CN110656099A - Xylanase mutant with high specific activity at 40 ℃ and construction method and application thereof - Google Patents

Abstract

The invention discloses a xylanase mutant with high specific activity at 40 ℃ and a construction method and application thereof, and relates to the fields of genetic engineering and genetic engineering. The invention takes an N-terminal truncated mutant XYL 10C-delta N of GH10 family high-temperature xylanase XYL10C derived from Bispora sp MEY-1 as a female parent, and adopts a molecular biology technology to perform amino acid site-directed mutagenesis and then express the amino acid site-directed mutagenesis. Under the modification condition, the specific activity of the xylanase variant at 40 ℃ is obviously improved compared with that of a wild type. The thermal stability is improved, the high-temperature granulation process of enzyme resistance is facilitated, and finally inactivation is avoided, so that conditions are created for the application of the compound feed in feed; the stability of the cellulose reducing sugar is improved in an acidic environment, the lignocellulose is degraded into reducing sugar, and the application prospect of the cellulose reducing sugar is widened.

Description

Xylanase mutant with high specific activity at 40 ℃ and construction method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a xylanase mutant with high specific activity at 40 ℃ and a construction method and application thereof.

Background

Cellulose, hemicellulose (xylan, mannan, xyloglucan, beta- (1,3/1,4) -glucan and the like, lignin, pectin and the like are main components constituting plant cell walls, and are also main components of non-starch polysaccharides, cellulose and xylan are the most abundant in plant cell wall polysaccharides, accounting for 40.6-51.2% and 28.5-37.2%, respectively (Pauly et al, 2008). xylan has a complex structure and a main chain connected by xylopyranose through beta-D-1, 4-xylosidic bonds and is provided with various substituents (Collins et al, 2005).

Xylanases are the most critical class of enzymes in the degradation process of xylan, and they are capable of degrading the β -1, 4-glycosidic bonds of the xylan backbone. Xylanases are very widely distributed in nature, with xylanases of microbial origin being the most commonly used material in practical applications and research today. Xylanases are distributed among glycoside hydrolases of families 5, 7, 8, 10, 11, 30 and 43, and the studies of xylanases of families 10 and 11 have been reported most so far. The catalytic activity of the xylanase of the 11 th family is relatively high, but the stability is generally poor; compared with xylanase of family 11, xylanase of family 10 has better thermal stability but lower catalytic efficiency, especially low catalytic activity under the condition of animal body temperature, and the application of xylanase of family 10 in feed industry is greatly limited.

For the last decade, research on family 10 xylanases has focused mainly on gene mining and thermostability of high temperature xylanases. And (4) improving the sexual function. Such as: bispora sp derived XYL10C (Luo et al, 2009) and its truncated mutants XYL10C- Δ N (You et al, 2018), glycophytolum trabeum derived xylanase GtXyn10(Wang et al, 2016), and the like. Xylanases similar to those mentioned above are resistant to the high temperature pelletization process, but do not exhibit catalytic activity in animals up to 20% of the maximum activity, and in this respect do not fully meet the criteria for feed addition. Therefore, in order to meet the requirements of the feed industry, it is necessary to improve the catalytic activity of high temperature xylanase under the condition of animal body temperature. On the one hand, new enzymes are obtained, and on the other hand, modification of enzymes derived from natural biological resources at the molecular level is required. Generally, molecular modification technology is adopted to improve the thermal stability of xylanase so as to adapt to the requirements of xylanase in industrial production. However, no systematic report is found on the influence mechanism and molecular improvement research of the catalytic activity of the high-temperature xylanase under the condition of animal body temperature.

The xylanase engineering strain with high specific activity and high catalytic efficiency is obtained mainly through mutagenesis, screening and enzyme molecule improvement. The mutagenesis is divided into natural mutagenesis and artificial mutagenesis, the probability of success of the natural mutagenesis is very small, the workload of the artificial mutagenesis is large, the beneficial mutation frequency is still low, and the direction and the property of the mutagenesis are difficult to control. The screening blindness is large, and the target strain is not easy to obtain. The improvement purpose of the enzyme molecules is strong, and the specific structure analysis of the enzyme molecules is modified, so that the purpose of improving the specific activity is achieved.

Disclosure of Invention

Aiming at the technical problems, the invention provides a xylanase mutant with high specific activity at 40 ℃, a construction method and application thereof, wherein the mutant is obtained by mutating amino acid sites on an active loop region of xylanase.

A xylanase mutant with high specific activity at 40 deg.C is prepared through selecting mutation site R based on the sequence SEQ ID No.11 of wild xylanase, and mutating.

The improvement is that when the mutation site R is M137E, the mutant is XYL delta N-M137E, and the nucleotide sequence is shown as SEQ ID NO. 1;

when the mutation site R is MF137/138DA, the mutant is XYL delta N-MF137/138DA, and the nucleotide sequence is shown as SEQ ID NO. 2;

when the mutation site R is MF137/138DS, the mutant is XYL delta N-MF137/138DS, and the nucleotide sequence is shown as SEQ ID NO. 3;

when the mutation site R is MF137/138QP and the xylanase mutant is XYL delta N-MF137/138QP, the nucleotide sequence is shown as SEQ ID NO. 4;

when the mutation site is RMF137/138SL, and the xylanase mutant is XYL delta N-MF137/138SL, the nucleotide sequence is shown as SEQ ID NO. 5.

The nucleotide sequence of SEQ ID No. 1:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCGAGTTCACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the nucleotide sequence of SEQ ID No. 2:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCGACGCCACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the nucleotide sequence of SEQ ID No. 3:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCGACTCTACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the nucleotide sequence of SEQ ID No. 4:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCCAGCCAACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the nucleotide sequence of SEQ ID No. 5:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCAGCTTGACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the amino acid sequence of the xylanase mutant encoding XYL delta N-M137E is shown in SEQ ID NO. 6; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138DA is shown in SEQ ID NO. 7; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138DS is shown in SEQ ID NO. 8; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138QP is shown as SEQ ID NO. 9; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138SL is shown in SEQ ID NO. 10.

The amino acid sequence of SEQ ID No. 6:

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFEFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

the amino acid sequence of SEQ ID No. 7:

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFDATEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

the amino acid sequence of SEQ ID No. 8:

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFDSTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

the amino acid sequence of SEQ ID No. 9:

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFQPTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

amino acid sequence of SEQ ID No. 10:

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFSLTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

the nucleic acid sequence of SEQ ID NO. 11:

TGGGGTCTTAATAATGCAGCTCGAGCCGATGGCAAGCTTTGGTTTGGAACTGCTGCAGATATCCCCGGTTTAGAGCAGGATGATCGCTATTACATGAAGGAATACAACAATACGCATGATTTTGGTGGTACCACACCCGCGAATATTATGAAATTCATGTTCACGGAGCCAGAGCAAAACGTTTTTAATTTCACCGGCGCGCAGGAGTTCCTGGACATTGCCTTTGCGTCGCACAAGCTTGTTCGTTGCCACAATCTTATCTGGCAATCCGAGCTTCCCACATGGGTTACTAACCCTACCACAAATTGGACAAACGAAACCTTGAGCAAGGTGCTACAAAATCATGTATATACTCTAGTCTCACATTTTGGAGATCAGTGCTATAGCTGGGATGTGGTTAACGAAGCCCTCTCTGATGACCCAGCCGGATCGTATCAAAACAATATCTGGTTCGACACTATTGGTCCCGAGTACGTTGCGATGGCATTCGAGTATGCCGAGAAAGCCGTCAAAGACCATAAGTTGAATGTTAAGCTCTACTACAATGACTACAACATTGAATATCCTGGGCCCAAATCTACAGCAGCACAGAATATTGTCAAGGAGCTTAAAGCAAGGAACATCCAAATAGATGGCGTCGGCCTTGAGTCCCACTTCATCGCTGGTGAAACTCCGTCTCAGGCTACGCAAATCACAAACATGGCTGATTTCACTTCTCTTGACATTGACGTTGCTGTTACCGAGCTCGATGTACGTCTTTATCTGCCTCCAAATGCTACCAGCGAGGCCCAGCAAGTTGCCGACTATTACGCCACCGTCGCAGCCTGTGCTGCAACAGAACGCTGTATCGGTATAACTGTCTGGGATTTTGACGATACATATTCATGGGTGCCCAGCACGTTCGCCGGCCAAGGGTATGCGGATCTGTTCTTCCAGCCAGACGGCCCCAACACTCCCCTAGTGAAAAAAGCGGCGTACGACGGTTGCCTACAGGCTTTGCAACATAAGGCGGAAAGTCCATGA

the construction method of the xylanase mutant with high specific activity at 40 ℃ comprises the following steps:

1) amplifying xylanase mutant sequence segments with high catalytic efficiency by adopting an over-lap PCR method;

2) cloning the sequence fragment of the xylanase mutant between restriction sites EcoR I and Not I of an expression vector pPIC9r to obtain a recombinant vector;

3) transforming the mutant recombinant vector into pichia pastoris GS115, and carrying out induced expression to obtain a mutant strain;

4) culturing the mutant strain, and inducing the expression of the recombinant xylanase;

5) recovering and purifying the expressed high specific activity xylanase mutant.

The specific activities of the obtained mutant XYL delta N-M137E, XYL delta N-MF137/138DS, XYL delta N-MF137/138DA, XYL delta N-MF137/138QP and XYL delta N-MF137/138SL at the temperature of 40 ℃ are respectively 1.8 times, 2.2 times and 1.7 times of that of the wild type; the optimal temperature of the enzymatic reaction is 80-85 ℃; the optimum pH value is between 4.0 and 5.0.

The application of the xylanase mutant with high specific activity at 40 ℃ in feed additives.

The application of the xylanase mutant with high specific activity at 40 ℃ in degradation of lignocellulose.

Has the advantages that:

compared with the prior art, the xylanase mutant provided by the invention has excellent properties and is suitable for being applied to lignocellulose degradation. The xylanase mutant has the optimum pH value of 4.0-5.0, is not changed much compared with a wild type, but has specific activity of 1.8 times, 2.2 times and 1.7 times of the wild type at 40 ℃, the temperature environment of the intestinal tract of an animal is about 40 ℃, and the catalytic activity of the enzyme at 40 ℃ is improved, so that the enzyme can better exert a degradation function in the digestive tract of the animal; the thermal stability at 90 ℃ is improved to different degrees compared with that of wild enzyme, an instant high-temperature process (80-90 ℃ for 10s) is carried out in the animal feed granulation process, the thermal stability of the enzyme at 90 ℃ is improved, the enzyme resistance high-temperature granulation process is facilitated, and finally inactivation is avoided; the optimum pH value is basically consistent with the mulberry bark degradation condition, stable activity can be kept in a low temperature range, and the mutant has excellent property as shown by the test result of the straw degradation cooperated with the cellulase. The xylanase which has higher enzyme activity under the conditions of acid pH and animal body temperature and is stable under the high temperature condition is considered as an enzyme for feed addition with excellent property in the feed industry, and has very wide application prospect.

Compared with the methods such as blind-mesh bacteria or artificial (natural) mutagenesis and the like, the enzyme molecule improvement shortens the time for modifying the enzymology property. The xylanase mutant which is stable in an acidic pH environment and in a medium-low temperature range and has high enzyme activity has wide application prospect when being applied to lignocellulose degradation to produce reducing sugar.

Drawings

FIG. 1 is an SDS-PAGE analysis of high specific activity xylanase mutants at 40 ℃ wherein M is a low molecular weight protein Marker; A-F are respectively purified wild enzyme XYL10C-N, mutant XYL delta N-M137E, XYL delta N-MF137/138DS, mutant XYL delta N-MF137/138DA, mutant XYL delta N-MF137/138QP and XYL delta N-MF137/138 SL;

FIG. 2 shows the optimum pH of the mutant xylanase with high specific activity at 40 ℃ compared with the wild type;

FIG. 3 shows the optimal temperature of the mutant xylanase with high specific activity at 40 ℃ and the wild type xylanase;

FIG. 4 shows the thermostability of the high specific activity xylanase mutant at 40 ℃ and the wild type at 90 ℃;

FIG. 5 shows the content determination of reducing sugar in mulberry twig bark treated by mixing different high specific activity xylanase mutants with cellulase;

FIG. 6 is a comparison of the synergy of high specific activity xylanase mutant and wild type with cellulase under the same conditions.

Detailed Description

The invention is further described below with reference to the accompanying drawings and specific embodiments.

1. Bacterial strain and carrier: the expression host Pichia pastoris GS115, expression plasmid vector pPIC9r was purchased from Invitrogen.

2. Enzymes and other biochemical reagents: the endonuclease was purchased from Fermentas, the ligase from Promaga, and the polygalacturonic acid from Sigma. Other reagents are domestic analytical pure reagents (all can be purchased from common biochemical reagents), mulberry bark is prepared by the mulberry bark self, and the specific method is as follows: and (3) crushing the mulberry twig bark, and mixing the crushed mulberry twig bark with 15% ammonia water according to a solid-liquid mass ratio of 1: 11, soaking, putting in an oven at 60 ℃ overnight, cleaning with sterile water to be neutral after 24 hours, and drying for use.

3. Culture medium:

1) LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;

2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;

4) MM solid medium: 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;

5) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin;

6) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V/V).

EXAMPLE 1 cloning of genes encoding high specific Activity xylanase mutants

GH10 family high temperature xylanase gene Xyl10C derived from Bispora sp.MEY-1 is taken as a male parent, mutation primers are designed at the loop region of xylanase, and the genes encoding xylanase mutants with increased catalytic efficiency, SEQ ID No.1(XYL delta N-M137E, 1023bp), SEQ ID No.2(XYL delta N-MF137/138DA, 1023bp), SEQ ID No.3(XYL delta N-MF137/138DS, 1023bp), SEQ ID No.4(XYL delta N-MF137/138QP, 1023bp) and SEQ ID No.5(XYL delta N-MF137/138SL, 1023bp) are amplified by an over-lap PCR method, and a mutation method and a cloning method reference (You, et al, 2016).

Gene specific primers for high specific activity xylanase mutants at 140 ℃ in table

Example 2 preparation of high specific Activity xylanase mutants

Carrying out double enzyme digestion (EcoR I + Not I) on the expression vector pPIC9r, simultaneously carrying out double enzyme digestion (EcoR I + Not I) on the gene encoding the xylanase mutant with high specific activity, connecting the cut gene segment encoding the mature xylanase mutant with high specific activity with the expression vector pPIC9r, obtaining the recombinant plasmid containing the xylanase mutant gene with high specific activity, and converting Pichia pastoris GS115 to obtain the recombinant yeast strain.

Taking a GS115 strain containing the recombinant plasmid, inoculating the strain into a 1L triangular flask of 300mL BMGY medium, and carrying out shake culture at 30 ℃ and 220rpm for 48 h; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 100mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ for induction culture at 220 rpm. 0.5mL of methanol is added every 12h, so that the concentration of the methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.

The method for determining the specific activity of the recombinant high specific activity xylanase mutant and the wild type at 40 ℃ is the classical DNS method (Miller et al, 1959). The operation method comprises the following steps: firstly diluting an enzyme solution to a proper dilution ratio, generally speaking, when the xylanase is measured by taking beechwood xylan as a substrate, the delta OD is between 0.6 and 1.0, mixing 100 mu l of the diluted enzyme solution with 900 mu l of the substrate with the concentration of 1 percent, wherein the substrate contains a buffer solution with proper pH, putting a glass tube at a required temperature for incubation for 10min after mixing, adding 1.5ml of DNS to stop the reaction, adding 100 mu l of the enzyme solution into a control group after adding 1.5ml of DNS, boiling the control group for five minutes in boiling water, cooling and measuring the absorbance at 540 nm. After the protein is quantified, the corresponding specific activity is calculated.

SDS-PAGE results (FIG. 1) show that the recombinant xylanase is expressed in Pichia pastoris. After the expressed xylanase is purified, the protein content of the xylanase reaches more than 95 percent of the total protein. The optimum pH value of the recombinant xylanase mutant with high catalytic efficiency is between 4.0 and 5.0 (figure 2), and the change is not large compared with that of a wild type; when the zelkova xylan is used as a substrate, the specific activities of the recombinant high specific activity xylanase mutant and the wild type at 40 ℃ are 1400U/mg, 1690U/mg, 1700U/mg, 1300U/mg and 760U/mg respectively, and the mutant is 1.8 times, 2.2 times and 1.7 times of the wild type respectively (Table 2).

TABLE 2 comparison of specific Activity and catalytic efficiency of wild enzymes and mutants

Example 3 Activity analysis of recombinant high specific Activity xylanase mutants and wild type

The DNS method: the specific method comprises the following steps: under the conditions of given pH and temperature, 1mL of reaction system comprises 100 mu L of enzyme solution and 900 mu L of substrate, the reaction is carried out for 10min, 1.5mL of LNNS is added to stop the reaction, and the reaction is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down xylan to 1. mu. mol reducing sugars per minute under the given conditions.

2. Determination of the Properties of the mutant and wild type recombinant high-specific-Activity xylanases

1) The method for measuring the optimum pH of the recombinant high specific activity xylanase mutant and the wild type comprises the following steps:

the recombinant high specific activity xylanase mutant purified in example 2 and the wild type were subjected to enzymatic reactions at different pH to determine their optimum pH. The substrate (beech xylan) was diluted with 0.1mol/L citric acid-disodium hydrogen phosphate buffer at different pH and xylanase activity measurements were performed at 80 ℃.

The results (FIG. 2) show that the optimum reaction pH of the recombinant xylanase mutant and the wild type is between 4.0 and 5.0, and the same action trend exists in the pH range of 3.0 to 6.5. The purpose of improving the specific activity at lower temperature without changing the optimum pH value is met.

2) The method for measuring the optimal temperature of the recombinant high specific activity xylanase mutant and the wild type comprises the following steps:

the optimal temperature of the recombinant high specific activity xylanase mutant and the wild type is determined by performing enzymatic reactions in a 0.1mol/L citrate-disodium hydrogen phosphate buffer (pH 3.5) buffer system at different temperatures. The results of the enzyme reaction optimum temperature measurement (figure 3) show that the optimum temperature of the recombinant xylanase mutant and the wild type is between 85 ℃ and 90 ℃, and the difference between the optimum temperature and the wild type is not obvious.

3) The thermal stability of the recombinant high specific activity xylanase mutant and the wild type at 90 ℃ is determined by the following method:

treating the high specific activity xylanase mutant and the wild type at 90 ℃ for 5, 10, 20 and 30min respectively, ensuring that the concentration of all the mutant and the wild type is 100ug/mL and the volume is 100ul during treatment, sampling at different time points, quickly placing on ice, and measuring the enzyme activity at 85 ℃ and pH 4.5 to measure the thermal stability of the high specific activity xylanase mutant and the wild type.

The determination result shows that the heat stability of all mutants at 90 ℃ is better than that of the wild type (figure 4), and the residual enzyme activity of the mutants is between 60% and 95% and is higher than that of the wild type (60%) when the mutants are treated for 5 min.

Example 4 degradation of Mulberry bark by recombinant high specific activity xylanase mutant and cellulase

1. Firstly, enzyme activity unit calibration is carried out on cellulase and all xylanase by taking CMC and beechwood xylan as substrates respectively at pH5.0 and 40 ℃. Selecting XYL 10C-delta N, XYL delta N-MF137/138DA and XYL delta N-MF137/138QP to be respectively mixed with cellulase according to the weight ratio of 1: 1 (10U in all) of the mulberry twig bark is degraded after being treated.

The experimental group comprises 10U of cellulase and 10U of xylanase, and the cellulase and the xylanase are respectively 10U. The control group was prepared by changing the enzyme solution to a 1% buffer of the same volume, pH5.0, for a reaction time of 24 hours, and sampling 1.2mL at 1 hour intervals for 16 times. Cooling in boiling water bath for 1min, centrifuging in a centrifuge tube at 12000r/min for 10min, and storing the supernatant at-20 deg.C. The total amount of reducing sugar in the sample is calculated by using a DNS method. The DNS method comprises the following steps: the specific method comprises the following steps: under the conditions of pH5.0 and 40 ℃, 1mL of reaction system comprises 100 μ L of enzyme solution and 900 μ L of substrate, the reaction is carried out for 10min, 1.5mL of DNS is added to stop the reaction, and the mixture is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down xylan to 1. mu. mol reducing sugars per minute under the given conditions.

2. The effect of the enzyme on the treatment of mulberry twigs was characterized by measuring the content of reducing sugars in the samples by the DNS method, and the results are shown in fig. 5. It can be seen that xylanase alone treated mulberry branches finally produced reducing sugars in an amount of 0.3-0.5. mu. mol/mL, wherein the amounts of reducing sugars produced by the mutant XYL.DELTA.N-MF 137/138QP, XYL.DELTA.N-MF 137/138DA and the wild-type enzyme at 15h were 0.5, 0.4 and 0.3. mu. mol/mL, respectively, and the amount of reducing sugars did not increase significantly after 2h of reaction. The amount of reducing sugars produced by the cellulase-treated mulberry branches alone was much greater than the amount of reducing sugars produced by the xylanase-treated mulberry branches alone, and the amount of reducing sugars produced increased gradually over time, reaching about 3.2. mu. mol/mL at 15 h.

The effect of synergistic treatment of mulberry branches by cellulase and xylanase is better, the amount of the generated reducing sugar is more than that of a treatment group with single addition of cellulase, and the amount of the reducing sugar is gradually increased along with the passage of time. The reducing sugar amount generated by co-treating mulberry twigs with the mutant and the cellulase is larger than the co-treating effect of the wild enzyme and the cellulase, particularly the combination of the mutant XYL delta N-MF137/138QP and the cellulase, the reducing sugar yield reaches 4.9 mu mol/mL at 15h, is 9.8 times of the treating effect of single xylanase, and is 1.5 times of the treating effect of single cellulase. The amount of reducing sugar produced by mulberry twigs treated with the combination of the mutant XYL delta N-MF137/138DA and cellulose at 15h was 4.3. mu. mol/mL, which was 10.8 times the effect of xylanase treatment alone and 1.3 times the effect of cellulase treatment alone. In conclusion, the synergistic effect of the mutant and the cellulase is greater than that of the single cellulase, and the single cellulase is greater than that of the single xylanase, so that the synergistic effect of the cellulase and the xylanase can better degrade mulberry branches, and more reducing sugar is generated.

To better compare the synergistic effects of different xylanases and cellulases, we compared the analysis by calculating the synergy. The synergy is expressed by DS, and the calculation formula is as follows:

DS＝Y1+2/Y1+Y2

y1: the amount of reducing sugars produced by the action of cellulase alone;

y2: the amount of reducing sugars produced by xylanase alone;

y1+ 2: the amount of reducing sugar produced by the synergistic action of xylanase and cellulase;

as shown in FIG. 6, from the 3h, the wild enzyme XYL10C- Δ N has a synergistic ratio with cellulase of more than 1. The synergy rates of the xylanase mutants XYL delta N-MF137/138DA and XYL delta N-MF137/138QP and the cellulase from the 1h are both more than 1 and are respectively 1.1 and 1.2. The highest synergy appears at 13h and 15h respectively, and DS reaches 1.3 and 1.5 respectively. The cellulase and the mutant have better synergistic effect, and the effect that 1+1 is more than 2 is achieved. The synergy of the mutant XYL delta N-MF137/138QP and the cellulase is the highest, and the synergy of the mutant XYL delta N-MF137/138DA and the cellulase is the second. This also confirms the results of the previous analysis, and further shows that the mutant MF137/138DA and cellulase synergistic treatment has the best effect on the treatment of mulberry bark and produces the highest amount of reducing sugar.

Sequence listing

<120> xylanase mutant with high specific activity at 40 ℃, and construction method and application thereof

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattcgagt tcacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 2

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattcgacg ccacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 3

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattcgact ctacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 4

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattccagc caacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 5

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattcagct tgacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 7

<211> 340

<212> PRT

<213> Amino acid (Amino acid)

<400> 7

Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly

1 5 10 15

Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met

20 25 30

Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn

35 40 45

Ile Met Lys Phe Glu Phe Thr Glu Pro Glu Gln Asn Val Phe Asn Phe

50 55 60

Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu

65 70 75 80

Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val

85 90 95

Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu

100 105 110

Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr

115 120 125

Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Ala Gly Ser

130 135 140

Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala

145 150 155 160

Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn

165 170 175

Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys

180 185 190

Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile

195 200 205

Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr

210 215 220

Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu

225 230 235 240

Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro

245 250 255

Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr

260 265 270

Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp

275 280 285

Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln

290 295 300

Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu

305 310 315 320

Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys

325 330 335

Ala Glu Ser Pro

340

<210> 7

<211> 340

<212> PRT

<213> Amino acid (Amino acid)

<400> 7

Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly

1 5 10 15

Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met

20 25 30

Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn

35 40 45

Ile Met Lys Phe Asp Ala Thr Glu Pro Glu Gln Asn Val Phe Asn Phe

50 55 60

Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu

65 70 75 80

Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val

85 90 95

Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu

100 105 110

Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr

115 120 125

Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Ala Gly Ser

130 135 140

Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala

145 150 155 160

Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn

165 170 175

Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys

180 185 190

Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile

195 200 205

Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr

210 215 220

Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu

225 230 235 240

Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro

245 250 255

Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr

260 265 270

Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp

275 280 285

Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln

290 295 300

Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu

305 310 315 320

Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys

325 330 335

Ala Glu Ser Pro

340

<210> 8

<211> 340

<212> PRT

<213> Amino acid (Amino acid)

<400> 8

Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly

1 5 10 15

Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met

20 25 30

Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn

35 40 45

Ile Met Lys Phe Asp Ser Thr Glu Pro Glu Gln Asn Val Phe Asn Phe

50 55 60

Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu

65 70 75 80

Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val

85 90 95

Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu

100 105 110

Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr

115 120 125

Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Ala Gly Ser

130 135 140

Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala

145 150 155 160

Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn

165 170 175

Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys

180 185 190

Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile

195 200 205

Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr

210 215 220

Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu

225 230 235 240

Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro

245 250 255

Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr

260 265 270

Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp

275 280 285

Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln

290 295 300

Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu

305 310 315 320

Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys

325 330 335

Ala Glu Ser Pro

340

<210> 9

<211> 340

<212> PRT

<213> Amino acid (Amino acid)

<400> 9

Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly

1 5 10 15

Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met

20 25 30

Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn

35 40 45

Ile Met Lys Phe Gln Pro Thr Glu Pro Glu Gln Asn Val Phe Asn Phe

50 55 60

Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu

65 70 75 80

Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val

85 90 95

Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu

100 105 110

Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr

115 120 125

Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Ala Gly Ser

130 135 140

Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala

145 150 155 160

Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn

165 170 175

Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys

180 185 190

Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile

195 200 205

Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr

210 215 220

Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu

225 230 235 240

Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro

245 250 255

Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr

260 265 270

Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp

275 280 285

Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln

290 295 300

Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu

305 310 315 320

Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys

325 330 335

Ala Glu Ser Pro

340

<210> 10

<211> 340

<212> PRT

<213> Amino acid (Amino acid)

<400> 10

Trp Gly Leu Asn Asn Ala Ala Arg Ala Asp Gly Lys Leu Trp Phe Gly

1 5 10 15

Thr Ala Ala Asp Ile Pro Gly Leu Glu Gln Asp Asp Arg Tyr Tyr Met

20 25 30

Lys Glu Tyr Asn Asn Thr His Asp Phe Gly Gly Thr Thr Pro Ala Asn

35 40 45

Ile Met Lys Phe Ser Leu Thr Glu Pro Glu Gln Asn Val Phe Asn Phe

50 55 60

Thr Gly Ala Gln Glu Phe Leu Asp Ile Ala Phe Ala Ser His Lys Leu

65 70 75 80

Val Arg Cys His Asn Leu Ile Trp Gln Ser Glu Leu Pro Thr Trp Val

85 90 95

Thr Asn Pro Thr Thr Asn Trp Thr Asn Glu Thr Leu Ser Lys Val Leu

100 105 110

Gln Asn His Val Tyr Thr Leu Val Ser His Phe Gly Asp Gln Cys Tyr

115 120 125

Ser Trp Asp Val Val Asn Glu Ala Leu Ser Asp Asp Pro Ala Gly Ser

130 135 140

Tyr Gln Asn Asn Ile Trp Phe Asp Thr Ile Gly Pro Glu Tyr Val Ala

145 150 155 160

Met Ala Phe Glu Tyr Ala Glu Lys Ala Val Lys Asp His Lys Leu Asn

165 170 175

Val Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile Glu Tyr Pro Gly Pro Lys

180 185 190

Ser Thr Ala Ala Gln Asn Ile Val Lys Glu Leu Lys Ala Arg Asn Ile

195 200 205

Gln Ile Asp Gly Val Gly Leu Glu Ser His Phe Ile Ala Gly Glu Thr

210 215 220

Pro Ser Gln Ala Thr Gln Ile Thr Asn Met Ala Asp Phe Thr Ser Leu

225 230 235 240

Asp Ile Asp Val Ala Val Thr Glu Leu Asp Val Arg Leu Tyr Leu Pro

245 250 255

Pro Asn Ala Thr Ser Glu Ala Gln Gln Val Ala Asp Tyr Tyr Ala Thr

260 265 270

Val Ala Ala Cys Ala Ala Thr Glu Arg Cys Ile Gly Ile Thr Val Trp

275 280 285

Asp Phe Asp Asp Thr Tyr Ser Trp Val Pro Ser Thr Phe Ala Gly Gln

290 295 300

Gly Tyr Ala Asp Leu Phe Phe Gln Pro Asp Gly Pro Asn Thr Pro Leu

305 310 315 320

Val Lys Lys Ala Ala Tyr Asp Gly Cys Leu Gln Ala Leu Gln His Lys

325 330 335

Ala Glu Ser Pro

340

<210> 11

<211> 1023

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggggtctta ataatgcagc tcgagccgat ggcaagcttt ggtttggaac tgctgcagat 60

atccccggtt tagagcagga tgatcgctat tacatgaagg aatacaacaa tacgcatgat 120

tttggtggta ccacacccgc gaatattatg aaattcatgt tcacggagcc agagcaaaac 180

gtttttaatt tcaccggcgc gcaggagttc ctggacattg cctttgcgtc gcacaagctt 240

gttcgttgcc acaatcttat ctggcaatcc gagcttccca catgggttac taaccctacc 300

acaaattgga caaacgaaac cttgagcaag gtgctacaaa atcatgtata tactctagtc 360

tcacattttg gagatcagtg ctatagctgg gatgtggtta acgaagccct ctctgatgac 420

ccagccggat cgtatcaaaa caatatctgg ttcgacacta ttggtcccga gtacgttgcg 480

atggcattcg agtatgccga gaaagccgtc aaagaccata agttgaatgt taagctctac 540

tacaatgact acaacattga atatcctggg cccaaatcta cagcagcaca gaatattgtc 600

aaggagctta aagcaaggaa catccaaata gatggcgtcg gccttgagtc ccacttcatc 660

gctggtgaaa ctccgtctca ggctacgcaa atcacaaaca tggctgattt cacttctctt 720

gacattgacg ttgctgttac cgagctcgat gtacgtcttt atctgcctcc aaatgctacc 780

agcgaggccc agcaagttgc cgactattac gccaccgtcg cagcctgtgc tgcaacagaa 840

cgctgtatcg gtataactgt ctgggatttt gacgatacat attcatgggt gcccagcacg 900

ttcgccggcc aagggtatgc ggatctgttc ttccagccag acggccccaa cactccccta 960

gtgaaaaaag cggcgtacga cggttgccta caggctttgc aacataaggc ggaaagtcca 1020

tga 1023

<210> 12

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tccgaattct ggggtcttaa taatgcag 28

<210> 13

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agtgcggccg ctcatggact ttccgcctta tgttg 35

<210> 14

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgaaattcga gttcacggag ccagag 26

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccgtgaactc gaatttcata atattc 26

<210> 16

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

attatgaaat tcgacgcaac ggagccaga 29

<210> 17

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgttgcgtcg aatttcataa tattcgcgg 29

<210> 18

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgaaattcga ctccacggag ccagagcaa 29

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggctccgtgg agtcgaattt cataatatt 29

<210> 20

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atgaaattcc agccaacgga gccagagca 29

<210> 21

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gctccgttgg ctggaatttc ataatattcg cg 32

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atgaaattca gcctaacgga gccag 25

<210> 23

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gctccgttag gctgaatttc ataatattcg 30

Claims (6)

1. A xylanase mutant with high specific activity at 40 ℃, which is characterized in that a mutation site R is selected to be mutated into a xylanase mutant based on the sequence SEQ ID NO.11 of xylanase wild type XYL 10C-delta N:

when the mutation site R is M137E, the xylanase mutant is XYL delta N-M137E, and the nucleotide sequence is shown as SEQ ID NO. 1;

when the mutation site R is MF137/138DA, the xylanase mutant is XYL delta N-MF137/138DA, and the nucleotide sequence is shown as SEQ ID NO. 2;

when the mutation site R is MF137/138DS, the xylanase mutant is XYL delta N-MF137/138DS, and the nucleotide sequence is shown as SEQ ID NO. 3;

when the mutation site R is MF137/138QP, the xylanase mutant is XYL delta N-MF137/138QP, and the nucleotide sequence is shown as SEQ ID NO. 4;

when the mutation site is RMF137/138SL, the xylanase mutant is XYL delta N-MF137/138SL, and the nucleotide sequence is shown as SEQ ID NO. 5.

2. The xylanase mutant with high specific activity at 40 ℃ according to claim 1, wherein the amino acid sequence encoding xylanase mutant XYL Δ N-M137E is shown in SEQ ID No. 6; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138DA is shown as SEQ ID NO. 7; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138DS is shown as SEQ ID NO. 8; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138QP is shown as SEQ ID NO. 9; the amino acid sequence of the coding xylanase mutant XYL delta N-MF137/138SL is shown in SEQ ID NO. 10.

3. A recombinant vector contains a recombinant expression vector with a nucleotide sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

4. A recombinant strain contains expression vectors with the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

5. The method for constructing the xylanase mutant with high specific activity at 40 ℃ based on the claim 1, which is characterized by comprising the following steps: 1) amplifying xylanase mutant sequence segments with high catalytic efficiency by adopting an over-lap PCR method; 2) cloning the sequence fragment of the xylanase mutant between restriction sites EcoR I and Not I of an expression vector pPIC9r to obtain a recombinant vector; 3) transforming the mutant recombinant vector into pichia pastoris GS115, and carrying out induced expression to obtain a mutant strain; 4) culturing the recombinant strain, and inducing the expression of the recombinant xylanase; 5) recovering and purifying the expressed high specific activity xylanase mutant.

6. Use of a high specific activity xylan mutant according to claim 1 at 40 ℃, a recombinant vector according to claim 3 or a recombinant strain according to claim 4 as a feed additive or for the degradation of lignocellulose.

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