WO2015007033A1

WO2015007033A1 - Mutant of xylanase xynas9-m with improved thermal stability and gene and use thereof

Info

Publication number: WO2015007033A1
Application number: PCT/CN2013/086988
Authority: WO
Inventors: 姚斌; 罗会颖; 王坤; 王亚茹; 孟昆; 石鹏君; 黄火清; 柏映国; 杨培龙; 赵珩; 马锐
Original assignee: 中国农业科学院饲料研究所
Priority date: 2013-07-16
Filing date: 2013-11-12
Publication date: 2015-01-22
Also published as: CN103343113B; CN103343113A

Abstract

Provided are a mutant of xylanase XynAS9-m with an improved thermal stability and a gene and use thereof. The mutant of xylanase XynAS9-m has an amino acid sequence with the valine at position 81 of xylanase mutated into proline, and the glycine at position 82 mutated into glutaminic acid as shown in SEQ ID No. 1. Further, the aspartic acid at position 185 of xylanase is mutated into proline, and the serine at position 186 is mutated into glutamic acid. The thermal stability of the mutant enzyme is significantly improved, showing potential application value in pulp making, biological energy sources and other industries.

Description

Thermodynamically improved xylanase XynAS9-m mutant and gene and application thereof

The invention belongs to the technical field of genetic engineering and enzymatic engineering, and specifically relates to a thermostable modified xylanase XynAS9-m mutant and a gene and application thereof. Background technique

Xylanase (endo-l, 4-β-xylanases, EC 3.2.1.8) is a generic term for a class of important industrial enzymes that degrade xylan into oligosaccharides and xylose. Hydrolyzing the β-1,4-glycosidic bond in the xylan molecule mainly by endo-cutting to form xylooligosaccharide and xylose, which is one of the most important hydrolase enzymes in the hemicellulose hydrolase system. Most of the glycanase belongs to the F/10 and G/11 families, and the xylanase of the tenth family has lower substrate specificity, faster hydrolysis rate, lower hydrolysis degree of hydrolysis products than the xylanase of the eleventh family. Advantages, therefore, have important application value in industry. In recent years, xylanase has been widely used in feed, pulp and paper, food, energy and other industries, especially in industrial papermaking. However, the optimum temperature of most xylanases is 45-55 ° C, and the thermal stability is poor, which can not meet the requirements of pulp brewing, and high temperature xylanase or heat-resistant enzyme can reduce the cost of enzyme preparation and improve the reaction. Catalytic efficiency, reduced reaction energy consumption, reduced contamination of bacteria and high tolerance to chemical denaturant and related metal interest. Based on this, finding effective ways and means to improve the stability and catalytic activity of xylanase in high temperature environment has become a top priority for the domestic xylanase industry.

With the development of protein engineering technology and molecular biology, the use of directed evolution and rational design

The artificial evolution and transformation of enzyme molecules has become a hot topic in the field of enzyme engineering. So far, many scholars have used this technology to successfully transform the thermal stability of proteins. The present invention uses a site-directed mutagenesis method to engineer xylanase to obtain xylanase XynAS9-m mutant V81P/G82E, respectively. And the xylanase XynAS9-m mutant V81P/G82E/D185P/S186E, the optimum temperature of the xylanase XynAS9- m was increased by 20 ° C, the Tm value was increased by 7 ° C, and its industrial expansion was expanded. Applications. Summary of the invention

The object of the present invention is to modify the xylanase by site-directed mutagenesis, so that the modified xylanase XynAS9-m mutant is more excellent in tolerance.

Another object of the present invention is to provide a gene encoding the above xylanase XynAS9-m mutant. Another object of the present invention is to provide a recombinant vector comprising the above xylanase XynAS9-m mutant gene.

The present invention also provides a host cell comprising the gene of the xylanase XynAS9-m mutant as described above or a recombinant vector as described above.

The present invention performs site-directed mutagenesis of a Streptomyces-derived xylanase XynAS9-m gene having an amino acid sequence as shown in SEQ ID NO. 1, which is derived from SEQ.

The nucleotide sequence shown by ID NO. 2 is encoded.

SEQ ID NO. 1

I FRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ I LGSEFSQI T VGNT KWQYT EP SRGRFDYT 1 01 AAEE IVDLAE SNGQSVRGHT LVWHNQLP SW VDDVPAGELL GV RDHI THE 151 VDHFKGRLI H WDWNEAFEE DGSRRQSVFQ QKI GD SYIAE AFKAARAADP 201 DVKLYYNDYN IEGI GPKSDA VYE VKSFKA QGIP I DGVG QAHLIAGQVP 251 ASLQENIRRF ADLGVDVALT ELD IR TLPR TAAKDAQQAT DYGAWEACL 301 WSRCVGI TV WDYTDKYSWV P SVFPGQGAA LPWDEDFAKK PAYHAIAAAL 351 NGGSPAPGGN CTATYRVTSQ WQGGFTAE IT VGNDHTAP IT GWTVTWTLS S 401 GQS I SHM 丽 G NLTVNGQDVT VRDVGYNGTL GGNGS TTFGF QGEGVADTPA 451 DVTCTPGRP S GT SA

SEQ ID NO. 2 i ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA

61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC 2丄 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC

1 S1 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC

241 GTCGGCAACA CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC

301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC

361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC

421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC

48] TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG

541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG

60i GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC

66i GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG

721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC

7S1 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC

841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG

901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC

961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG

1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC

1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT

1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC

1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC

1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC

13S1 GGGACTTCGG CGTAG

A xylanase XynAS9-m mutant having improved thermostability according to the present invention, an amino acid sequence such as The 81st proline of the xylanase shown by SEQ ID NO. 1 is mutated to proline and the 82th glycine is mutated to glutamic acid. Preferably, the aspartic acid at position 185 is mutated to guanidine. The acid, more preferably, the serine at position 186 is mutated to glutamic acid.

According to a specific embodiment of the present invention, two thermo-stable xylanase XynAS9-m mutants were obtained by site-directed mutagenesis, and named as V81P/G82E, V81P/G82E/D185P/S186E, BP: V81P/G82E is the 81th proline mutated to proline and the 82th glycine is mutated to glutamic acid; V81P/G82E/D185P/S186E is the 81st proline mutated to proline, the 82th glycine The mutation is glutamic acid, the aspartic acid at position 185 is mutated to proline and the serine at position 186 is mutated to glutamic acid.

Therefore, the thermostability-improved xylanase XynAS9-m mutant according to the present invention

V81P/G82E, wherein the 81th proline is mutated to proline, and the 82th glycine is mutated to glutamic acid, and the amino acid sequence thereof is shown in SEQ ID NO.

SEQ ID NO.3

1 FRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT PENT KWQYT EPSRGRFDYT

101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GV RDHITHE

151 VDHFKGRLIH WDWNEAFEE DGSRRQSVFQ QKIGDSYIAE AFKAARAADP

201 DVKLYYNDYN IEGIGPKSDA VYE VKSFKA QGIPIDGVG QAHLIAGQVP

251 ASLQENIRRF ADLGVDVALT ELDIR TLPR TAAKDAQQAT DYGAWEACL

301 WSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL

351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS

401 GQSISHM 丽 G NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA 451 DVTCTPGRPS GTSA Therefore, the thermostability-improved xylanase XynAS9-m mutant V81P/G82E/D185P/S186E according to the present invention, wherein the 81th proline is mutated to proline The 82th glycine is mutated to glutamic acid, the aspartic acid at position 185 is mutated to proline and the serine at position 186 is mutated to glutamic acid, and the amino acid sequence thereof is as shown in SEQ ID N0.4:

MFRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT PENTMKWQYT EPSRGRFDYT 101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GVMRDHITHE 151 VDHFKGRLIH WDWNEAFEE DGSRRQSVFQ QKIGPEYIAE AFKAARAADP 201 DVKLYYNDYN IEGIGPKSDA VYEMVKSFKA QGIPIDGVGM QAHLIAGQVP 251 ASLQENIRRF ADLGVDVALT ELDIRMTLPR TAAKDAQQAT DYGAWEACL 301 WSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL 351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS 401 GQSISHM Li G NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA 451 DVTCTPGRPS GTSA The present invention also provides a xylanase XynAS9-m mutant gene sequence encoding the above heat stability improvement, the nucleotide sequence of which is shown in SEQ ID NO. SEQ ID NO. 5

1 ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA

61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC

121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC

181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC

241 CCCGAAKKCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC

301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC

361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC

421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC

481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG

541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG

601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC

661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG

721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC

781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC

841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG

901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC

961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG

1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC

1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT

1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC

1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC

1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC

1381 GGGACTTCGG CGTAG

SEQ ID NO. 6

1 ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA

61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC

121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC

181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC

241 CCCGAAkkCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC

301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC

361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC

421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC

481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG

541 CAGAAGATCG QCCCCGAGlk CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG

601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC

661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG

721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC

781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC

841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG

901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC

961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG

1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC

1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT

1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC

1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC 1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC 1381 GGGACTTCGG CGTAG The above-described cDNA molecule encoding the thermostable modified xylanase XynAS9-m mutant was inserted into the vector in a suitable orientation and the correct reading frame. Between the sites, the nucleotide sequence is operably linked to the expression control sequence. A preferred vector of the invention is pPIC9, which inserts the engineered xylanase gene between the EcoRI and Not I restriction sites on plasmid pPIC9 such that the nucleotide sequence is downstream of and protected by the AOX1 promoter. The recombinant yeast expression plasmid of each mutant was obtained by regulation. A preferred host strain of the invention is Pichia pastoris GS115.

Compared with the wild type, the thermal stability of the two xylanase XynAS9-m mutants V81P/G82E and V81P/G82E/D185P/S186E of the present invention is significantly improved, and the optimum temperature is increased by 20 ° compared with 70 ° C, respectively. At 20 °C and 20 °C, the Tm values increased by 6.84 °C and 6.99 °C, respectively, showing potential applications in pulp brewing, bioenergy and other industries. DRAWINGS

Figure 1. Schematic diagram of Overlap-PCR of xylanase XynAS9-m mutation;

Figure 2 is a map of pPIC9 vector;

Figure 3 shows the map of pPIC9-XynAS9-m recombinant vector

Figure 4 is a graph showing the activity of xylanase at different temperatures before and after the transformation;

Figure 5 is the enzyme activity curve of xylanase before and after modification at different temperatures for different time; Figure 6 is the enzyme activity curve of xylanase before and after modification at different pH;

Figure 7 is a graph showing the tolerance of xylanase to different pH before and after modification. DETAILED DESCRIPTION Test Materials and Reagents

1. Strains and vectors: Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitragen.

2. Enzymes and other biochemical reagents: Endonuclease was purchased from TaKaRa, and ligase was purchased from Invitragen. Purchased from Sigma, all other domestically produced reagents (both available from common biochemical reagents).

3. Culture medium: (1) The medium of i3⁄4fltop/wra sp. is potato medium: 1000 mL of 200 g of potato juice, 10 g of glucose, 25 g of agar, pH 5.0.

(2) E. coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

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

(4) BMMY medium: Divided by 0.5% methanol instead of glycerol, the rest of the ingredients are the same as BMGY, H4.0 o

Note: The molecular biology experimental methods not specified in the following examples are all carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or according to the kit and Product manuals are carried out. Example 1

1. Obtaining the mutated gene - the gene sequence of the xylanase XynAS9-m derived from Streptomyces sp. S9

(SEQ ID NO. 2) was modified, and the mutation was introduced by Overlap PCR and sequenced to obtain a mutant gene.

The mutation includes six PCR primers: S9 BF, S9 BR, V81P/G82E-F, V81P/G82E-R, D185P/S186E-F, D185P/S186E-R.

The primer sequences are as follows:

S9BF: 5 '- TAGAATTCGACACCGCCACCCTGGGCGAACT-3 '

S9 BR: 5 '-TATGCGGCCGCCTACGCCGAAGTCCCGGACGGC-3 '

V81P/G82E-F: 5'-GCCAGATCACCcccgaaAACACCATGAAGT-3 '

V81P/G82E-R: 5- ACTTC ATGGTGTTttcgggGGTG ATCTGGC-3 '

D185P/S186E-F: 5 '-AGAAGATCGGCcccgagTACATCG-3 ';

D185P/S186E-R: 5 '-CGATGTActcgggGCCGATCTTCT-3 '

Underlined for restriction enzyme sites EcoRI and NotI, lowercase letters indicate mutant bases. Overlap extension PCR is performed by three PCR reactions. Take the "A3⁄4>-m- ρ/Υί^ plasmid as a template and the mutant V81P/G82E as an example:

PCR reaction system:

PCR1 2*Pfu mix 25.0 μΐ

S9BF 0.5 ul

V81P/G82E-R 0.5 ul

ddH ₂ 0 22.0 μΐ

Template 2単

Total volume 50.0 μΐ

PCR1 program settings:

95. C, 5 min;

94 ° C, 30 sec; 60 ° C, 30 sec; 72 ° C, 1 min, 30 cycles;

72 ° C, 10 min; 10 ° C, hold.

The PCR amplification product was electrophoresed on a 1.2% agarose gel, and then recovered by a DNA recovery kit, and the solution was bathed at 2 (^LddH20, designated as V81P/G82E_R.

PCR 2

2*Pf _U mix PCR2 : 25.0 μΐ

S9BR 0.5 ul

V81P/G82E-F 0.5 ul

ddH ₂ 0 22.0 μΐ

Template 2単

Total volume 50.0 μΐ

PCR2 program settings:

95. C, 5 min;

94. C, 30 sec; 60. C, 30 sec; 72. C, 1 min, 30 cycles;

72 ° C, 10 min; 10 ° C, hold.

The PCR amplification product was electrophoresed on a 1.2% agarose gel, and then recovered by a DNA recovery kit, and the solution was bathed at 2 (^LddH20, designated as V81P/G82E_F.

PCR3

The PCR1 and PCR2 reaction products are mixed and used as a template.

2*Pfu mix 25.0 μΐ

S9BF 0.5 ul

S9BR 0.5 ul ddH ₂ 0 22.0 μΐ

Template 2単

Total volume 50.0 μΐ PCR program settings:

95. C, 5 min;

94. C, 30 sec; 60. C, 30 sec,; 72. C, 2 min, 30 cycles;

72 ° C, 10 min; 10 ° C, hold.

The PCR amplification product was electrophoresed on a 1.2% agarose gel, and the 1.2 kb band was cut out and recovered by a DNA recovery kit. The solution was dissolved in 2 (VL ddH ₂ 0. The strand was confirmed by DNA sequencing. Mold xylanase gene.

2. The connection of the xylanase gene XynAS9-m to the expression vector

The above-obtained mutant, -m and the expression vector pPIC9 were digested with restriction endonuclease EcoR I/Not I, respectively, and the conditions were as follows:

PCR fragment digestion system (50μυ plasmid pPIC9 digestion system (50μυ)

PCR fragment 20 pPIC9 20

10*H 5μί 10*Buffer 5μί

BSA 5μί BSA 5μί

Triton 5μί Triton 5μί

EcoR I 1 Generation EcoR I 1 Generation

Not I 1 generation Not I 1 generation

DdH ₂ 0 13 generation DdH ₂ 0 13 generation

The mixture was digested with a 37 ° C water bath for 2 h, and two target fragments were separately recovered after electrophoresis, and dissolved in 20 nL of ddH ₂ O. The ligation was carried out using T4 DNA ligase, and the ligation system was as follows:

PCR fragment 5μΙ pPIC9 2 μΐ

T4 DNA ligase Ιμί

10*Buffer 1.5 L

DdH ₂ Q 5.5

The total volume of 15.0 was connected at room temperature for 20-30 min. The electrophoresis results showed that there were about 9 kb fragments, and the two bands of 1.2 kb and 8 kb were found by double digestion with EcoR I/Not I, indicating that the ligation was successful and the constructs contained V81P. /G82E, V81P/G82E/D185P/S186E mutant pPIC9-XynAS9-m vector (XynAS9-m is the code for each xylanase gene after site-directed mutagenesis)

3. Screening of Pichia pastoris GS115 and engineering bacteria by XynAS9-m expression vector Preparation of Receptor Competent Cells: Pichia pastoris GS 115 strain was streaked on YPD plates, cultured at 30 ° C for 48 h, and single colonies grown vigorously in 20 mL YPD liquid medium, 30 ° C, 200 Incubate for 48 h at rpm shaker; inoculate activated fresh GS 115 solution in 200% YPD at 200 rpm, incubate at 30 ° C until OD _{6 (K)} is about 1.0-1.3, place on ice Pre-cooling; Transfer the bacterial solution to a pre-cooled centrifuge tube, centrifuge at 5 ° C, 5000 rpm for 5 min, discard the supernatant, and resuspend the cells with 200 mL of pre-cooled sterile water; resuspend the bacteria at 4. C, centrifuge at 5000 rpm for 5 min, discard the supernatant and resuspend with 100 mL of pre-cooled sterile water; centrifuge the resuspended cells at 4 ° C, 5000 rpm for 5 min, with 20 mL of pre-cooled 1 mol / The precipitate was resuspended in L sorbitol solution; the resuspended cells were precipitated at 4. C, centrifuge at 5000 rpm for 5 min, resuspend with 60 (^L pre-cooled 1 mol/L sorbitol solution; resuspend the cell suspension in 80 ml to 1.5 mL pre-cooled EP tube, and Store at -70 ° C, spare.

Electroporation conversion: The electrorotator was pre-warmed in advance, and the linearized plasmid was purified and dissolved in 10 μL of sterile water. 80 prepared GS 115 competent cells were mixed with it, and then transferred to pre-cooled. In the electric rotor; adjust the parameters of the instrument to a voltage of 2 kV, electric shock; immediately after the electric shock is completed. 0.5-1 mL of pre-cooled 1 mol/L sorbitol, mixed and transferred to 1.5 ml EP tube, allowed to stand in a 30 ° C incubator for 30 min, centrifuged at 5000 rpm for 3 min, discarded part of the supernatant, and the remaining liquid After being mixed, it is coated on an MD plate, and cultured in an inverted culture at 30 ° C for 2-3 days;

Screening of yeast transformants: Monoclonal sterilized toothpicks grown on MD plates were picked up to MM by number, and then placed on the corresponding numbered MD plates; the two plates were placed in a 30 ° C incubator for cultivation 2 day. Normally grown transformants were picked by number in yeast tubes containing 3 mL of BMGY medium, which was sterilized by moist heat and wrapped in eight layers of gauze, and placed in a 30 ° C, 220 rpm shaker. 48 h; The culture solution of the shaker cultured for 48 h was centrifuged at 4400 rpm for 10 min, and the supernatant was removed. 1 mL of BMMY medium containing 0.5% methanol was added to the centrifuge tube, and the culture was induced at 30 ° C and 220 rpm. After induction culture for 48 h, the bacterial solution was centrifuged at 12000 rpm for 3 min, and the supernatant was assayed for activity, and the transformant having xylanase activity was selected therefrom;

Expression of the target gene in shake flask levels in Pichia pastoris: A positive strain containing higher enzyme activity was inoculated into a 1 L flask of 400 mL BMGY medium and placed at 30. C, 220 rpm shaker culture for 48 h; then centrifuge the culture solution at 5000 rpm for 5 min, discard the supernatant, resuspend the pellet with 200 mL of BMMY medium containing 0.5% methanol, and place again at 30 ° C, 220 rpm Induction culture. 1 mL of methanol was added every 12 h to keep the methanol concentration in the bacterial solution at 0.5%. The induced solution was centrifuged and the supernatant was the crude enzyme solution of the mutant XynAS9-m. The crude enzyme solution was passed through a 6KDa hollow fiber column and lOKDa. After the ultrafiltration membrane is concentrated, it is further concentrated by acetone precipitation, and then purified by a desalting column and an anion column to obtain a wild type. Protein molecular weight.

4. Determination of mutant xylanase activity according to the present invention

Activity unit of one xylanase (U) Definition: The amount of enzyme required to decompose xylan per minute to release the reducing sugar of 1 μηιοΐ ϋ-xylose under certain conditions.

Determination of pH and pH stability of the recombinant enzyme reaction:

The purified recombinant mutant xylanase was subjected to enzymatic reaction at 60 ° C in different pH substrates to determine its optimum pH. The buffer used was: Mcllvaine buffer (0.2 M disodium hydrogen phosphate / 0.1 M citric acid) at pH 2.0-7.0, 0.1 mol/L Tris-HCl buffer at pH 8.0-9.0, and Gly at pH 10.0-12.0. - NaOH buffer. The results (Fig. 6) showed that the optimum pH of each mutant enzyme did not change much, and the range of action was basically the same, except that the enzyme activity was different in the buffer exchange.

Determination of pH stability: The concentrated pure enzyme solution was treated at 37 ° C for 1 h in buffers of different pH values, and appropriately diluted with the buffer of the optimum pH to determine the residual enzyme under optimal conditions. active. The results (Fig. 7) showed that the pH tolerance of the mutant enzyme was almost identical to that of the wild type, except that the mutant enzymes V81P/G82E and V81P/G82E/D185P/S186E were stable at pH 3.0. Amino acid substitution does not affect its optimum pH and pH tolerance.

Determination of the optimum temperature and thermal stability of the recombinant enzyme reaction:

The activity was determined in the pH optimum buffer and at different temperatures (40-90 ° C) to determine the optimum temperature. The heat resistance was measured after treatment at different temperatures for various times, and then the residual enzyme activity was measured under respective optimum conditions. The results (Fig. 4, 5) showed that the optimum temperature of the mutant enzyme was increased by 10-20 compared with the original enzyme, and after treatment at 65 °C for 60 min, only 9.3% of the enzyme activity remained in the wild type, while the mutant enzyme V81P/G82E Nearly 83% of the enzyme activity was left with V81P/G82E/D185P/S186E, and 47% of the enzyme activity of D185P/S186E remained. The remaining enzyme activity of V81P/G82E and V81P/G82E/D185P/S186E was wild type after 60 minutes of treatment at 70 °C. 12 times, that is, the mutant enzyme is 64% and the wild type is 5.9%. In addition, after treatment under other high temperature conditions, the mutant enzyme always maintains higher residual enzyme activity than the wild type, and the experimental results show that the thermal stability is significantly improved. .

Recombinase specific activity, Km value and Vmax determination of recombinase

Using different concentrations of xylan (0.5, 0.75, 1.0, 2.0, 4.0, 5.0, 6.0, 8.0, 10.0 mg/ml) as the substrate, the enzyme activity was determined under the optimal conditions, and the double reciprocal mapping method was used to calculate Corresponding reaction speed, m value and Vmi«.

A standard curve was drawn according to the method of the Bio-Rad kit. The protein concentration was divided into 2.0, 1.5, 1.0, 0.75, 0.5, 0.25 and 0.125 mg/ml, using 5 uL (protein) and 250 uL (color development). The reaction system of the liquid) was reacted at room temperature for 10-60 min, and the absorption value was measured at OD ₅₉₅ to prepare a standard curve. Determination of specific activity: First, calculate the content of the target protein by the standard curve, followed by the enzyme activity of the recombinant enzyme under the optimal conditions, and the ratio of the enzyme activity to the protein concentration is the specific activity of the enzyme. Specific activity is defined as: The number of enzyme activity units per milligram of enzyme protein.

Table 1 compares the kinetic parameters and specific activities of xylanase before and after transformation.

Different mutation sites Specific activity Km ^cat kcat/ Km V3⁄4iax

-1

Enzyme U.mg-1s ml.s '.mg" ¹ U.mg" ¹ wild type enzyme 630.27 1.03 790.20 760.33 950.82

D 185P/S 186E 90.73 4.02 184.42 45.84 221.91

V81P/G82E 206.93 1.05 212.46 202.51 255.64

V81P/G82E/

160.37 2.21 235.59 106.48 283.47

D 185P/S 186E

Claims

Rights request

1. A xylanase XynAS9-m mutant, characterized in that the valine at position 81 of the xylanase whose amino acid sequence is shown in SEQ ID NO. 1 is mutated to proline, and the valine at position 82 is proline. Mutation of glycine to glutamic acid.

2. The xylanase XynAS9-m mutant according to claim 1, characterized in that the aspartic acid at position 185 of the xylanase whose amino acid sequence is shown in SEQ ID NO. 1 is mutated to proline. Acid.

3. The xylanase XynAS9-m mutant according to claim 1 or 2, characterized in that the serine at position 186 of the xylanase whose amino acid sequence is shown in SEQ ID NO. 1 is mutated to glutamine acid.

4. A xylanase mutant gene, characterized in that it encodes the xylanase XynAS9-m mutant described in claim 1, 2 or 3.

5. The xylanase mutant gene according to claim 4, wherein the nucleotide sequence of the gene is as shown in SEQ ID NO. 5 or 6.

6. A recombinant vector comprising the xylanase mutant gene of claim 4.

7. A recombinant strain comprising the xylanase mutant gene of claim 4.

8. A method for preparing xylanase XynAS9-m with improved stability, characterized by comprising the following steps:

1) Transform host cells with the recombinant vector of claim 6 to obtain a recombinant strain;

2) Cultivate the recombinant strain and induce the expression of the recombinant site-directed mutated xylanase XynAS9-m; and

3) Recover and purify the expressed site-directed mutated xylanase XynAS9-m.

9. Application of the xylanase XynAS9-m mutant described in claim 1, 2 or 3.