CN113943723B - Xylanase mutant with improved thermostability, preparation and application thereof - Google Patents

Xylanase mutant with improved thermostability, preparation and application thereof Download PDF

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CN113943723B
CN113943723B CN202010693613.3A CN202010693613A CN113943723B CN 113943723 B CN113943723 B CN 113943723B CN 202010693613 A CN202010693613 A CN 202010693613A CN 113943723 B CN113943723 B CN 113943723B
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CN113943723A (en
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周志华
邢宏观
邹根
刘春燕
柴顺星
刘睿
严兴
李新良
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Center for Excellence in Molecular Plant Sciences of CAS
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Abstract

The invention provides xylanase mutants with improved thermostability, preparation and application thereof. The invention determines the amino acid locus related to the thermal stability, and obtains the mutant with obviously improved thermal stability by site-directed transformation. The invention also provides nucleic acid for encoding the mutant, a construct for expressing the mutant, a xylanase mutant library for covering a plurality of mutants, a method for degrading xylan by using the mutants and the like.

Description

Xylanase mutant with improved thermostability, preparation and application thereof
Technical Field
The invention belongs to the fields of protein engineering and genetic engineering, and relates to xylanase mutants with improved thermal stability and better specific activity, and preparation and application thereof.
Background
Hemicellulose is the most abundant non-cellulose heteropolysaccharide present on earth and is the main constituent of plant cells and cell walls. The hemicellulose is efficiently converted into other valuable byproducts, so that the method has good economic benefit and plays a positive role in environmental protection.
The main component of hemicellulose in plant cells and cell walls is xylan. Xylan is the main structural polysaccharide in plant cells, the second most abundant polysaccharide in nature, and accounts for about one third of all renewable organic carbon on earth.
Xylan, which is linked by Xylose molecules (D Xylose) to a backbone with β -1, 4-glycosidic linkages; arabinofuranosidyl, glucuronyl, acetyl, etc. are linked into a branched chain. The raw materials rich in xylan in nature are widely available, and comprise agriculture, forestry, industrial waste, municipal solid waste and the like, such as hardwood, cork, straw, wheat bran and the like. The amount of xylan contained in different plants is also different, the xylan contained in the hard material is more than that in the soft material, the hard material can occupy 15-30% of dry weight, and the soft material generally occupies 7-12% of dry weight. In some annual plants such as wheat, cotton seed hulls, the xylan content is as high as 30% or more.
Xylanases are glycosidases (O-glycoside hydrolases, EC3.2.1. X) which catalyze the endo-hydrolysis of 1, 4-. Beta.D-xyloside bonds (. Beta. -1, 4-glycosidic bonds) in xylans. Xylanases can be largely divided into the GH10 family and the GH11 family, depending on their structure and catalytic domain. The xylanase GH10 family comprises endo-1, 4-beta-xylanases (EC 3.2.1.8), endo-1, 3-beta-xylanases (EC 3.2.1.32) and cellobiohydrolases (EC 3.2.1.91), with endo-1, 4-beta-xylanases being the dominant. Compared with GH10 family, GH11 family can only act on D-xylose, can be called as xylanase in the true sense, and has the characteristics of low molecular weight, high activity and the like.
In industry, xylanases have found wide application. In the food industry, xylanases are used in the processing of fruits, vegetables, plants, etc., to facilitate the infusion process; in the paper industry, xylanases are used to facilitate pulp processing; in the textile industry, xylanases are used in enzymatic hydrolysis of textiles to reduce or replace chemical mixing processes to reduce environmental pollution; in the feed industry, xylanases are added to the feed of monogastric animals as well as ruminants to improve the digestibility and nutritional value of the feed; in the aspect of the bioenergy field, xylanase can be applied to industrial production for converting lignocellulose into fuel ethanol together with other cellulases and hemicellulases, and along with research and development of five-carbon sugar fermentation paths and strains in recent years, the process for producing fuel ethanol by fermenting xylan hydrolysate xylose by utilizing bacteria, yeast and filamentous fungi is mature, and the role of xylanase in the bioenergy field is becoming more important.
Xylanases in industrial applications are mostly bacterial or fungal in origin, and they are mostly mesophilic. However, in the production process, high temperature treatment is often required, which results in a considerable decrease in xylanase performance. The xylanase mutant strain with excellent heat stability is obtained, so that the xylanase mutant strain has wider application and higher efficiency. Therefore, there is a need in the art to develop a novel enzyme that is adaptable to different high temperature conditions and maintains desirable enzyme activities
Disclosure of Invention
The invention aims at providing xylanase mutants with improved thermostability, preparation and application thereof.
In a first aspect of the invention, there is provided a method of improving the thermostability of a xylanase comprising: the amino acid sequence of the xylanase is mutated to correspond to the amino acid sequence shown in the wild-type xylanase (SEQ ID NO: 2), and the mutation is selected from the group consisting of the following sites or combinations thereof: 14 th, 28 th, 29 th, 30 th, 31 st, 41 st, 52 th, 131 th, 133 th, 192 th and 203 th.
In a preferred embodiment, the mutation at position 14 is His (H); mutation at position 28 to Val (V); mutation at position 29 to Ser (S) or Ala (a); mutation at position 30 to Ser (S) or Pro (P); mutation at position 31 to His (H); mutation at position 41 to Arg (R); mutation at position 52 to Leu (L); mutation at position 131 to Cys (C); mutation at position 133 to Glu (E); mutation at position 192 to Asp (D); and/or, mutation at position 203 to Val (V).
In another aspect of the invention, there is provided a xylanase mutant, which is: (a) The amino acid sequence corresponds to the wild-type xylanase, a mutant protein at a site or combination of sites selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 st, 41 st, 52 th, 131 th, 133 th, 192 th, 203 th; (b) A protein derived from (a) having the function of (a) and obtained by substitution, deletion or addition of one or more (e.g., 1 to 20; preferably 1 to 15; more preferably 1 to 10, e.g., 5, 3) amino acid residues of the amino acid sequence of the protein (a), but corresponding to the amino acid at position 14, 28, 29, 30, 31, 41, 52, 131, 133, 192 or 203 of the wild-type xylanase, which is the same as the amino acid obtained by the mutation of the corresponding position of the protein (a); (c) The protein derived from (a) has 80% or more homology (preferably 85% or more; more preferably 90% or more; more preferably 95% or more such as 98% or 99%) with the amino acid sequence of the protein (a) and has the function of the protein (a), but the amino acid corresponding to the 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th or 203 th position of the wild-type xylanase is the same as the amino acid after the mutation at the corresponding position of the protein (a).
In a preferred embodiment, the mutation at position 14 is His (H); mutation at position 28 to Val (V); mutation at position 29 to Ser (S) or Ala (a); mutation at position 30 to Ser (S) or Pro (P); mutation at position 31 to His (H); mutation at position 41 to Arg (R); mutation at position 52 to Leu (L); mutation at position 131 to Cys (C); mutation at position 133 to Glu (E); mutation at position 192 to Asp (D); and/or, mutation at position 203 to Val (V).
In another preferred embodiment, the xylanase mutant comprises a protein selected from the group consisting of: corresponding to wild-type xylanase:
(1) Mutation at position 14 to His and mutation at position 28 to Val; the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 th mutation is Asp (D), the 203 th mutation is Val (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L);
(2) Mutation of His at position 14, val at position 28, ser at position 29, pro at position 30, his at position 31, leu at position 52, glu at position 133, asp at position 192, val at position 203, arg at position 41 (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L/K41R);
(3) Mutation of His at position 14, val at position 28, ser at position 29, val at position 203, pro at position 30, his at position 31, leu at position 52, cys at position 131, glu at position 133, asp at position 192 (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L/Q131C);
(4) Mutation of His at position 14, val at position 28, ser at position 29, val at position 203, pro at position 30, his at position 31, glu at position 133, asp at position 192 (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H);
(5) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 133 to Glu, mutation at position 192 to Asp (L28V/K133E/G192D/Q14H/D29S/A203V/S30P)
(6) The 29 th mutation is Ser, the 28 th mutation is Val, the 203 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 nd mutation is Leu, the 133 th mutation is Glu, the 192 th mutation is Asp (L28V/K133E/G192D/Q14/D29S/A203V/S30P/T31H/A52L);
(7) Mutation of His at position 14, val at position 28, val at position 203, pro at position 30, his at position 31, leu at position 52, glu at position 133, asp at position 192 (L28V/K133E/G192D/Q14H/D29/A203V/S30P/T31H/A52L);
(8) Mutation of His at position 14, val at position 28, ser at position 29, pro at position 30, his at position 31, leu at position 52, glu at position 133, asp at position 192 (L28V/K133E/G192D/Q14H/D29S/A203/S30P/T31H/A52L);
(9) The 29 th mutation is Ser, the 28 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, and the 192 th mutation is Asp (L28V/K133E/G192D/Q14/D29S/A203/S30P/T31H/A52L);
(10) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Ser, mutation at position 203 to Val, mutation at position 133 to Glu, mutation at position 192 to Asp (L28V/N30S/K133E/G192D/Q14H/D29S/A203V); or (b)
(11) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ala, mutation at position 30 to Ser, mutation at position 203 to Val, mutation at position 133 to Glu, mutation at position 192 to Asp (L28V/N30S/K133E/G192D/Q14H/D29A/A203V); or (b)
(12) Val at position 28, ser at position 30, glu at position 133, asp at position 192 (L28V/N30S/K133E/G192D (Xyn 370)).
In a preferred embodiment, the xylanase mutant comprises a protein selected from the group consisting of: 14,SEQ ID NO:16,SEQ ID NO:18,SEQ ID NO:12,SEQ ID NO:10,SEQ ID NO:20,SEQ ID NO:22,SEQ ID NO:24,SEQ ID NO:26,SEQ ID NO:8,SEQ ID NO:6 or 4.
In another aspect of the invention, there is provided an isolated polynucleotide encoding a xylanase mutant as defined in any one of the preceding claims; preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 13,SEQ ID NO:15,SEQ ID NO:17,SEQ ID NO:11,SEQ ID NO:9,SEQ ID NO:19,SEQ ID NO:21,SEQ ID NO:23,SEQ ID NO:25,SEQ ID NO:7,SEQ ID NO:5 or SEQ ID NO. 4.
In another aspect of the invention, there is provided a vector comprising said polynucleotide.
In another aspect of the invention there is provided a genetically engineered host cell comprising said vector, or said polynucleotide integrated in the genome.
In a preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cells comprise escherichia coli cells, bacillus subtilis cells and the like; preferably, the eukaryotic cells include mold cells, yeast cells, insect cells, plant cells, fungal cells, mammalian cells, or the like.
In another aspect of the present invention, there is provided a method for preparing a xylanase mutant as defined in any one of the preceding, the method comprising:
(i) Culturing said host cell;
(ii) Collecting a culture containing said xylanase mutant;
(iii) Isolating the xylanase mutant from the culture.
In another aspect of the present invention, there is provided a composition for degrading xylan comprising an effective amount of: a xylanase mutant as defined in any one of the preceding claims; or said host cell or a culture or lysate thereof; a pharmaceutically or industrially acceptable carrier.
In a further aspect of the invention there is provided the use of a xylanase mutant as defined in any one of the preceding or a composition as defined in the preceding for degrading xylan; preferably, the xylanase mutant cleaves a beta-1, 4-glycosidic bond, thereby hydrolyzing xylan.
In another aspect of the present invention, there is provided a method of degrading xylan, the method comprising: degrading xylan using a xylanase mutant as defined in any one of the preceding claims or said composition; preferably, the xylanase mutant cleaves a beta-1, 4-glycosidic bond, thereby hydrolyzing xylan.
In a preferred embodiment, the degradation or cleavage is a degradation or cleavage at elevated temperature; preferably, the high temperature is 45-95 ℃; preferably 50 to 90 ℃ (e.g. 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃ or 90 ℃).
In another preferred embodiment, the substrate for degradation of the xylanase mutant or composition is xylan or a xylan-containing material.
In another preferred embodiment, the xylan includes (but is not limited to): beech xylan, corncob xylan, oat hull xylan.
In another preferred embodiment, the xylan-containing material includes (but is not limited to): industrial products, agricultural, forestry, industrial waste and municipal solid waste; preferably, including (but not limited to): pulp, feed, food, straw, hardwood, softwood, straw, and bran.
In a further aspect of the invention there is provided the use of a xylanase mutant as defined in any one of the preceding or a composition comprising: as a food additive; as plant (including, for example, fruit or vegetable) processing additives; as an additive in the papermaking process; as an additive in textile processing; as a feed additive; and/or for the conversion of lignocellulose to fuel ethanol.
In another aspect of the invention there is provided a library of xylanase mutants or polynucleotides encoding them, the library comprising: at least 5, preferably at least 10, xylanase mutants of any of the preceding claims or polynucleotides encoding the same; more preferably it comprises said xylanase mutant or a polynucleotide encoding it.
In a further aspect of the invention there is provided the use of a library of said xylanase mutants or polynucleotides encoding them for providing an appropriate xylanase mutant for degrading xylan, depending on the temperature of the reaction system in which the xylan is to be degraded.
In another aspect of the present invention, there is provided a kit for degrading xylan comprising: a xylanase mutant or combination of mutants as defined in any of the preceding; said host cell; said composition; or a library of said xylanase mutants or polynucleotides encoding the same.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIGS. 1-1 to 1-13, and summary of wild-type and mutant sequence information.
Detailed Description
The inventor starts from wild xylanase from GH11 family, combines long-term research experience of the inventor on the xylanase through directed evolution error-prone PCR technology, determines amino acid sites related to heat stability, and obtains mutants with obviously improved heat stability through site-directed transformation.
As used herein, "xylose" refers to a monosaccharide that contains five carbon atoms. Molecular formula C4H9O4CHO. The "xylan" is a polymer of "xylose".
As used herein, unless otherwise indicated, the terms "xylanase mutant" and "mutant xylanase" are used interchangeably to refer to an enzyme (polypeptide/protein) that is formed after mutation of some sites identified by the inventors as having a correlation with the thermostability of the enzyme, corresponding to a xylanase prior to mutation, preferably corresponding to the amino acid sequence shown in SEQ ID NO:2, the mutation being selected from the following group of sites or combinations thereof: 14 th, 28 th, 29 th, 30 th, 31 st, 41 st, 52 th, 131 th and 203 th.
If desired, the xylanase prior to mutation (wild-type) may be a "protein having the amino acid sequence shown in SEQ ID NO. 2. The mutation site of the mutant in the present invention is based on the sequence shown in SEQ ID NO. 2, unless otherwise specified.
In the present invention, unless otherwise indicated, the identification of a xylanase mutant refers to the "amino acid substituted at the original amino acid position" to the mutated amino acid, e.g., Q14H, in the xylanase mutant, indicating that the amino acid at position 14 is substituted with H from Q of the parent xylanase.
As used herein, "isolated xylanase" refers to a xylanase mutant that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. The skilled person will be able to purify the xylanase mutants using standard protein purification techniques. Substantially pure proteins can produce a single main band on a non-reducing polyacrylamide gel.
As used herein, "improving thermostability" refers to a statistically significant increase in thermostability of a mutated xylanase, or referred to as a significant increase, as compared to a xylanase starting polypeptide prior to modification. For example, after a heat treatment for a certain period of time at the same treatment temperature, the residual enzyme activity of the mutant xylanase with improved thermostability is significantly improved by 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 100% or more, 150% or more, etc., than that of the enzyme before modification.
As used herein, the term "xylanase mutant or library of polynucleotides encoding same" refers to a collection of polypeptides or polynucleotides comprising a series of mutant xylanases provided herein. The assembly of a plurality of xylanase mutants having different stability or different temperature adaptation or polynucleotides encoding them into a library facilitates the selection of the appropriate xylanase or nucleic acid encoding it by the skilled person depending on the desired reaction conditions.
The mutant proteins of the invention may be chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, and mammalian cells) using recombinant techniques.
The invention also includes fragments, derivatives and analogues of the xylanase mutants. As used herein, the terms "fragment," "derivative" and "analog" refer to proteins that retain substantially the same biological function or activity of the native xylanase mutants of the invention. The protein fragments, derivatives or analogues of the invention may be (i) proteins having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) proteins having a substituent in one or more amino acid residues, or (iii) proteins in which an additional amino acid sequence is fused to the protein sequence (such as a leader or secretory sequence or a sequence used to purify the protein or a proprotein sequence, or fusion proteins). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known to those skilled in the art in view of the definitions herein. However, it is certain that at least one of the above-described mutations according to the present invention, preferably the mutation corresponding to the amino acid sequence shown in SEQ ID NO. 2, is present in the amino acid sequence of the xylanase mutant as defined herein, and is selected from the group consisting of the amino acid sequence at position 14, position 28, position 29, position 30, position 31, position 41, position 52, position 131, position 133, position 192 and/or position 203.
In the present invention, the term "xylanase mutant" also includes (but is not limited to): deletion, insertion and/or substitution of several (usually 1-20, more preferably 1-10, still more preferably 1-8, 1-5, 1-3, or 1-2) amino acids, and addition or deletion of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of xylanase mutants. However, in these variants, it is certain that at least one of the mutations described above according to the invention is present, preferably the mutation is one corresponding to the amino acid sequence shown in SEQ ID NO. 2, selected from the group consisting of amino acids 14, 28, 29, 30, 31, 41, 52, 131, 133, 192 and/or 203.
In the present invention, the term "xylanase mutant" also includes (but is not limited to): a derivative protein having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, such as 98% or more, 99% or more, sequence identity to the amino acid sequence of the xylanase mutant, which retains its protein activity. Likewise, in these derived proteins, it is certain that there is at least one mutation as described above according to the invention, preferably a mutation corresponding to the amino acid sequence shown in SEQ ID NO. 2, selected from the group consisting of the amino acid at position 14, position 28, position 29, position 30, position 31, position 41, position 52, position 131, position 133, position 192 and/or position 203.
The invention also provides analogues of the xylanase mutants. These analogs may differ from the xylanase mutants by differences in amino acid sequence, by differences in modified forms that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, by site-directed mutagenesis or other known techniques of molecular biology. Analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.
As a preferred mode of the present invention, in the preferred stability-related site, the 14 th site is mutated to His (H); mutation at position 28 to Val (V); mutation at position 29 to Ser (S) or Ala (a); mutation at position 30 to Ser (S) or Pro (P); mutation at position 31 to His (H); mutation at position 41 to Arg (R); mutation at position 52 to Leu (L); mutation at position 131 to Cys (C); mutation at position 133 to Glu (E); mutation at position 192 to Asp (D); and/or, mutation at position 203 to Val (V).
In a specific embodiment of the invention, the inventors used error-prone PCR technology to make a first round of random mutations of wild Xyn11 (the nucleic acid sequence is shown as SEQ ID NO:1, the protein sequence is shown as SEQ ID NO: 2) from xylanase 11 family, the random mutation library capacity is 10000, and the preferred mutant Xyn68 is obtained. The preferred Xyn68 is used as a template, error-prone PCR is utilized to carry out second round random mutation on the Xyn68, the mutation library capacity is 20000, and the preferred mutant Xyn370 (the nucleic acid sequence is shown as SEQ ID NO:3, and the protein sequence is shown as SEQ ID NO: 4) is obtained. Xyn370 includes mutations compared to the wild type: L28V/N30S/K133E/G192D.
The preferred mutants obtained in the two rounds above (including Xyn68 and Xyn370, but not limited to Xyn68 and Xyn 370) were sequenced and the mutation sites were found to be mostly concentrated in Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, a187D, G192D, a203V.
The mutation sites of Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, A187D, G192D and A203V are singly introduced into the wild type Xyn11 one by one, and the Q14H, D29A and A203V have the effect of improving the heat stability of the wild type Xyn11
Q14H, D29A, V203 are introduced into the preferred mutant Xyn370 in a single, two or three combination mode to obtain the preferred mutant Xyn370-Q14H/D29A/A203V (the nucleic acid sequence is shown as SEQ ID NO:5, the protein sequence is shown as SEQ ID NO: 6)
Saturation mutation is carried out on the 14 th amino acid residue of the preferred Xyn370-Q14H/D29A/A203V mutant, and the residue Q is replaced by 18 other amino acid residues except H; performing saturation mutation on the 29 th amino acid residue, and replacing the residue D with 18 other amino acids except A; the 203 th amino acid residue is saturated mutated and replaced by 18 other amino acids except A to obtain a preferred mutant Xyn370-Q14H/D29S/A203V (the nucleic acid sequence is shown as SEQ ID NO:7, the protein sequence is shown as SEQ ID NO: 8)
And carrying out homology modeling on the preferred mutant Xyn370-Q14H/D29S/A203V by using an online server to obtain a 3D structure diagram of the preferred mutant Xyn 370-Q14H/D29S/A203V.
And predicting the B-factor value of the 3D three-dimensional structure of the preferred mutant Xyn370-Q14H/D29S/A203V by using an online server to obtain the B-factor value information of each residue of the preferred mutant Xyn 370-Q14H/D29S/A203V.
The residues with higher B-factor values in the preferred mutant Xyn370-Q14H/D29S/A203V are selected as follows: 30S, 31T, 41K, 52A, 53V, 108G, 109V, 110Q, 131Q, 170Q, 171K.
The 30S, 31T, 41K, 52A, 53V, 108G, 109V, 110Q, 131Q, 170Q and 171K in the preferred mutant Xyn370-Q14H/D29S/A203V are respectively subjected to saturation mutation, a saturation mutation library is constructed, and S30P, T31H, K41R, A52L, Q C mutation residues are screened from the saturation mutation library, so that the thermal stability of the preferred mutant Xyn370-Q14H/D29S/A203V is improved.
S30P, T31H, K3541R, A52L, Q C is respectively introduced into the preferred mutants Xyn370-Q14H/D29S/A203V in an iterative combination mode of S30P, S30P/T31H, S30P/T31H/A52L/K41R, S30P/T31H/A52L/Q131C to obtain the preferred mutants Xyn370-Q14H/D29S/A203V/S30P (the nucleic acid sequence is shown as SEQ ID NO:9, the protein sequence is shown as SEQ ID NO: 10); preferably mutant Xyn370-Q14H/D29S/A203V/S30P/T31H (nucleic acid sequence shown as SEQ ID NO:11, protein sequence shown as SEQ ID NO: 12); preferably mutant Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L (nucleic acid sequence shown as SEQ ID NO:13, protein sequence shown as SEQ ID NO: 14); xyn370-Q14H/D29S/A203V-S30P/T31H/A52L/K41R (nucleic acid sequence shown as SEQ ID NO:15, protein sequence shown as SEQ ID NO: 16); preferably, the mutant Xyn370-Q14H/D29S/A203V/S30P/T31H/Q131C (the nucleic acid sequence is shown as SEQ ID NO:17, and the protein sequence is shown as SEQ ID NO: 18).
Through a series of transformation, the inventor obtains a plurality of preferable mutant strains with greatly improved thermostability, but discovers that the specific enzyme activities of the preferable mutant strains are inevitably reduced while improving the thermostability, and in order to balance the relationship between the thermostability and the specific enzyme activities, three possible sites which have influence on the enzyme activities of the preferable mutant strains Xyn 370-Q14/D29S/A203V/A31H/A52L are respectively subjected to back mutation on the basis of the preferable mutant strains Xyn 370-Q14/A29S/A203V/S30P/T31H/A52L, and the preferable mutant strains Xyn 370-Q30P/A31H/A52L with different degrees of the enzyme activities are obtained, wherein the nucleic acid sequences are shown as SEQ ID NO:19, and the protein sequences are shown as SEQ ID NO: 20; xyn370-Q14H/D29/A203V/S30P/T31H/A52L (nucleic acid sequence shown as SEQ ID NO:21, protein sequence shown as SEQ ID NO: 22); xyn370-Q14H/D29S/A203/S30P/T31H/A52L (nucleic acid sequence shown as SEQ ID NO:23, protein sequence shown as SEQ ID NO: 24); xyn370-Q14/D29S/A203/S30P/T31H/A52L (nucleic acid sequence shown as SEQ ID NO:25, protein sequence shown as SEQ ID NO: 26).
The invention also provides polynucleotide sequences encoding the xylanase mutants of the invention or conservative variant proteins thereof.
The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
Polynucleotides encoding the mature proteins of the mutants include: a coding sequence encoding only the mature protein; coding sequences for mature proteins and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature protein, and non-coding sequences.
The "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or may include additional coding and/or non-coding sequences.
The invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors or xylanase mutant coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
The polynucleotide sequences of the present invention may be used to express or produce recombinant xylanase mutants by conventional recombinant DNA techniques. Generally, there are the following steps:
(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a xylanase mutant of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) Host cells cultured in a suitable medium;
(3) Isolating and purifying the protein from the culture medium or the cells.
In the present invention, the xylanase mutant polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.
Methods well known to those skilled in the art can be used to construct expression vectors containing xylanase mutant-encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
In the present invention, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells such as mold cells, yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: coli, bacillus subtilis, streptomycete, agrobacterium; eukaryotic cells such as yeast, plant cells, and the like. In a specific embodiment of the present invention, E.coli is used as the host cell.
It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. In a preferred form of the invention, the expression vector used is pET28a; the microbial host cells transformed by the expression vector are all escherichia coli.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
The xylanase provided by the invention can act on the interior of a xylan long-chain molecule, and act on beta-1, 4-xyloside bonds in the interior of a xylan main chain in an inscribed manner, so that macromolecular polyxylose is hydrolyzed into simple sugar (such as xylooligosaccharide).
As used herein, "simple sugar" refers broadly to the general term for a class of sugars formed after a xylan chain has been cut, which has a lower chain length than before the cut. For example, the simple sugar contains 1 to 50 xylose, preferably 1 to 30 xylose; more preferably, 1 to 15 xylose; more preferably 1-10 xylose, such as 2,3,4,5,6,7,8,9 xylose. The simple sugar comprises: monosaccharides, xylobioses, xylotrioses, xylotetraoses, and the like. In the present invention, the simple sugar refers to: xylo-oligosaccharide, a small amount of xylose and xylo-oligosaccharide.
After obtaining the xylanase mutant enzyme of the invention, the skilled person can conveniently apply the enzyme to act as a hydrolysis substrate, in particular xylan, according to the teachings of the invention. As a preferred mode of the present invention, there is also provided a method for degrading xylan, the method comprising: the xylanase mutant enzyme of the invention is used for treating substrates to be hydrolyzed, wherein the substrates comprise, but are not limited to, beech xylan, corncob xylan, oat husk xylan and the like. In an alternative mode of the invention, the xylanase of the invention is subjected to degradation or cleavage of a substrate at high temperature; preferably, the high temperature is 45-95 ℃; preferably 50 to 90℃such as 55℃60℃65℃70℃75℃80℃85℃or 90 ℃.
The invention also provides a composition comprising an effective amount of a xylanase mutant of the invention and a pharmaceutically or industrially acceptable carrier or excipient. Such vectors include (but are not limited to): water, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The effective amount of xylanase mutants in the composition can be determined by one skilled in the art, depending on the actual use of the composition.
Substances which modulate the enzymatic activity of the xylanase mutants of the invention may also be added to the composition. Any substance having a function of enhancing the enzymatic activity is usable. For example, some substances that may increase the enzymatic activity of the xylanase mutants of the invention are selected and validated from the group consisting of: k (K) + 、Mg 2+ 、Cu 2+ 、Co 2+ 、K + 、Mn 2+ 、Cu 2+ 、Co 2+ 、Ni 2+ 、Zn 2+ 、Fe 3+ EDTA, and the like.
The xylanase mutant, the composition containing the xylanase mutant, the cell expressing the xylanase mutant and the like can be contained in a kit so as to facilitate the expansion of application or commercial application. Preferably, the kit may further comprise instructions for use to direct the use of the subject.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.
Materials and methods
Culture medium and culture medium formula
LB liquid medium: yeast extract 5.0g/L, peptone 10.0g/L, naCl 10.0g/L.
LB solid medium: yeast extract 5.0g/L, peptone 10.0g/L, naCl 10.0g/L, agar 2g/L.
Protein expression purification
Host BL21 containing xylanase wild type or preferably mutant expression vector is selected into LB test tube, shaking overnight at 37 ℃ and 180r/min, transferring into LB triangular flask, standing on ice for 10min when shaking to OD600 to 0.4-0.6 at 37 ℃, adding IPTG to final concentration 40uM, and inducing for 16h at 110 r/min. After the induction, the cells were collected by centrifugation, and the cells were washed with lysis buffer (50 mM NaH 2 PO 4 300mm nacl,10mm imidazole, ph=8.0) were resuspended and pressed. Crude enzyme was purified by Ni column from Saint of Helianthus tuberosus, washed with 20mM imidazole, eluted with 200mM imidazole, and the eluate was collected and subjected to protein SDS-PAGE electrophoresis.
Preparation method of reagent DNS used in xylanase enzyme activity determination
Xylanase enzyme activity assays include wild-type and mutant assays. 10g NaOH was weighed and dissolved in approximately 400ml ddH 2 In O, 10g of dinitrosalicylic acid, 2g of phenol, 0.5g of anhydrous sodium sulfite and 200g of sodium potassium tartrate tetrahydrate are weighed again and dissolved in about 300ml of ddH 2 In O, the two solutions are mixed, the volume is fixed to 1 liter, and the mixture is preserved in a dark place.
Substrate preparation for xylanase enzyme activity
Substrate selection: fagus xylan (from Sigma), beech xylan (from Megazyme, corncob xylan, oat hull xylan).
Beech xylan from Sigma was used as an example, the remainder being the same.
0.5g beech xylan (from Sigma) was weighed into 20mL 0.1% NaOH, stirred in a 70℃water bath for 1.5h, then added with 0.2M acetate buffer (pH=6.0) to 45mL, adjusted to pH 6.0 with glacial acetic acid, and finally fixed to a volume of 50mL with 0.2M acetate buffer to give a final concentration of 1% (W/V).
Xylanase enzyme activity determination method
Substrate selection: fagus xylan (from Sigma), fagus xylan (from Megazyme), corncob xylan, oat hull xylan.
Beech xylan from Sigma was used as an example, the remainder being the same.
50. Mu.L of 1% Fagus xylan (from Sigma) +50. Mu.L of enzyme solution which has been diluted by a suitable factor are mixed and reacted at 37℃for 10min, then 100. Mu.L of DNS is added to terminate the reaction, the comparison is that 100. Mu.L of DNS is added to the reaction system and then the enzyme solution is added, color reaction is carried out at 95℃for 10min, and finally the absorbance at 540nm is measured by an enzyme-labeling instrument and converted into corresponding enzyme activity units.
Definition of enzyme Activity Unit (U)
1U is the amount of enzyme required to catalyze the hydrolysis of xylan to 1. Mu. Mol xylose per minute.
Example 1 first round mutant library construction and screening
Random mutation library construction: random mutation (reference GeneMorph II Random Mutagenesis Kit for specific PCR reaction program) is carried out on xylanase wild type coding gene Xyn11 (SEQ ID NO: 1) by using GeneMorph II Random Mutagenesis Kit error-prone PCR kit, the mutated target gene fragment gel is recovered, then recombined into pET28a vector by Vazyme ClonExpress II One Step Cloning Kit, electrically transformed into escherichia coli BL21 competence and coated with LB solid plate containing Kana antibiotics, and incubated overnight at 37 ℃. Transformants incubated overnight were picked with toothpick into LB liquid medium 96 well plates containing Kana antibiotics, incubated at 37℃for 12h, transferred to new LB liquid 96 well plates at 1% inoculum size, added IPTG to a final concentration of 2mM, and induced at 37℃for 12h.
Screening of random mutation libraries: 96-well plates induced for 12h at 37℃were centrifuged, medium removed, and 50ul PBS buffer was added to each well for resuspension, followed by treatment for 2.5h at 65 ℃. After the heat treatment, 50ul of 1% beech xylan was added, the reaction was stopped by adding 100ul of DNS at 37℃for 10 minutes, developed at 95℃for 10 minutes, and then transferred to a 96-well ELISA plate for reading OD540.OD540 was higher than wild type and marked as positive transformants, and the protection was selected for further verification. The round of library covered 10000 transformants.
Thermal stability validation of preferred mutants: from 10000 transformants, the inventors selected 5 preferred mutants with greatly improved thermostability compared with wild type Xyn11, and expressed and purified the 5 preferred mutants for measuring thermostability. The residual enzyme activities (%) of the preferred mutants in the first round of random mutant library after heat treatment at 65℃for different times are shown in Table 1, and it can be seen that the preferred mutant Xyn68 has the best thermostability and is selected as the most preferred mutant for this round.
TABLE 1
EXAMPLE 2 construction and screening of a second round random mutation library
The first round of preferred mutant Xyn68 is taken as a template, geneMorph II Random Mutagenesis Kit error-prone PCR kit is utilized to randomly mutate Xyn68 coding genes, and the subsequent construction method is not different from the first round of random mutation library construction method. The screening method was identical to the first round of screening method except that the 65℃treatment for 2.5h was changed to 80℃for 30min, and the round of library covered 20000 transformants. The preferred mutants were purified and then subjected to heat stability verification, and the preferred mutants were subjected to heat treatment at 70℃for various times and the residual enzyme activity (%) results are shown in Table 2, xyn370 being the preferred mutant for this round.
TABLE 2
Example 3 site directed mutagenesis
Sequencing and analysis of preferred mutants (including Xyn68 and Xyn370, but not limited to Xyn68 and Xyn 370) in the above two rounds of random mutagenesis, the present inventors found that amino acid sites such as Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, A187D, G192D, A203V appeared more frequently, presumably to have an increasing effect on xylan thermostability, and then the above sites were subjected to site-directed mutagenesis one by one (site-directed mutagenesis procedure was referred to Hieff Mut TM site-directed mutagenesis kit) was introduced into xylanase wild-type Xyn 11. Xylanase wild type is not subjected to heat treatment at 70 ℃ after 14H,29A and 203V mutation sites are introducedThe residual enzyme activities (%) after the same time are shown in Table 3.
TABLE 3 Table 3
As can be seen from Table 3, Q14H, D29A, A203V has an increasing effect on the thermal stability of wild type Xyn 11.
To further determine whether these three amino acid sites have an important effect on xylanase thermostability, the inventors introduced these three sites as single mutations as well as double mutations, in combination with the three mutations, into the preferred mutant Xyn370 in the second round of random mutation. The residual enzyme activities (%) of xylanase mutant Xyn370 after introduction of 14H,29A,203V mutation sites and heat treatment at 75℃for different times are shown in Table 4.
TABLE 4 Table 4
As can be seen from Table 4, the heat stability of the three mutant Xyn370-Q14H/D29A/A203V is significantly improved compared with the wild type Xyn11 and the starting strain Xyn 370. The Xyn370-A203V/Q14H double mutant has a certain improvement of thermal stability compared with the wild type and single mutant.
Example 4, preferred mutant Xyn370-Q14H/D29A/A203V saturation mutation
By Hieff Mut TM site-directed mutagenesis kit site-directed mutagenesis kit saturation mutagenesis was performed on the 14 th, 29 th and 203 th positions of the preferred mutant Xyn370-Q14H/D29A/A203V in example 4: substitution of amino acid residue Q at position 14 with 18 other amino acid residues other than H; substitution of amino acid residue D at position 29 with 18 other amino acids than a; the 203 th amino acid residue V is replaced by 18 other amino acids except A. The residual enzyme activities (%) of xylanase mutants Xyn370-Q14H/D29A/A203V after the 29 th saturation mutation were treated at 80℃for different times are shown in Table 5.
TABLE 5
As can be seen from Table 5, the thermostability of the mutant Xyn370-Q14H/D29S/A203V was further significantly improved after replacing residue D at position 29 with S, so that the preferred mutant Xyn370-Q14H/D29S/A203V was obtained in example 4.
Example 5 rational design of preferred mutant Xyn370-Q14H/D29S/A203V
Logging in a protein structure database protein datebank (https:// www.rcsb.org /), selecting a crystal structure with high homology with the three-dimensional structure of the preferred mutant Xyn370-Q14H/D29S/A203V as a template after BLAST comparison, and then carrying out homology modeling by using an online server to obtain the structure diagram of the preferred mutant Xyn 370-Q14H/D29S/A203V. And predicting the B-factor value of the preferred mutant Xyn370-Q14H/D29S/A203V three-dimensional structure model by using an online server to obtain the B-factor value of each residue. Preferred mutants Xyn370-Q14H/D29S/A203V have high B-factor amino acid residues as shown in Table 6.
TABLE 6
Residue number 30 31 41 52 53 108 109 110 130 170
B-factor value 29.21 30.22 32.17 30.87 30.883 33.92 35.29 33.34 30.71 30.36
According to Table 6, residues with higher B-factor values were selected for saturation mutagenesis to construct a mutation library.
The saturation mutagenesis method was consistent with example 4 above and the library screening method was consistent with example 1 or example 2 above. The inventors further screened from the mutant library that the mutation at site S30P, T31H, K3541R, A L, Q C had an increasing effect on the thermostability of the preferred mutant Xyn370-Q14H/D29S/A203V, and subsequently introduced the preferred mutant Xyn370-Q14H/D29S/A203V in an iterative combination of S30P, S30P/T31H, S30P/T31H/A52L, S30P/T31H/A52L/K41R, S30P/T31H/A52L/Q131C, respectively, and expressed and purified to determine the thermostability. Preferably, the residual enzyme activities (%) of xylanase mutants Xyn370-Q14H/D29S/A203V after introduction of different mutation sites and after different times of heat treatment are shown in Table 7 (in the table, xyn370-Q14H/D29S/A203V is abbreviated as Xyn 370-A29S).
TABLE 7
As can be seen from Table 7, the heat stability of the mutant Xyn370-Q14H/D29S/A203V/S30P, xyn370-Q14H/D29S/A203V-S30P/T31H, xyn370-Q14H/D29S/A203V-S30P/T31H/A52L, xyn370-Q14H/D29S/A203V-S30P/T31H/A52L/K41R, xyn370-Q14H/D29S/A203V/S30P/T31H/A52L/Q131C is improved to a different extent compared with that of the starting strain in example 4, example 5 or example 6, wherein the heat stability of the mutant Xyn 370-Q14H/D29S/A203V-S30P/A30P/B30H/A52L, xyn 370-Q14H/S29S 30S/A30S/S29V, xyn 370-Q14H/S29S/A15V/S30S/S30S 15C is significantly improved, and the residual heat stability of the mutant Xyn 370-Q31H/S52L/Q131C is significantly improved at about 20% after heat treatment of the left heat treatment of only 20% and the left heat stability of the mutant is significantly improved to about 80.50.50.50.35.3% compared with the starting strain in example 4, example 5 or example 6.
EXAMPLE 6 specific enzyme Activity determination of xylanase mutant and Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L back mutation
The specific enzyme activities of the wild type and the preferred mutant strain were measured at 37℃using beech (purchased from Sigma) xylan as a substrate, the units of U/mg, and the specific enzyme activities of the xylanase mutant strain are shown in Table 8 (Xyn 370-Q14H/D29S/A203V in the tables are abbreviated as Xyn 370-A29S).
TABLE 8
As can be seen from Table 8, the specific enzyme activity of the preferred mutants is reduced, and in the modification of the thermostability of the protein, the improvement of thermostability is often unavoidable at the expense of the enzyme activity. In order to balance the relationship between enzyme activity and thermostability, the present inventors performed reversion of Q14H, D29S, A203V of the preferred mutant Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L to Q14, D29, A203V, respectively, and measured the residual enzyme activity change and specific enzyme activity after various time treatments at 85 ℃. The residual enzyme activities (%) and specific enzyme activities (U/mg) of Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L back mutations after heat treatment at 85℃for different times are shown in Table 9.
TABLE 9
As can be seen from Table 9, after Q14H, D29S and A203V were reverted to Q14, D29 and A203 respectively, the heat stability was reduced, the decrease in D29 heat stability was most obvious and the effect of A203 was minimal compared with the starting strain Xyn 370-Q14H/D29S/A203V/S30P/T31H/A52L. However, compared with the original strain, the specific enzyme activity of the strain is increased, wherein Q14 is increased maximally, and the enzyme activity of xylanase is increased from 593.2 +/-2.7 to 1158.8+/-24.4 of the original strain, so that three sites of Q14, D29 and A203 are proved to have great influence on the enzyme activity of xylanase.
Subsequently, the inventors constructed mutant Xyn370-Q14/D29S/A203/S30P/T31H/A52L, which had a further increased enzyme activity than that of the mutant Xyn 370-Q14/D29S/A203/S31P/T31H/A52L, by simultaneously back-mutating Q14 and A203 (1554.7.+ -. 156.8, table 9).
In summary, the sequence information of the wild type and mutant are summarized in FIGS. 1-1 to 1-13.
The mutant strain can be reasonably selected in practical application, the relation between the heat stability and the enzyme activity can be better balanced, and the proper mutant strain is selected according to the practical production requirement so as to achieve the purpose.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> China academy of sciences and Intelligent technology Excellent innovation center
<120> xylanase mutant with improved thermostability, preparation and use thereof
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cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgccg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 4
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn Gln His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Asp Ser Thr Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Ala Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 5
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcgccagc accggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccgccgtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 6
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ala Ser Thr Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 7
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcagc accggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccgccgtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 8
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Ser Thr Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 9
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg accggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccgccgtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 10
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro Thr Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 11
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccgccgtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 12
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 13
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 14
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 15
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
cgcggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 16
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Arg Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 17
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc tgcggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 18
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Cys Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 19
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc agcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 20
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn Gln His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 21
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcgacccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgtcg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 22
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Asp Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Val Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 23
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc atcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgccg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 24
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn His His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Ala Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225
<210> 25
<211> 687
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
atggccggcc agcgcctgtc ggtcggcggc ggccagaacc agcacaaggg cgtctttgac 60
ggcttttcct acgagatttg ggtcagcccg catggcggct cgggcagcat gaccctcggc 120
aagggcgcca cgttcaaggc cgagtggtcc gccctggtca accgcggcaa cttcctcgcc 180
cgccgcggcc tggactttgg ctcgaccaag aaggccacgg cctacgagta catcggcctg 240
gactacgagg cctcgtaccg ccagacggcc tccgcctcgg gcaacagccg cctctgcgtc 300
tacggctggt tccagaaccg cggcgtccag ggcgtccccc tggtcgagta ctacatcatt 360
gaggactggg tcgactgggt ccccgacgcc cagggcgaga tggtcaccat cgacggcgcc 420
cagtacaaga ttttccagat ggaccacacc ggccccacga tcaacggtgg caacgagacg 480
ttcaagcagt actttagcgt ccgccagcag aagcgcacca gcggccacat cacggtctcc 540
gaccacttta aggcctgggc caaccagggc tgggacattg gcaacctcta cgaggtcacc 600
ctgaacgccg agggctggca gagctccggc gtcgccgacg tcaccaagct ggacgtctac 660
accaccaagc agggctccgc cccccgc 687
<210> 26
<211> 229
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Met Ala Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn Gln His Lys
1 5 10 15
Gly Val Phe Asp Gly Phe Ser Tyr Glu Ile Trp Val Ser Pro His Gly
20 25 30
Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu
35 40 45
Trp Ser Ala Leu Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu
50 55 60
Asp Phe Gly Ser Thr Lys Lys Ala Thr Ala Tyr Glu Tyr Ile Gly Leu
65 70 75 80
Asp Tyr Glu Ala Ser Tyr Arg Gln Thr Ala Ser Ala Ser Gly Asn Ser
85 90 95
Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val
100 105 110
Pro Leu Val Glu Tyr Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro
115 120 125
Asp Ala Gln Gly Glu Met Val Thr Ile Asp Gly Ala Gln Tyr Lys Ile
130 135 140
Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr
145 150 155 160
Phe Lys Gln Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His
165 170 175
Ile Thr Val Ser Asp His Phe Lys Ala Trp Ala Asn Gln Gly Trp Asp
180 185 190
Ile Gly Asn Leu Tyr Glu Val Thr Leu Asn Ala Glu Gly Trp Gln Ser
195 200 205
Ser Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln
210 215 220
Gly Ser Ala Pro Arg
225

Claims (27)

1. A method of increasing the thermostability of a xylanase comprising: a mutation in the amino acid sequence of the xylanase, corresponding to a wild-type xylanase, said mutation being selected from the group consisting of:
(1) Mutation at position 14 to His and mutation at position 28 to Val; the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 th mutation is Asp, and the 203 th mutation is Val;
(2) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 rd mutation is Asp, the 203 th mutation is Val, and the 41 st mutation is Arg;
(3) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ser, the 203 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 131 th mutation is Cys, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(4) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 133 to Glu, mutation at position 192 to Asp;
(5) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 133 to Glu, mutation at position 192 to Asp;
(6) The 29 th mutation is Ser, the 28 th mutation is Val, the 203 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(7) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, mutation at position 192 to Asp;
(8) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, mutation at position 192 to Asp;
(9) The 29 th mutation is Ser, the 28 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(10) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Ser, mutation at position 203 to Val, mutation at position 133 to Glu, mutation at position 192 to Asp;
(11) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ala, the 30 th mutation is Ser, the 203 th mutation is Val, the 133 th mutation is Glu, and the 192 th mutation is Asp; or (b)
(12) Mutation at position 28 to Val, mutation at position 30 to Ser, mutation at position 133 to Glu, mutation at position 192 to Asp;
the amino acid sequence of the wild xylanase is shown as SEQ ID NO. 2.
2. A xylanase mutant, wherein said xylanase mutant is selected from the group consisting of proteins of: corresponding to the wild-type xylanase enzyme, the enzyme,
(1) Mutation at position 14 to His and mutation at position 28 to Val; the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 th mutation is Asp, and the 203 th mutation is Val;
(2) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 rd mutation is Asp, the 203 th mutation is Val, and the 41 st mutation is Arg;
(3) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ser, the 203 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 131 th mutation is Cys, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(4) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 133 to Glu, mutation at position 192 to Asp;
(5) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 133 to Glu, mutation at position 192 to Asp;
(6) The 29 th mutation is Ser, the 28 th mutation is Val, the 203 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(7) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, mutation at position 192 to Asp;
(8) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, mutation at position 192 to Asp;
(9) The 29 th mutation is Ser, the 28 th mutation is Val, the 30 th mutation is Pro, the 31 st mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, and the 192 th mutation is Asp;
(10) Mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Ser, mutation at position 203 to Val, mutation at position 133 to Glu, mutation at position 192 to Asp;
(11) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ala, the 30 th mutation is Ser, the 203 th mutation is Val, the 133 th mutation is Glu, and the 192 th mutation is Asp; or (b)
(12) Mutation at position 28 to Val, mutation at position 30 to Ser, mutation at position 133 to Glu, mutation at position 192 to Asp;
the amino acid sequence of the wild xylanase is shown as SEQ ID NO. 2.
3. An isolated polynucleotide encoding the xylanase mutant of claim 2.
4. The polynucleotide of claim 3, wherein the polynucleotide has the nucleotide sequence set forth in SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 11, SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 7, SEQ ID NO. 5 or SEQ ID NO. 3.
5. A vector comprising the polynucleotide of claim 4.
6. A genetically engineered host cell comprising the vector of claim 5, or having integrated into its genome the polynucleotide of claim 4.
7. A method of preparing a xylanase mutant according to claim 2, comprising:
(i) Culturing the host cell of claim 6;
(ii) Collecting a culture containing the xylanase mutant of claim 2;
(iii) Isolating the xylanase mutant from the culture.
8. A composition for degrading xylan, comprising an effective amount of: the xylanase mutant of claim 2; or, the host cell of claim 6 or a culture or lysate thereof containing the xylanase mutant of claim 2; and
a pharmaceutically or industrially acceptable carrier.
9. Use of a xylanase mutant according to claim 2 for degrading xylan.
10. The use according to claim 9, wherein the xylanase mutant cleaves a β -1, 4-glycosidic bond, thereby hydrolyzing xylan.
11. Use of the composition of claim 8 for degrading xylan.
12. The use according to claim 11, wherein the xylanase mutant cleaves a β -1, 4-glycosidic bond, thereby hydrolyzing xylan.
13. The use according to any one of claims 9 to 12, wherein the xylan-degrading substance comprises: industrial products, agricultural, forestry, industrial waste and municipal solid waste.
14. The use according to any one of claims 9 to 12, wherein the xylan-degrading substance comprises: pulp, feed, food, straw, hardwood, softwood, straw, and bran.
15. Use of the xylanase mutant of claim 2 or the composition of claim 8 comprising:
as a food additive;
as a plant processing additive;
as an additive in the papermaking process;
as an additive in textile processing;
as a feed additive; and/or
Is applied to the conversion of lignocellulose into fuel ethanol.
16. A method of degrading xylan, said method comprising: degrading xylan with the xylanase mutant of claim 2 or the composition of claim 8.
17. The method of claim 16, wherein the xylanase mutant cleaves a β -1, 4-glycosidic bond, thereby hydrolyzing xylan.
18. The method of claim 16 or 17, wherein the degradation or cleavage is a degradation or cleavage at elevated temperature; the high temperature is 45-95 ℃.
19. The method of claim 18, wherein the elevated temperature is 50 to 90 ℃.
20. The method of claim 16, wherein the substrate degraded by the xylanase mutant or composition is xylan or a xylan-containing material.
21. The method of claim 20, wherein the xylan-containing material comprises: industrial products, agricultural, forestry, industrial waste and municipal solid waste.
22. The method of claim 20, wherein the xylan-containing material comprises: pulp, feed, food, straw, hardwood, softwood, straw, and bran.
23. A library of xylanase mutants or polynucleotides encoding the same, the library comprising: the xylanase mutant of claim 2, or at least 5 of the polynucleotides encoding the same.
24. The library of claim 23, wherein the library comprises: the xylanase mutant of claim 2, or at least 10 of the polynucleotides encoding the same.
25. The library of claim 24, comprising the xylanase mutant of claim 2 or a polynucleotide encoding the same.
26. Use of a library of xylanase mutants or polynucleotides encoding them according to claim 23 for providing an appropriate xylanase mutant for degrading xylan, depending on the temperature of the reaction system in which the xylan is to be degraded.
27. A kit for degrading xylan comprising:
the xylanase mutant or combination of mutants of claim 2;
the host cell of claim 6;
the composition of claim 8; or (b)
A library of xylanase mutants or polynucleotides encoding the same according to claim 23.
CN202010693613.3A 2020-07-17 2020-07-17 Xylanase mutant with improved thermostability, preparation and application thereof Active CN113943723B (en)

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