阅读说明：本技术 一种热稳定性和比活力提高的低温β-木糖苷酶突变体及其编码基因和应用 (Low-temperature beta-xylosidase mutant with improved thermal stability and specific activity and coding gene and application thereof ) 是由 李中媛 张同存 刘仲琦 李爽 赵军旗 于 2019-09-16 设计创作，主要内容包括：本发明涉及一种热稳定性和比活力提高的低温β-木糖苷酶突变体,所述突变体为低温β-木糖苷酶突变体G110S,其氨基酸序列为SEQ ID NO.1；或者,所述突变体为低温β-木糖苷酶突变体Q201R,其氨基酸序列为SEQ ID NO.2；或者,所述突变体为低温β-木糖苷酶突变体loop2,其氨基酸序列如SEQ ID NO.3。本发明中稳定性提高和比活力上升的低温β-木糖苷酶突变体更适合工业应用,在食品、饲料、造纸等领域具有更广泛的应用前景,从而扩大了低温β-木糖苷酶在食品、医药、造纸、饲料等领域提供了应用潜力。(The invention relates to a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity, which is a low-temperature beta-xylosidase mutant G110S, and the amino acid sequence of the mutant is SEQ ID NO. 1; or the mutant is a low-temperature beta-xylosidase mutant Q201R, and the amino acid sequence of the mutant is SEQ ID NO. 2; or the mutant is a low-temperature beta-xylosidase mutant loop2, and the amino acid sequence of the mutant is shown as SEQ ID NO. 3. The low-temperature beta-xylosidase mutant with improved stability and increased specific activity is more suitable for industrial application and has wider application prospect in the fields of food, feed, paper making and the like, so that the application potential of the low-temperature beta-xylosidase in the fields of food, medicine, paper making, feed and the like is expanded.)

1. A low temperature beta-xylosidase mutant with improved thermostability and specific activity, characterized by: the mutant is a low-temperature beta-xylosidase mutant G110S, and the amino acid sequence of the mutant is SEQ ID No. 1;

or the mutant is a low-temperature beta-xylosidase mutant Q201R, and the amino acid sequence of the mutant is SEQ ID NO. 2;

or the mutant is a low-temperature beta-xylosidase mutant loop2, and the amino acid sequence of the mutant is shown as SEQ ID NO. 3.

2. A gene encoding the low temperature β -xylosidase mutant of claim 1 having increased thermostability and specific activity.

3. The gene encoding the low-temperature β -xylosidase mutant with increased thermostability and specific activity according to claim 2, characterized in that: the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant G110S is SEQ ID NO. 4;

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant Q201R is SEQ ID NO. 5;

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant loop2 is SEQ ID NO. 6.

4. A recombinant vector, recombinant cell or recombinant engineered bacterium carrying the gene encoding the mutant of claim 2 or 3.

5. A method for improving the thermal stability and specific activity of low-temperature beta-xylosidase is characterized by comprising the following steps: in the method, the amino acid sequence of the low-temperature beta-xylosidase is subjected to mutation at the following sites: G110S, Q201R, and loop 2.

6. The method of improving the thermostability and specific activity of a low temperature β -xylosidase according to claim 5, characterized in that: the method comprises the following steps:

① amplifying the gene sequence of the beta-xylosidase mutant with high thermal stability and specific activity by over-lap PCR;

② cloning the sequence fragment of the beta-xylosidase mutant with high thermal stability and specific activity to the expression vector pPIC 9;

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

7. The method of improving the thermostability and specific activity of a low temperature β -xylosidase according to claim 6, characterized in that: the mutant is G110S, Q201R or loop 2.

8. The use of the low-temperature β -xylosidase mutant with improved thermostability and specific activity according to claim 1 in the industrial field.

Technical Field

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity, and a coding gene and application thereof.

Background

Xylan is widely distributed and is the main component of hemicellulose in plant cell walls. Xylan is a heterogeneous polysaccharide, a main chain is formed by connecting beta-1, 4-glycosidic bonds with xylose units, the structure is complex, and the degradation process needs a plurality of glycosidase hydrolases to complete together; among them, β -xylosidase and α -L-arabinosidase are essential for the complete degradation of xylan.

Beta-xylosidase (EC 3.2.1.37) is an exoglycosidase, which has the ability to hydrolyze the non-reducing end of xylan oligosaccharides to xylose, it catalyzes mainly alkyl and aryl glycosides and hydrolyzes xylooligosaccharides from the non-reducing end in an exo-manner, xylooligosaccharide above xylobiose, and the hydrolysis product is xylose. Beta-xylosidase is one of the component enzymes of the hemicellulase complex, and is one of the constituent enzymes of the hemicellulase complex. The beta-xylosidase plays an important role in the enzymolysis of hemicellulose, and can be used for completely decomposing xylan under the synergistic action of the beta-xylosidase and xylanase in the enzymolysis of the hemicellulose. The beta-xylosidase is widely applied to various fields of biological medicines, foods, paper making, feeds and the like: the loss of active ingredients, flavor substances and nutrient ingredients in food is reduced, the cholesterol level is reduced, the bioavailability of calcium is increased, and the absorption and utilization of the nutrient ingredients of the feed by animals are improved.

In order to further promote the application of the beta-xylosidase in the industrial field, further improvement of the existing properties of the beta-xylosidase is needed, such as maintaining good activity in more extreme environments, possessing higher enzyme activity and the like. Wherein, because the low-temperature xylosidase has poor thermal stability and low specific activity, the residual activity of the wild strain is only 1.79 percent after being processed for 1 hour at 40 ℃, the specific activity is 4.85U/mg, and because the reaction temperature is about 50 ℃ in the industry, the application of the wild low-temperature beta-xylosidase is not favorable. Therefore, the method has wide application value for improving the thermal stability and specific activity of the low-temperature beta-xylosidase.

Through searching, the following patent publications related to the patent application of the invention are found:

1. compared with the existing mutant PGL-S1, the alkaline pectinase mutant (CN105316310B) with improved specific enzyme activity and thermal stability has the advantages that the specific enzyme activity of the mutant PGL- (GS)3-S1 is improved by 6 times, and the half-life period at 60 ℃ is improved by 1.3 times. The alkaline pectinase of the invention can catalyze the alpha-1, 4 glycosidic bond cracking of polygalacturonic acid through trans-elimination under alkaline conditions, and can be widely applied to industries of food, textile, paper making and the like.

2. A maltooligosyl trehalose synthase mutant (CN108753746A) with improved thermostability, which is obtained by mutating an amino acid site of the maltooligosyl trehalose synthase and has higher thermostability than that of a parent maltooligosyl trehalose synthase. The half-life period of the mutant of the maltooligosyl trehalose synthase is increased by 41 hours at 50 ℃ compared with that of the wild type, and is 2 times of that of the wild type, namely the stability of the mutant of the maltooligosyl trehalose synthase is improved by 2 times compared with that of the wild type.

3. A lipase mutant with improved thermal stability and a preparation method and application thereof (CN109468301A), wherein the amino acid sequence of the lipase mutant is shown as SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7 or SEQ ID NO.9 or SEQ ID NO.11 or SEQ ID NO. 13. According to the invention, the thermal stability of the rhizopus oryzae lipase ROL is improved greatly by performing multi-sequence comparison and disulfide bond prediction results, and the principle of improving the thermal stability is explained at a molecular level by means of a computer simulation technology. The thermal stability of the lipase mutant provided by the invention is obviously improved, and the lipase mutant has higher industrial application value by combining with high Sn 1,3 selectivity.

4. A lipase mutant with improved thermostability (CN108841805A) is disclosed, which is prepared from Aspergillus oryzae (Aspergillus oryzae) lipase as parent through site-directed mutagenesis. In the amino acid sequence of this mutant, the amino acid mutations involved are: gly57Glu/Leu156 Cys. The temperature (T) at which the mutant loses 50 percent of enzyme activity after being subjected to heat preservation for 10min at different temperatures and the half-life period (T) at 50 ℃ are used for expressing, so that the thermal stability of the mutant is improved, and the mutant has higher practical application value and wide market prospect.

5. Phytase mutants YkAPPA-L396V and YeAPPA-L396V as well as coding genes and application thereof (CN106011102A), and relates to phytase mutants YkAPPA-L396V and YeAPPA-L396V as well as coding genes and application thereof. Obtained by mutating leucine at position 396 of phytase to valine. Compared with the wild type, the two phytase mutants of YkAPPA-L396V and YeAPPA-L396V have obviously improved pepsin resistance, and are beneficial to development and application of feed enzymes.

By contrast, the present patent application is substantially different from the above patent publications.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity, and a coding gene and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity is a low-temperature beta-xylosidase mutant G110S, and the amino acid sequence of the mutant is SEQ ID No. 1;

SEQ ID NO.1

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSTSDFKEV TDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVSDKPEGPFT ADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVSGE GVAAQGPRAAKLTDDMHQFESPAQELLILDPETKEPILGDDHARRFFEAAWMHKHNGKY YFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCELSK GVDHLRSVKAKEIFYDDQGRIITTKAD；

or the mutant is a low-temperature beta-xylosidase mutant Q201R, and the amino acid sequence of the mutant is SEQ ID NO. 2;

SEQ ID NO.2

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSTSDFKEV TDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVGDKPEGPFT ADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVSGE GVAAQGPRAAKLTDDMHQFESPARELLILDPETKEPILGDDHARRFFEAAWMHKHNGKY YFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCELSK GVDHLRSVKAKEIFYDDQGRIITTKAD；

or the mutant is a low-temperature beta-xylosidase mutant loop2, and the amino acid sequence of the mutant is shown in SEQ ID No. 3:

SEQ ID NO.3

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSLDSLDPP SEVTDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVGDKPEG PFTADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVS GEGVAAQGPRAAKLTDDMHQFESPAQELLILDPETKEPILGDDHARRFFEAAWMHKHNG KYYFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCEL SKGVDHLRSVKAKEIFYDDQGRIITTKAD。

a gene encoding the low temperature β -xylosidase mutant with improved thermostability and specific activity as described above.

Moreover, the nucleotide sequence of the gene coding the low-temperature beta-xylosidase mutant G110S is SEQ ID NO. 4;

SEQ IQ NO.4

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCACGTCGGACTTCAAGG AGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCGAGCAAGC AGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACTTCCCGGC ACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGAGTGACAAGCCCGAGGG CCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCGGCCAGT TTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGGCCAGCT CCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACCCAAGGA AGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACAGACGAC ATGCACCAATTCGAGTCGCCAGCCCAAGAGCTCCTCATTCTGGACCCAGAGACCAAGG AACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGATGCACAA GCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCTGCTACG CCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGAGCCCGT GCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATATCTCTTC TTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGGCCAAGG AGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant Q201R is SEQ ID NO. 5;

SEQ IQ NO.5

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCACGTCGGACTTCAAGG AGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCGAGCAAGC AGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACTTCCCGGC ACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGGGTGACAAGCCCGAGGG CCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCGGCCAGT TTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGGCCAGCT CCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACCCAAGGA AGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACAGACGAC ATGCACCAATTCGAGTCGCCAGCCCGAGAGCTCCTCATTCTGGACCCAGAGACCAAGG AACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGATGCACAA GCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCTGCTACG CCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGAGCCCGT GCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATATCTCTTC TTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGGCCAAGG AGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant loop2 is SEQ ID NO. 6;

SEQ IQ NO.6

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCCTCGACTCCCTCGACCC CCCTTCCGAGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCG AGCAAGCAGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACT TCCCGGCACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGGGTGACAAGCC CGAGGGCCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCG GCCAGTTTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGG CCAGCTCCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACC CAAGGAAGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACA GACGACATGCACCAATTCGAGTCGCCAGCCCAAGAGCTCCTCATTCTGGACCCAGAGA CCAAGGAACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGAT GCACAAGCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCT GCTACGCCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGA GCCCGTGCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATAT CTCTTCTTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGG CCAAGGAGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

a recombinant vector, a recombinant cell or a recombinant engineering bacterium carrying the gene encoding the mutant as described above.

A method for improving the thermal stability and specific activity of low-temperature beta-xylosidase, wherein the amino acid sequence of the low-temperature beta-xylosidase is mutated at the following sites: G110S/Q201R/loop 2.

Moreover, the steps are as follows:

① amplifying the gene sequence of the beta-xylosidase mutant with high thermal stability and specific activity by over-lap PCR;

② cloning the sequence fragment of the beta-xylosidase mutant with high thermal stability and specific activity to the expression vector pPIC 9;

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

Furthermore, the mutant is G110S, Q201R or loop 2.

The use of the low-temperature beta-xylosidase mutant with improved thermostability and specific activity as described above in the industrial field.

The invention has the advantages and positive effects that:

1. the low-temperature beta-xylosidase mutant with improved thermal stability and specific activity has the advantages that the residual activity of the low-temperature beta-xylosidase mutant G110S is improved to 37% from the basic disappearance of wild type after the low-temperature beta-xylosidase mutant is treated at 45 ℃ for 10 min; meanwhile, the specific activity is improved to 7.02U/mg from 4.85U/mg, and kinetic parameters Km, Vmax and Kcat are respectively 12.8mmol/L, 1.24mmol/L and 19.26s^-1(ii) a After the mutant Q201R is treated at 45 ℃ for 10min, the residual activity is improved to 41 percent, the specific activity is improved to 6.29U/mg, and the kinetic parameters Km, Vmax and Kcat are respectively 20.2mmol/L, 1.46mmol/L and 15.61s^-1(ii) a After the mutant loop2 is treated at 45 ℃ for 10min, the residual activity is improved to 41 percent, the specific activity is improved to 6.83U/mg, and the kinetic parameters Km, Vmax and Kcat are respectively 17.28mmol/L, 1.49 mu mol/min/mg and 24.98s^-1And still maintain its low temperature characteristics; therefore, the low-temperature beta-xylosidase mutant with improved stability and increased specific activity is more suitable for industrial application and has wider application prospect in the fields of food, feed, paper making and the like, so that the application potential of the low-temperature beta-xylosidase in the fields of food, medicine, paper making, feed and the like is expanded.

2. According to the method, the G110S/Q201R/loop2 site mutation is carried out on the amino acid sequence of the wild type low-temperature beta-xylosidase, so that the thermal stability and specific activity of the low-temperature beta-xylosidase can be obviously improved, the problems of poor thermal stability and low specific activity of the conventional low-temperature beta-xylosidase are solved, and the low-temperature beta-xylosidase mutant which is good in thermal stability, high in specific activity and still keeps the low-temperature characteristic is obtained by adopting a site-specific mutation mode.

Drawings

FIG. 1 is a protein electrophoresis diagram of low temperature beta-xylosidase wild enzyme AX543 and mutant loop2/G110S/Q201R and no-load plasmid (note: M is protein maker, 1 is empty vector pPIC9 protein, 2 is wild type AX543 protein, 3 is loop2 mutant protein, 4 is G110S mutant protein, 5 is Q201R mutant protein.);

FIG. 2 is a line graph showing the optimal temperature curves of the low-temperature beta-xylosidase mutant G110S and the wild enzyme AX543 in the present invention;

FIG. 3 is a line graph showing the residual enzyme activity of beta-xylosidase mutant G110S and wild enzyme AX543 at a low temperature of 45 ℃ in the present invention;

FIG. 4 is a line graph showing the optimal temperature curves of the low-temperature beta-xylosidase mutant Q201R and the wild enzyme AX543 in the invention;

FIG. 5 is a line graph showing the residual enzyme activity of beta-xylosidase mutant Q201R and wild enzyme AX543 at a low temperature of 45 ℃ in the present invention;

FIG. 6 is a line graph showing the optimal temperature curves of the low-temperature beta-xylosidase mutant loop2 and the wild enzyme AX543 in the present invention;

FIG. 7 is a line graph showing the residual enzyme activity of beta-xylosidase mutant loop2 and wild enzyme AX543 at 45 deg.C in accordance with the present invention;

FIG. 8 is a bar graph showing the specific activity of the low temperature beta-xylosidase mutant G110S/Q201R/loop2 and the wild enzyme AX543 under optimal reaction conditions in the present invention.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A low-temperature beta-xylosidase mutant with improved thermal stability and specific activity is a low-temperature beta-xylosidase mutant G110S, and the amino acid sequence of the mutant is SEQ ID No. 1;

SEQ ID NO.1

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSTSDFKEV TDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVSDKPEGPFT ADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVSGE GVAAQGPRAAKLTDDMHQFESPAQELLILDPETKEPILGDDHARRFFEAAWMHKHNGKY YFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCELSK GVDHLRSVKAKEIFYDDQGRIITTKAD；

or the mutant is a low-temperature beta-xylosidase mutant Q201R, and the amino acid sequence of the mutant is SEQ ID NO. 2;

SEQ ID NO.2

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSTSDFKEV TDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVGDKPEGPFT ADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVSGE GVAAQGPRAAKLTDDMHQFESPARELLILDPETKEPILGDDHARRFFEAAWMHKHNGKY YFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCELSK GVDHLRSVKAKEIFYDDQGRIITTKAD；

or the mutant is a low-temperature beta-xylosidase mutant loop2, and the amino acid sequence of the mutant is shown in SEQ ID No. 3:

SEQ ID NO.3

MPPLITSIYTADPSAHVFNDKIYIYPSHDRETDIAFNDNGDQYDMADYHVFSLDSLDPP SEVTDHGVVLKTEDVPWASKQLWAPDAAHKNGKYYLYFPARDKEGIFRIGVAVGDKPEG PFTADPEPIKGSYSIDPASFVDDDGQAYLYFGGLWGGQLQCYQKGDDTYDPEWQGPKEVS GEGVAAQGPRAAKLTDDMHQFESPAQELLILDPETKEPILGDDHARRFFEAAWMHKHNG KYYFSYSTGDTHFLCYAVGDSPMGPFTYGGKILEPVLGWTTHHSIVEYKGKTYLFFHDCEL SKGVDHLRSVKAKEIFYDDQGRIITTKAD。

a gene encoding the low temperature β -xylosidase mutant with improved thermostability and specific activity as described above.

Preferably, the nucleotide sequence of the gene encoding the low-temperature beta-xylosidase mutant G110S is SEQ ID NO. 4;

SEQ IQ NO.4

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCACGTCGGACTTCAAGG AGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCGAGCAAGC AGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACTTCCCGGC ACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGAGTGACAAGCCCGAGGG CCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCGGCCAGT TTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGGCCAGCT CCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACCCAAGGA AGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACAGACGAC ATGCACCAATTCGAGTCGCCAGCCCAAGAGCTCCTCATTCTGGACCCAGAGACCAAGG AACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGATGCACAA GCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCTGCTACG CCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGAGCCCGT GCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATATCTCTTC TTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGGCCAAGG AGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant Q201R is SEQ ID NO. 5;

SEQ IQ NO.5

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCACGTCGGACTTCAAGG AGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCGAGCAAGC AGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACTTCCCGGC ACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGGGTGACAAGCCCGAGGG CCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCGGCCAGT TTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGGCCAGCT CCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACCCAAGGA AGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACAGACGAC ATGCACCAATTCGAGTCGCCAGCCCGAGAGCTCCTCATTCTGGACCCAGAGACCAAGG AACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGATGCACAA GCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCTGCTACG CCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGAGCCCGT GCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATATCTCTTC TTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGGCCAAGG AGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

the nucleotide sequence of the gene for coding the low-temperature beta-xylosidase mutant loop2 is SEQ ID NO. 6;

SEQ IQ NO.6

ATGCCGCCCCTCATTACCTCCATCTACACAGCTGATCCCTCAGCCCACGTCTTCAA TGACAAGATCTACATCTACCCGTCTCACGACCGCGAGACGGACATTGCCTTCAACGAC AATGGCGACCAGTATGACATGGCCGACTACCATGTCTTCTCCCTCGACTCCCTCGACCC CCCTTCCGAGGTGACCGACCACGGCGTCGTGCTCAAGACAGAGGACGTGCCGTGGGCG AGCAAGCAGCTCTGGGCCCCCGACGCGGCGCACAAGAACGGAAAATACTACCTCTACT TCCCGGCACGAGACAAGGAAGGCATCTTTCGGATTGGCGTGGCCGTGGGTGACAAGCC CGAGGGCCCCTTCACCGCCGACCCAGAGCCCATCAAGGGCAGCTACTCCATCGACCCG GCCAGTTTCGTCGACGATGACGGCCAGGCCTACCTCTACTTTGGCGGTCTCTGGGGTGG CCAGCTCCAATGCTACCAAAAGGGCGACGACACGTACGACCCGGAGTGGCAAGGACC CAAGGAAGTCTCTGGCGAGGGCGTCGCAGCGCAGGGCCCTCGCGCCGCCAAGTTGACA GACGACATGCACCAATTCGAGTCGCCAGCCCAAGAGCTCCTCATTCTGGACCCAGAGA CCAAGGAACCCATTCTCGGCGACGACCACGCGCGCCGCTTCTTTGAAGCCGCGTGGAT GCACAAGCACAATGGCAAGTACTACTTCTCCTACTCGACGGGCGACACCCACTTCCTCT GCTACGCCGTGGGCGACTCACCCATGGGTCCGTTCACGTATGGCGGCAAGATCCTGGA GCCCGTGCTGGGCTGGACGACGCACCACTCGATTGTCGAGTACAAGGGCAAGACATAT CTCTTCTTCCACGACTGTGAGCTGAGCAAGGGTGTGGACCACCTCAGGAGCGTCAAGG CCAAGGAGATCTTTTACGACGATCAGGGTAGGATCATCACGACAAAGGCAGAT

a recombinant vector, a recombinant cell or a recombinant engineering bacterium carrying the gene encoding the mutant as described above.

A method for improving the thermal stability and specific activity of low-temperature beta-xylosidase, wherein the amino acid sequence of the low-temperature beta-xylosidase is mutated at the following sites: G110S, Q201R, and loop 2.

Preferably, the steps are as follows:

① amplifying the gene sequence of the beta-xylosidase mutant with high thermal stability and specific activity by over-lap PCR;

② cloning the sequence fragment of the beta-xylosidase mutant with high thermal stability and specific activity to the expression vector pPIC 9;

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

Preferably, the mutants are G110S, Q201R and loop 2.

The low-temperature beta-xylosidase mutant having improved thermostability and specific activity as described above can be applied in the industrial field.

The related specific embodiments of the invention:

1. test materials and reagents

Strains and vectors: expression host Pichia pastoris GS115, expression plasmid vector pPIC 9.

Enzymes and other biochemical reagents:endonuclease was purchased from Fermentas, Inc., T₄Ligase was purchased from Promega and polymerase from total gold biotechnology limited, beijing.

2. Culture medium:

(1) YPD liquid medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) anhydrous glucose;

(2) MD solid medium: 1.34% (w/v) YNB, 2% (w/v) glucose, 4X10^-5% biotin (w/v), 2% (w/v) agar;

(3) BMGY medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 100mM phosphoric acid, 1.34% (w/v) YNB, 4X10^-5Percent (w/v) biotin, 1% (w/v) glycerol;

(4) BMMY medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 100mM phosphoric acid, 1.34% YNB, 4X10^-5% biotin (w/v), 0.5% (v/v) methanol;