embodiment, the starting sequence has a homology of 80% or more, for example 85% or more, 90% or more or 95% or more, with SEQ ID NO. 1, in order to be able to determine the specific amino acid position corresponding to SEQ ID NO. 1. In a further embodiment, the wild-type formaldehyde converting enzyme (amino acid sequence SEQ ID NO:23, nucleotide sequence SEQ ID NO: 24) is used as starting sequence, and the mutation or substitution according to the invention is still carried out at the corresponding site corresponding to SEQ ID NO:1, resulting in the corresponding formaldehyde converting mutein.
In the present invention, "start protein" or "start sequence" refers to formaldehyde protease or its amino acid sequence before modification or mutation of the present invention. The expression "XaY" is used herein to denote mutation or substitution of amino acids, wherein a denotes the position of the amino acid in SEQ ID NO. 1, X denotes the wild-type amino acid species at the a-position in SEQ ID NO. 1, and Y denotes the amino acid species after mutation at the a-position in SEQ ID NO. 1. For example, "H281Y" means that the histidine H corresponding to position 281 of SEQ ID NO. 1 is replaced by tyrosine Y in comparison with SEQ ID NO. 1.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods. The experimental methods used in the following examples are conventional methods unless otherwise specified.
EXAMPLE 1 construction of Formaldehyde converting muteins (Formaldehyde converting mutants)
The formaldehyde-converting mutein is a mutant BFD1 (hereinafter abbreviated as original BFD1 in the present invention) of a benzoic acid decarboxylase (benzoylformate decarboxylases, BFD) derived from Pseudomonas putida (pseudomonas putida), the nucleotide sequence of which is SEQ ID NO:2, and restriction enzyme sites NdeI and XhoI are added to both ends of the nucleotide sequence, and the resultant sequence is ligated to the region of the multicloning site of plasmid pET28a with T4 DNA ligase to construct recombinant plasmid pET28a-BFD1. Then, a primer was designed using pET28a-bfd1 as a template (upstream primer: 5'-CCGCGCGGCAGCCATATG-3')
(SEQ ID NO: 21) downstream primer: 5'-GGTGGTGGTGGTGGTGCTCGAGTTATT-3' (SEQ ID NO: 22)), error-prone PCR was performed to randomly mutate the gene of interest. After the PCR is completed, the target gene is recovered by glue, the mutated target gene is connected to a vector pET28a, and then the connected vector is transformed into escherichia coli BL21 Gold (DE 3), and the incubator at 37 ℃ is cultured until monoclonal grows.
EXAMPLE 2 screening of Formaldehyde-converting muteins
For the mutant clone obtained in example 1, a single clone was picked up in a 96-well plate containing LB medium containing kanamycin sulfate antibiotic, cultured overnight in a shaker, and the above mutant was replicated in a 96-well plate containing LB medium of isopropyl-. Beta. -D-thiogalactoside (IPTG) and cultured for a certain period of time. After completion of the culture, the cells were collected by centrifugation, and then washed with 200. Mu.L of potassium phosphate buffer (50 mM K 2HPO4 and KH 2PO4,5mM MgSO4, pH 7.4) to collect the cells.
The cells were resuspended in 50. Mu.L of potassium phosphate buffer, and an equal volume of formaldehyde solution (potassium phosphate buffer containing 50mM or 30mM or 20mM or 200mM formaldehyde and 1mM thiamine pyrophosphate (TPP)) was added thereto, followed by reaction at 30℃for 3 hours. After completion of the reaction, 90. Mu.L of the supernatant was centrifuged and placed in a 96-well plate, 60. Mu.L of tool enzyme buffer 1 (0.3 mg/mL galactose oxidase, 36U/mL horseradish peroxidase) was added, followed by 50. Mu.L of tool enzyme buffer 2 (3.2 mM 2,2' -biazo-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS)) and detection was performed under light absorption at 410nm for 20 minutes.
Compared with the original BFD1, the obtained strain is a beneficial mutant strain with higher catalytic efficiency, and then the loci and amino acids of the corresponding mutation are found out through gene sequencing. Through three rounds of mutant library screening, eighteen mutants with remarkably improved enzyme activity and stable activity are finally obtained. The name of the eighteen mutants and the corresponding mutant amino acids are shown in Table 1 through gene sequencing.
TABLE 1 mutant names and corresponding mutant amino acids
EXAMPLE 3 expression of original BFD1 and mutants thereof in E.coli
The inoculating needle picks up positive bacteria of original BFD1 and 18 mutants, respectively inoculating in 5mL LB culture medium, culturing at 37 ℃ overnight, then inoculating in 25mL LB culture medium with 1% (V/V) inoculum size, culturing at 37 ℃ at 200 r/min. When OD 600 reached 0.6, 0.1mM IPTG was added and expression was induced at 30 ℃. After the induction was completed, the above-mentioned cultured mutant cells were collected into centrifuge tubes, respectively, and then the cells were resuspended and washed with potassium phosphate buffer. Centrifuging, and storing the thallus in a refrigerator at-80deg.C.
Example 4 original BFD1 and mutant Activity detection thereof
The cells of example 3 were removed, resuspended in potassium phosphate buffer, sonicated in an ice bath, and the supernatant collected by centrifugation. Mixing 50 mu L of cell heavy suspension or supernatant with 50 mu L of formaldehyde solution, reacting for 3 hours at 30 ℃, taking out 90 mu L of reaction solution, adding 60 mu L of tool enzyme buffer 1, adding 50 mu L of tool enzyme buffer 2, and detecting for 20 minutes under the condition of absorbing light of 410 nm. The original BFD1 and mutant whole cell enzyme activities were calculated, and the percentage of mutant enzyme activities relative to the original BFD1 (100% of the original BFD1 and starting strain whole cell enzyme activities) was shown in the following table.
TABLE 2 Activity of whole cells of original BFD1 and mutants at 30mM formaldehyde concentration
TABLE 3 Activity of mutant V3 and further mutants of whole cells at 20mM formaldehyde concentration
TABLE 4 cell disruption supernatants of mutant V10 and further mutants thereof were active at 20mM formaldehyde concentration
TABLE 5 cell disruption supernatants of mutant V11 and further mutants thereof were active at 20mM formaldehyde concentration
TABLE 6 cell disruption supernatants of mutant V11 and further mutants thereof were active at 200mM formaldehyde concentration
EXAMPLE 5 original BFD1 and mutant thermal stability detection thereof
The cells of example 3 were removed, resuspended in potassium phosphate buffer, sonicated in an ice bath, and the supernatant collected by centrifugation. 50. Mu.L of the cell supernatant was heated at 55℃for 30min, 50. Mu.L of 50mM formaldehyde solution was added after the completion of the heating, the reaction was carried out at 30℃for 3h, 90. Mu.L of the reaction solution was taken out after the completion of the reaction, 60. Mu.L of the tool enzyme buffer 1 was added, and then 50. Mu.L of the tool enzyme buffer 2 was added, followed by detection at 410nm of the absorbed light for 20min. After heating the cell supernatant, the activity of the mutant is better than the original BFD 1. The relative residual activity of the original BFD1 and its mutants is shown in table 7 (100% relative residual activity of the original BFD 1).
TABLE 7 original BFD1 and mutant relative residual Activity
EXAMPLE 6 purification of original BFD1 transformation and mutants thereof
The original BFD1 and mutant protein V8 cells collected in example 3 were resuspended in 25mL of potassium phosphate buffer, sonicated in an ice bath, and the supernatant collected by centrifugation, and the expressed mutants were affinity-purified using a Ni 2+ column, desalted using a desalting column after elution with imidazole, and the purified proteins were stored in potassium phosphate buffer for use.
EXAMPLE 7 reaction of Formaldehyde conversion to 1, 3-dihydroxyacetone
The protein concentrations of the original BFD1 and mutant V8 purified in example 6 were diluted to 15mg/mL with potassium phosphate buffer. A certain volume of pure enzyme was taken in the reaction system, and then an equal amount of formaldehyde solution (600 mM formaldehyde, 1mM TPP,50mM K 2HPO4 and KH 2PO4,5mM MgSO4, pH 7.4) was added thereto, and reacted at 30℃for 1 hour. After the reaction is finished, adding an equal volume of acetonitrile into a certain volume of reaction liquid to terminate the reaction, centrifuging the reaction liquid through a 0.22 mu m filter membrane, and carrying out HPLC detection under the conditions of: chromatographic column: aminex HPX-87H,300 mm. Times.7.8 mm (Bio-Rad); mobile phase: 50%0.005M H 2SO4 solution, 50% H 2 O; ultraviolet absorption wavelength: 210nm; flow rate: 0.5mL/min; column temperature: 65 ℃; sample injection amount: 5. Mu.L.
The HPLC detection result is shown in FIG. 1, and analysis shows that the original BFD1 and the mutant V8 can catalyze formaldehyde to generate 1, 3-dihydroxyacetone.
The conversion of formaldehyde to 1, 3-dihydroxyacetone was calculated for both the original BFD1 and mutant V8 as shown in Table 8 below.
TABLE 8 conversion of raw BFD1 and mutant V8 to 1, 3-dihydroxyacetone by catalyzing formaldehyde
Example 8 alkali metal hydroxide and alkaline earth metal hydroxide catalyzed conversion of mutein-catalyzed 1, 3-dihydroxyacetone to lactic acid
The reaction solution in example 7 was centrifuged using an ultrafiltration tube to remove the enzyme in the reaction system, 17.5mL of the solution was slowly added to 17.5mL of 2M NaOH or KOH solution, the reaction was carried out at room temperature for 24 hours, and after the completion of the reaction, diluted sulfuric acid was added to adjust the pH of the solution to 1.0 to 2.0. Passing through 0.22 μm organic filter membrane, detecting target product lactic acid by HPLC, and detecting conditions by HPLC: chromatographic column: aminex HPX-87H,300 mm. Times.7.8 mm (Bio-Rad); mobile phase: 0.010M H 2SO4; a differential detector; flow rate: 0.5mL/min; column temperature: 35 ℃; sample injection amount: 20. Mu.L.
As shown in FIG. 2, the analysis shows that the alkali metal hydroxide (sodium hydroxide and potassium hydroxide) can further catalyze the mutant V8 to catalyze the conversion of the 1, 3-dihydroxyacetone generated by formaldehyde into lactic acid.
The reaction solution in example 7 was centrifuged using an ultrafiltration tube to remove the enzyme in the reaction system, 17.5mL of the solution was slowly added to 17.5mL of a calcium hydroxide (250 mM) suspension, the reaction was carried out at room temperature for 24 hours, and after the completion of the reaction, diluted sulfuric acid was added to adjust the pH of the solution to 1.0 to 2.0. Passing through 0.22 μm organic filter membrane, detecting target product lactic acid by HPLC, and detecting conditions by HPLC: chromatographic column: aminex HPX-87H,300 mm. Times.7.8 mm (Bio-Rad) mobile phase: 50%0.005M H 2SO4 solution, 50% H 2 O, UV absorption wavelength: 210nm, flow rate: 0.5mL/min, column temperature: 65 ℃, sample injection amount: 5. Mu.L.
The HPLC detection result is shown in FIG. 3, and analysis shows that alkaline earth metal hydroxide (calcium hydroxide) can further catalyze mutant V8 to catalyze the conversion of formaldehyde-generated 1, 3-dihydroxyacetone into lactic acid.
The calculated conversion of lactic acid produced from formaldehyde by catalysis with mutant V8, and alkali metal hydroxides (sodium hydroxide, potassium hydroxide) and alkaline earth metal hydroxides (calcium hydroxide) is shown in table 9 below.
TABLE 9 conversion of formaldehyde to lactic acid
Example 9 Activity detection of wild-type protein and its single amino acid site mutant of original BFD1
The amino acid mutation site S26F, L43Q, F L, R86A, G87A, G109S, A204V, H Y, A322T, F397S, M460 463R, V467A, V473A, S A screened in example 1 was designed as a primer and single point mutation was performed in the wild type protein (amino acid sequence of SEQ ID NO:23, nucleotide sequence of SEQ ID NO: 24) sequence of the original BFD1, and after the mutation was completed and sequencing was successful, the mutants were plated.
17 Strains of the mutant positive bacteria and the original BFD1 wild type protein strain are respectively inoculated into 5mL of LB culture medium, cultured overnight at 37 ℃, then inoculated into 25mL of LB culture medium with 1% (V/V) inoculum size, and cultured at 37 ℃ and 200 r/min. When OD 600 reached 0.6, 0.1mM IPTG was added and expression was induced at 30 ℃. After the induction was completed, the above-mentioned cultured mutant cells were collected into centrifuge tubes, respectively, and then the cells were resuspended and washed with potassium phosphate buffer. Centrifuging, and storing the thallus in a refrigerator at-80deg.C.
Taking out the thalli, re-suspending by using potassium phosphate buffer solution, performing ice bath ultrasonic sterilization, and centrifugally collecting the supernatant. Mixing 50 mu L of supernatant with 50 mu L of 50mM formaldehyde solution, reacting for 3 hours at 30 ℃, taking out 90 mu L of reaction solution, adding 60 mu L of tool enzyme buffer 1, adding 50 mu L of tool enzyme buffer 2, and detecting for 20 minutes under the condition of absorbing light of 410 nm. The wild-type protein of original BFD1 and the mutant whole-cell enzyme activities were calculated, and the percentage of mutant enzyme activities relative to the wild-type protein of original BFD1 is shown in the following table (100% of wild-type protease activity of original BFD 1).
Table 10 mutant names and corresponding mutant amino acids and Activity at 50mM Formaldehyde concentration
EXAMPLE 10 structural analysis of Formaldehyde-converting muteins
The present invention resolves the crystal structure of mutant V8 (PDB ID:6M 2Y), and by structural analysis, it was found that the combined mutations H281Y and S26F formed a pie interaction (FIG. 4), which greatly helped to increase the activity of formaldehyde-converting muteins comprising the H281Y and S26F mutations.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Institute of Tianjin Industrial biotechnology, national academy of sciences
<120> Formaldehyde conversion muteins and their use
<130> CPCN21111638
<150> 2020101134418
<151> 2020-02-24
<160> 24
<170> PatentIn version 3.3
<210> 1
<211> 528
<212> PRT
<213> Artificial sequence
<400> 1
Met Ala Ser Val His Gly Thr Thr Tyr Glu Leu Leu Arg Arg Gln Gly
1 5 10 15
Ile Asp Thr Val Phe Gly Asn Pro Gly Ser Asn Glu Leu Pro Phe Leu
20 25 30
Lys Asp Phe Pro Glu Asp Phe Arg Tyr Ile Leu Ala Leu Gln Glu Ala
35 40 45
Cys Val Val Gly Ile Ala Asp Gly Tyr Ala Gln Ala Ser Arg Lys Pro
50 55 60
Ala Phe Ile Asn Leu His Ser Ala Ala Gly Thr Gly Asn Ala Met Gly
65 70 75 80
Ala Leu Ser Asn Ala Arg Thr Ser His Ser Pro Leu Ile Val Thr Ala
85 90 95
Gly Gln Gln Thr Arg Ala Met Ile Gly Val Glu Ala Gly Glu Thr Asn
100 105 110
Val Asp Ala Ala Asn Leu Pro Arg Pro Leu Val Lys Trp Ser Tyr Glu
115 120 125
Pro Ala Ser Ala Ala Glu Val Pro His Ala Met Ser Arg Ala Ile His
130 135 140
Met Ala Ser Met Ala Pro Gln Gly Pro Val Tyr Leu Ser Val Pro Tyr
145 150 155 160
Asp Asp Trp Asp Lys Asp Ala Asp Pro Gln Ser His His Leu Phe Asp
165 170 175
Arg His Val Ser Ser Ser Val Arg Leu Asn Asp Gln Asp Leu Asp Ile
180 185 190
Leu Val Lys Ala Leu Asn Ser Ala Ser Asn Pro Ala Ile Val Leu Gly
195 200 205
Pro Asp Val Asp Ala Ala Asn Ala Asn Ala Asp Cys Val Met Leu Ala
210 215 220
Glu Arg Leu Lys Ala Pro Val Trp Val Ala Pro Ser Ala Pro Arg Cys
225 230 235 240
Pro Phe Pro Thr Arg His Pro Cys Phe Arg Gly Leu Met Pro Ala Gly
245 250 255
Ile Ala Ala Ile Ser Gln Leu Leu Glu Gly His Asp Val Val Leu Val
260 265 270
Ile Gly Ala Pro Val Phe Arg Tyr His Gln Tyr Asp Pro Gly Gln Tyr
275 280 285
Leu Lys Pro Gly Thr Arg Leu Ile Ser Val Thr Cys Asp Pro Leu Glu
290 295 300
Ala Ala Arg Ala Pro Met Gly Asp Ala Ile Val Ala Asp Ile Gly Ala
305 310 315 320
Met Ala Ser Ala Leu Ala Asn Leu Val Glu Glu Ser Ser Arg Gln Leu
325 330 335
Pro Thr Ala Ala Pro Glu Pro Ala Lys Val Asp Gln Asp Ala Gly Arg
340 345 350
Leu His Pro Glu Thr Val Phe Asp Thr Leu Asn Asp Met Ala Pro Glu
355 360 365
Asn Ala Ile Tyr Leu Asn Glu Ser Thr Ser Thr Thr Ala Gln Met Trp
370 375 380
Gln Arg Leu Asn Met Arg Asn Pro Gly Ser Tyr Tyr Phe Cys Ala Ala
385 390 395 400
Gly Gly Leu Gly Phe Ala Leu Pro Ala Ala Ile Gly Val Gln Leu Ala
405 410 415
Glu Pro Glu Arg Gln Val Ile Ala Val Ile Gly Asp Gly Ser Ala Asn
420 425 430
Tyr Ser Ile Ser Ala Leu Trp Thr Ala Ala Gln Tyr Asn Ile Pro Thr
435 440 445
Ile Phe Val Ile Met Asn Asn Gly Thr Tyr Gly Met Leu Arg Trp Phe
450 455 460
Ala Gly Val Leu Glu Ala Glu Asn Val Pro Gly Leu Asp Val Pro Gly
465 470 475 480
Ile Asp Phe Arg Ala Leu Ala Lys Gly Tyr Gly Val Gln Ala Leu Lys
485 490 495
Ala Asp Asn Leu Glu Gln Leu Lys Gly Ser Leu Gln Glu Ala Leu Ser
500 505 510
Ala Lys Gly Pro Val Leu Ile Glu Val Ser Thr Val Ser Pro Val Lys
515 520 525
<210> 2
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 2
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 3
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 3
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 4
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 4
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgcttgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 5
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 5
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 6
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 6
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactacct ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 7
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 7
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgctc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 8
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 8
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatccagg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 9
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 9
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctctcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 10
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 10
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctagtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 11
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 11
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ttatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 12
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 12
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactc ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 13
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 13
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgtcggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtccgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 14
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 14
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgc tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 15
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 15
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtgc ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atgacttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 16
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 16
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctagtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgtcggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtccgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 17
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 17
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctagtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactc ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 18
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 18
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctggtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtacg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg ttgctccggt taaataa 1587
<210> 19
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 19
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctagtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atgacttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactc ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 20
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 20
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttttaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgctcgtac ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctagtgaa accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
taccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactc ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtatg 1380
ctgcgtcggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtccgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence
<400> 21
ccgcgcggca gccatatg 18
<210> 22
<211> 27
<212> DNA
<213> Artificial sequence
<400> 22
ggtggtggtg gtggtgctcg agttatt 27
<210> 23
<211> 528
<212> PRT
<213> Artificial sequence
<400> 23
Met Ala Ser Val His Gly Thr Thr Tyr Glu Leu Leu Arg Arg Gln Gly
1 5 10 15
Ile Asp Thr Val Phe Gly Asn Pro Gly Ser Asn Glu Leu Pro Phe Leu
20 25 30
Lys Asp Phe Pro Glu Asp Phe Arg Tyr Ile Leu Ala Leu Gln Glu Ala
35 40 45
Cys Val Val Gly Ile Ala Asp Gly Tyr Ala Gln Ala Ser Arg Lys Pro
50 55 60
Ala Phe Ile Asn Leu His Ser Ala Ala Gly Thr Gly Asn Ala Met Gly
65 70 75 80
Ala Leu Ser Asn Ala Trp Asn Ser His Ser Pro Leu Ile Val Thr Ala
85 90 95
Gly Gln Gln Thr Arg Ala Met Ile Gly Val Glu Ala Leu Leu Thr Asn
100 105 110
Val Asp Ala Ala Asn Leu Pro Arg Pro Leu Val Lys Trp Ser Tyr Glu
115 120 125
Pro Ala Ser Ala Ala Glu Val Pro His Ala Met Ser Arg Ala Ile His
130 135 140
Met Ala Ser Met Ala Pro Gln Gly Pro Val Tyr Leu Ser Val Pro Tyr
145 150 155 160
Asp Asp Trp Asp Lys Asp Ala Asp Pro Gln Ser His His Leu Phe Asp
165 170 175
Arg His Val Ser Ser Ser Val Arg Leu Asn Asp Gln Asp Leu Asp Ile
180 185 190
Leu Val Lys Ala Leu Asn Ser Ala Ser Asn Pro Ala Ile Val Leu Gly
195 200 205
Pro Asp Val Asp Ala Ala Asn Ala Asn Ala Asp Cys Val Met Leu Ala
210 215 220
Glu Arg Leu Lys Ala Pro Val Trp Val Ala Pro Ser Ala Pro Arg Cys
225 230 235 240
Pro Phe Pro Thr Arg His Pro Cys Phe Arg Gly Leu Met Pro Ala Gly
245 250 255
Ile Ala Ala Ile Ser Gln Leu Leu Glu Gly His Asp Val Val Leu Val
260 265 270
Ile Gly Ala Pro Val Phe Arg Tyr His Gln Tyr Asp Pro Gly Gln Tyr
275 280 285
Leu Lys Pro Gly Thr Arg Leu Ile Ser Val Thr Cys Asp Pro Leu Glu
290 295 300
Ala Ala Arg Ala Pro Met Gly Asp Ala Ile Val Ala Asp Ile Gly Ala
305 310 315 320
Met Ala Ser Ala Leu Ala Asn Leu Val Glu Glu Ser Ser Arg Gln Leu
325 330 335
Pro Thr Ala Ala Pro Glu Pro Ala Lys Val Asp Gln Asp Ala Gly Arg
340 345 350
Leu His Pro Glu Thr Val Phe Asp Thr Leu Asn Asp Met Ala Pro Glu
355 360 365
Asn Ala Ile Tyr Leu Asn Glu Ser Thr Ser Thr Thr Ala Gln Met Trp
370 375 380
Gln Arg Leu Asn Met Arg Asn Pro Gly Ser Tyr Tyr Phe Cys Ala Ala
385 390 395 400
Gly Gly Leu Gly Phe Ala Leu Pro Ala Ala Ile Gly Val Gln Leu Ala
405 410 415
Glu Pro Glu Arg Gln Val Ile Ala Val Ile Gly Asp Gly Ser Ala Asn
420 425 430
Tyr Ser Ile Ser Ala Leu Trp Thr Ala Ala Gln Tyr Asn Ile Pro Thr
435 440 445
Ile Phe Val Ile Met Asn Asn Gly Thr Tyr Gly Ala Leu Arg Trp Phe
450 455 460
Ala Gly Val Leu Glu Ala Glu Asn Val Pro Gly Leu Asp Val Pro Gly
465 470 475 480
Ile Asp Phe Arg Ala Leu Ala Lys Gly Tyr Gly Val Gln Ala Leu Lys
485 490 495
Ala Asp Asn Leu Glu Gln Leu Lys Gly Ser Leu Gln Glu Ala Leu Ser
500 505 510
Ala Lys Gly Pro Val Leu Ile Glu Val Ser Thr Val Ser Pro Val Lys
515 520 525
<210> 24
<211> 1587
<212> DNA
<213> Artificial sequence
<400> 24
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgcttggaa ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctctgctg accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtgct 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587

Claims (6)

1. A formaldehyde-converting mutein capable of catalyzing the conversion of formaldehyde, said formaldehyde-converting mutein having an amino acid sequence of V473A single point mutation corresponding to SEQ ID No. 23.
2. A polynucleotide encoding the formaldehyde conversion mutein of any one of claim 1.
3. A transgenic cell line or recombinant bacterium comprising the formaldehyde-converting mutein of claim 1.
4. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the polynucleotide of claim 2.
5. Use of the formaldehyde-converting mutein of claim 1, the transgenic cell line or recombinant bacterium of claim 3 or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of claim 4 for catalyzing the condensation of formaldehyde to 1, 3-dihydroxyacetone.
6. Use of a formaldehyde-converting mutein according to claim 1, a transgenic cell line or recombinant bacterium according to claim 3 or a recombinant vector, an expression cassette, a transgenic cell line or recombinant bacterium according to claim 4, respectively, in combination with an alkali metal hydroxide or an alkaline earth metal hydroxide for catalyzing the production of lactic acid from formaldehyde.
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