CN103451171B

CN103451171B - Xylanase XynAS9-m mutants D185P/S186E with improved thermal stability as well as gene and application thereof

Info

Publication number: CN103451171B
Application number: CN201310326839.XA
Authority: CN
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: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2013-07-31
Filing date: 2013-07-31
Publication date: 2015-04-22
Anticipated expiration: 2033-07-31
Also published as: CN103451171A

Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to xylanase XynAS9-m mutants D185P/S186E and V81P/G82E/D185P/S168E with improved thermal stability as well as genes and application thereof. Aspartic acid at the 185th site of xylanase which is shown by an amino acid sequence such as SEQ ID NO.1 is mutated into proline, and serine at the 186th site is mutated into glutamic acid to obtain a mutant; and further, valine at the 81st site is mutated into proline, glycine at the 82nd site is mutated into glutamic acid, the aspartic acid at the 185th site is mutated into proline, and the serine at the 186th site is mutated into glutamic acid to obtain mutants V81P/G82E/D185P/S186E. The thermal stability of two mutant enzymes is obviously improved, the optimal temperatures are respectively increased by 10 DEG C and 20 DEG C compared with 70 DEG C, the Tm values are respectively increased by 1.2 DEG C and 6.99 DEG C, and the potential application values of the xylanase XynAS9-m mutants in the industries of paper pulp brewing, biological energy sources and the like are revealed.

Description

The zytase XynAS9-m mutant D185P/S186E of a kind of thermostability improvement and gene thereof and application

Technical field

The invention belongs to genetically engineered and technical field of enzyme engineering, particular content relates to zytase XynAS9-m mutant D185P/S186E and gene and the application of the improvement of a kind of thermostability.

Background technology

Zytase (endo-1,4-β-xylanases, EC3.2.1.8) is the important industrial enzymes of a class, and degradation of xylan becomes the general name of the class of enzymes of oligose and wood sugar.Main with the β-1 in internal-cutting way hydrolyzed xylan molecule, 4-glycosidic link, generate xylo-oligosaccharide and wood sugar, it is one of lytic enzyme of most critical in hydrolysis of hemicellulose enzyme system, the zytase of cloning at present belongs to F/10 and G/11 family mostly, and the zytase of the tenth family has the advantages such as substrate specificity is low, hydrolysis rate fast, the hydrolysate polymerization degree is low compared with the zytase of the 11 family, therefore industrially has important using value.Zytase was used widely in the industries such as feed, pulping and paper-making, food, the energy in recent years, the application especially on commercial paper.But the optimum temperuture of zytase is 45-55 DEG C mostly at present, and poor heat stability, can not meet paper pulp brewage in requirement, and high-temperature xylanase or thermostable enzyme have the cost reducing zymin, improve catalytic reaction efficiency, reduce energy consumption of reaction, reduce the pollution of miscellaneous bacteria and to chemical denaturant and associated metal interest, there is higher tolerance.Find effective way and means based on this, improve the task of top priority that the stability of zytase in hot environment and catalytic activity have become domestic zytase industry.

Along with protein engineering and molecular biological development, the means of orthogenesis and design and rational are used to carry out artificial evolution to enzyme molecule and transform the focus having become current enzyme engineering area research.Up to the present, existing many scholars use this technology successfully to transform the thermostability of albumen, the method transformation zytase of the present invention's application rite-directed mutagenesis, obtain zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E, the optimum temperuture of zytase XynAS9-m is improve 10 DEG C and 20 DEG C respectively compared with 70 DEG C, Tm value improves 1.2 DEG C and 6.99 DEG C respectively, show brewage at paper pulp, the industrial potential using value such as bioenergy.

Summary of the invention

The object of the invention is to be transformed zytase by the method for rite-directed mutagenesis, make improved zytase character in tolerance more excellent.

Another object of the present invention is to provide the gene of zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E of coding said mutation.

Another object of the present invention is to provide the recombinant vectors comprising said gene.

Present invention also offers a kind of host cell, it contains the gene of foregoing zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E or foregoing recombinant vectors.

The zytase XynAS9-m gene of the present invention to Streptomyces carries out rite-directed mutagenesis, the maturation protein of this zytase XynAS9-m has the aminoacid sequence as shown in SEQ ID NO.1, and this maturation protein is nucleotide sequence coded by such as shown in SEQ ID NO.2.

SEQ ID NO.1

1 MFRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT VGNTMKWQYT EPSRGRFDYT

101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GVMRDHITHE

151 VDHFKGRLIH WDVVNEAFEE DGSRRQSVFQ QKIGDSYIAE AFKAARAADP

201 DVKLYYNDYN IEGIGPKSDA VYEMVKSFKA QGIPIDGVGM QAHLIAGQVP

251 ASLQENIRRF ADLGVDVALT ELDIRMTLPR TAAKDAQQAT DYGAVVEACL

301 VVSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL

351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS

401 GQSISHMWNG NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA

451 DVTCTPGRPS GTSA

SEQ ID NO.2

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 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

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

1021CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC

1141GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT

1201GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC

1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC

1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC

1381 GGGACTTCGG CGTAG

This experiment adopts the method for rite-directed mutagenesis, obtain the mutant that 2 thermostabilitys improve, respectively called after D185P/S186E and V81P/G82E/D185P/S186E, that is: D185P/S186E is that the Aspartic acid mutations of the 185th is proline(Pro) and the mutant serine of the 186th is L-glutamic acid; V81P/G82E/D185P/S186E is the 81st valine mutation is proline(Pro), and the 82nd glycine mutation is L-glutamic acid, and the Aspartic acid mutations of the 185th is proline(Pro) and the mutant serine of the 186th is L-glutamic acid.

Therefore the zytase XynAS9-m mutant D185P/S186E that improves of thermostability according to the present invention, its aminoacid sequence is as shown in SEQ ID NO.3

SEQ ID NO.3

1 MFRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT VGNTMKWQYT EPSRGRFDYT

101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GVMRDHITHE

151 VDHFKGRLIH WDVVNEAFEE DGSRRQSVFQ QKIG PEYIAE AFKAARAADP

201 DVKLYYNDYN IEGIGPKSDA VYEMVKSFKA QGIPIDGVGM QAHLIAGQVP

251 ASLQENIRRF ADLGVDVALT ELDIRMTLPR TAAKDAQQAT DYGAVVEACL

301 VVSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL

351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS

401 GQSISHMWNG NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA

451 DVTCTPGRPS GTSA

The aminoacid sequence of mutant V81P/G82E/D185P/S186E is as shown in SEQ ID NO.4

1 MFRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK

51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT PENTMKWQYT EPSRGRFDYT

101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GVMRDHITHE

151 VDHFKGRLIH WDVVNEAFEE DGSRRQSVFQ QKIG PEYIAE AFKAARAADP

201 DVKLYYNDYN IEGIGPKSDA VYEMVKSFKA QGIPIDGVGM QAHLIAGQVP

251 ASLQENIRRF ADLGVDVALT ELDIRMTLPR TAAKDAQQAT DYGAVVEACL

301 VVSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL

351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS

401 GQSISHMWNG NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA

451 DVTCTPGRPS GTSA

Present invention also offers the gene order of zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E of the above-mentioned thermostability improvement of coding, its nucleotide sequence is as shown in SEQ ID NO.5,6

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 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

481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG

541 CAGAAGATCG GCCCCGAGTA 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

1021CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC

1141GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT

1201GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC

261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC

1321CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC

1381GGGACTTCGG 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 CCCGAAAACA 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 GCCCCGAGTA 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

1021CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC

1081TGCACCGCTA 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 cDNA molecule of zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E of above-mentioned encoding heat stable improvement is inserted between the restriction enzyme site of described carrier with suitable orientation and correct reading frame, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.The preferred carrier of the present invention is pPIC9, between EcoRI and the NotI restriction enzyme site that the xylanase gene of transformation is inserted on plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulate and control by it, obtain the expression of recombinant yeast plasmid of each mutant.The preferred Host Strains of the present invention is Pichia pastoris GS115.

Compared to wild-type, the thermostability of two zytase XynAS9-m mutant D185P/S186E and V81P/G82E/D185P/S186E of the present invention significantly improves, optimum temperuture improves 10 DEG C and 20 DEG C respectively compared with 70 DEG C, Tm value improves 1.2 DEG C and 6.99 DEG C respectively, show brewage at paper pulp, the industrial potential using value such as bioenergy.

Accompanying drawing explanation

The Overlap-PCR schematic diagram that Fig. 1 zytase XynAS9-m suddenlys change;

Fig. 2 is pPIC9 Vector map;

Fig. 3 is pPIC9-XynAS9-m recombinant vectors collection of illustrative plates;

Fig. 4 transforms forward and backward zytase enzyme at different temperatures graphic representation alive;

Fig. 5 transforms forward and backward zytase enzyme after treatment of different temperature different time graphic representation alive;

Fig. 6 transforms the enzyme of forward and backward zytase under different pH graphic representation alive;

Fig. 7 transforms forward and backward zytase to the tolerability curves figure of different pH.

Embodiment

Test materials and reagent

1, bacterial strain and carrier: yeast expression vector pPIC9 and bacterial strain GS115 is purchased from Invitrogen company.

2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase enzyme is purchased from Invitrogen company.Available from Sigma, other is all domestic reagent (all can buy from common biochemical Reagent Company and obtain).

3, substratum:

(1) Phialophora sp. substratum is potato culture: 1000mL200g potato liquor, 10g glucose, 25g agar, pH5.0.

(2) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).

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

(4) BMMY substratum: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY, pH4.0.

Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, all carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.

Embodiment 1

1, the acquisition of mutator gene:

Transform with the gene order (SEQ IDNO.2) deriving from the zytase XynAS9-m of streptomycete Streptomyces sp.S9, introduce sudden change by the mode of Overlap PCR, and it is checked order, obtain mutator gene.

Sudden change comprise six PCR primer: S9BF, S9BR, V81P/G82E-F, V81P/G82E-R,

D185P/S186E-F、D185P/S186E-R。

Primer sequence is as follows:

S9BF:5’-TA GAATTCGACACCGCCACCCTGGGCGAACT-3’

S9BR：5’-TAT GCGGCCGCCTACGCCGAAGTCCCGGACGGC-3’

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

V81P/G82E-R:5-ACTTCATGGTGTTttcgggGGTGATCTGGC-3’

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

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

Underscore represents restriction enzyme site EcoRI and NotI, and what lowercase represented is that mutating alkali yl Overlap extension PCR method has been come by 3 PCR reactions.With XynAS9-m-pPIC9 plasmid for template, for mutant V81P/G82E:

PCR reaction system:

PCR1

PCR1 program setting:

95℃，5min；

94 DEG C, 30sec; 60 DEG C, 30sec; 72 DEG C, 1min, 30 circulations;

72℃，10min；10℃，hold。

Pcr amplification product, after the agarose gel electrophoresis of 1.2%, reclaims test kit with DNA and carries out glue recovery, be dissolved in 20 μ LddH2O, called after V81P/G82E-R.

PCR2

PCR2 program setting:

95℃，5min；

94 DEG C, 30sec; 60 DEG C, 30sec; 72 DEG C, 1min, 30 circulations;

72℃，10min；10°，hold。

Pcr amplification product, after the agarose gel electrophoresis of 1.2%, reclaims test kit with DNA and carries out glue recovery, be dissolved in 20 μ LddH2O, called after V81P/G82E-F.

PCR3

For template after mixing with PCR1 and PCR2 reaction product

PCR program setting:

95℃，5min；

94 DEG C, 30sec; 60 DEG C, 30sec; 72 DEG C, 2min, 30 circulations;

72℃，10min；10℃，hold。

Pcr amplification product, after the agarose gel electrophoresis of 1.2%, cuts the band of 1.2kb effect, reclaims test kit and carries out glue recovery, be dissolved in 20 μ L ddH with DNA ₂o.The xylanase gene (SEQ ID NO.4) of the streptomycete suddenlyd change is defined as by DNA sequencing.

2, the connection of xylanase gene XynAS9-m and expression vector

The XynAS9-m suddenly change above-mentioned acquisition and expression vector pPIC9 carries out restriction enzyme EcoRI/Not I double digestion respectively, and enzyme tangent condition is as follows:

37 DEG C of water-bath enzymes cut process 2h, reclaim two object fragments respectively, be dissolved in 20 μ LddH after electrophoresis ₂o.Connect with T4DNA ligase enzyme, linked system is as follows:

Room temperature connects 20-30min, electrophoresis detection result shows the fragment of about 9kb, and find that there is about 1.2kb and 8kb two band with after EcoR I/Not I double digestion, successful connection is described, build comprised three mutant respectively pPIC9-XynAS9-m carrier (XynAS9-m be Fixedpoint mutation modified after the code name of each xylanase gene)

3, XynAS9-m expression vector transforms the screening of Pichia pastoris GS115 and engineering bacteria

The preparation of acceptor competent cell: by Pichia pastoris GS115 bacterial strain at the flat lining out of YPD, cultivate 48h for 30 DEG C, picking grows healthy and strong single bacterium colony in 20mL YPD liquid nutrient medium, 30 DEG C, 200rpm shaking table cultivation 48h; By activation fresh GS115 bacterium liquid in 0.1% ratio be inoculated in the YPD of 200mL, 200rpm, 30 DEG C are cultured to OD ₆₀₀be about 1.0-1.3, place precooling on ice; Be transferred in the centrifuge tube of precooling by bacterium liquid, 4 DEG C, the centrifugal 5min of 5000rpm, abandon supernatant, with the resuspended thalline of the sterilized water of 200mL precooling; By resuspended thalline at 4 DEG C, the centrifugal 5min of 5000rpm, abandons supernatant postprecipitation and uses the sterilized water of 100mL precooling resuspended again; By resuspended thalline at 4 DEG C, the centrifugal 5min of 5000rpm, by the resuspended precipitation of 1mol/L Sorbitol Solution USP of 20mL precooling; By resuspended bacterial sediment at 4 DEG C, the centrifugal 5min of 5000rpm, resuspended with the Sorbitol Solution USP of the 1mol/L of 600 μ L precoolings; Resuspended cell suspending liquid is dispensed in the EP pipe of the precooling of 1.5mL with 80 μ L respectively, and is kept at-70 DEG C, for subsequent use.

Electroporated: electroporation is opened preheating in advance, 10 μ L sterilized waters will be dissolved in after linearizing plasmid purification, get the GS115 competent cell that 80 μ L have prepared and mix with it, after gone in the electric revolving cup of precooling; The parameter of adjustment electroporation is voltage 2kV, electric shock; Add immediately after having shocked by electricity.The 1mol/L sorbyl alcohol of 0.5-1mL precooling, is transferred in the EP pipe of 1.5ml after mixing, leave standstill 30min in 30 DEG C of thermostat containers after, the centrifugal 3min of 5000rpm, discards part supernatant, coats on MD flat board after remaining liq mixing, is inverted for 30 DEG C and cultivates 2-3 days;

The screening of yeast transformant: the mono-clonal sterilizing toothpick grown on MD flat board is chosen by number on MM, then put on the MD flat board of corresponding numbering; Two flat boards are placed in 30 DEG C of incubators to cultivate 2 days.The transformant of picking normal growth is inoculated in the yeast pipe that 3mL BMGY substratum is housed by number, and this yeast pipe needs to shift to an earlier date moist heat sterilization and eight layers of gauzes parcel, is placed on 30 DEG C, 220rpm shaking table cultivation 48h; Bacterium liquid shaking table being cultivated 48h is placed in the centrifugal 10min of 4400rpm, removes supernatant, adds the BMMY substratum that 1mL contains 0.5% methyl alcohol in centrifuge tube, at 30 DEG C, 220rpm inducing culture.After inducing culture 48h, bacterium liquid is placed in the centrifugal 3min of 12000rpm, gets supernatant detection of active, therefrom filter out the transformant with xylanase activity;

The expression of goal gene shaking flask level in pichia spp: the positive strain containing high enzyme vigor is inoculated in the 1L triangular flask of 400mL BMGY substratum, is placed in 30 DEG C, 48h cultivated by 220rpm shaking table; After by centrifugal for nutrient solution 5000rpm 5min, abandon supernatant, precipitate that to contain the BMMY substratum of 0.5% methyl alcohol with 200mL resuspended, and be again placed in 30 DEG C, inducing culture under 220rpm condition.1mL methyl alcohol is added every 12h, the methanol concentration in bacterium liquid is made to remain on 0.5%, induced liquid supernatant after centrifugal is the crude enzyme liquid of sudden change XynAS9-m, this crude enzyme liquid is after 6KDa hollow fiber column and 10KDa ultra-filtration membrane concentrate, concentrate with acetone precipitation further, then obtain the molecular weight of albumen consistent with wild-type after desalting column and anion column purifying.

4, the mensuration of mutant xylanases enzyme activity of the present invention

Activity unit (U) definition of 1 zytase: under certain conditions, per minute decomposes the enzyme amount required for reducing sugar that xylan discharges 1 μm of ol D-wood sugar.

The mensuration of recombinase reaction optimal pH and pH stability:

By recombination mutation zytase good for purifying at 60 DEG C, in the substrate of different pH, carry out zymetology reaction, to measure its optimal pH.Damping fluid used is: the McIlvaine damping fluid (0.2M Sodium phosphate dibasic/0.1M citric acid) of pH2.0 – 7.0, the Tris-HCl damping fluid of the 0.1mol/L of pH8.0 – 9.0, and the Gly-NaOH damping fluid of pH10.0 – 12.0.Result (Fig. 6) shows the optimal pH not too large change of each mutant enzyme, and sphere of action is basically identical, and just in damping fluid exchanges for, enzyme activity is variant.

PH Stability Determination: process 1h at the pure enzyme liquid after concentrated is placed in 37 DEG C in the damping fluid of different pH value, do suitably dilution with the damping fluid of respective optimal pH, measure residual enzyme activity under optimum condition.Result (Fig. 7) shows, and the pH tolerance of mutant enzyme is almost consistent with wild-type, and just mutant enzyme V81P/G82E and V81P/G82E/D185P/S186E is stable when pH3.0.Amino acid whose replacement does not affect its optimal pH and pH tolerance.

The mensuration of recombinase reaction optimum temperuture and thermostability:

In the damping fluid of optimal pH and under differing temps, (40 – 90 DEG C) measures active in determine optimum temperuture.

Heat-tolerance Determination is after processing different time at different temperatures, then under respective optimum condition, measure its residual enzyme vigor.Result (Fig. 4, 5) show, mutant enzyme improves 10-20 DEG C respectively compared with the optimum temperuture of protoenzyme, and after 65 DEG C of process 60min, wild-type only remains the enzyme work of 9.3%, and mutant enzyme V81P/G82E and V81P/G82E/D185P/S186E remains the enzyme work of 83% nearly, the enzyme of D185P/S186E residue 47% is lived, after 70 DEG C of process 60min, V81P/G82E and V81P/G82E/D185P/S186E residual enzyme vigor is 12 times of wild-type, namely mutant enzyme is 64% and wild-type is 5.9%, under these other hot conditionss external after process, mutant enzyme remains the residual enzyme vigor higher than wild-type, experimental result display thermostability truly has and significantly improves.

The mensuration recombinase of recombinase specific activity, Km value and Vmax

Be substrate with the xylan of different concns (0.5,0.75,1.0,2.0,4.0,5.0,6.0,8.0,10.0mg/ml), under optimum condition, measure enzymic activity, utilize double-reciprocal plot method to calculate corresponding speed of response, Km value and Vmax.

According to the method for Bio-Rad test kit, drawing standard curve.Select protein concentration to divide and be written as 2.0,1.5,1.0,0.75,0.5,0.25 and 0.125mg/ml, adopt 5uL(albumen) and 250uL(nitrite ion) reaction system, room temperature reaction 10-60min, at OD ₅₉₅its absorption value drawing standard curve of lower mensuration.Secondly the measuring method of Rate activity: first calculate the content of target protein by typical curve is that the enzyme recording recombinase under optimum condition is lived, and is the specific activity of enzyme with the live ratio of number and protein concentration of enzyme.Rate activity is defined as: the enzyme activity unit number that every milligram of zymoprotein has.

Table 1 is the forward and backward kinetic parameter of zytase of transformation and the comparison of Rate activity

Claims

1. the zytase XynAS9-m mutant D185P/S186E of a rite-directed mutagenesis, it is characterized in that, the Aspartic acid mutations of the 185th of the zytase of aminoacid sequence as shown in SEQ ID NO.1 is proline(Pro) and the mutant serine of the 186th is L-glutamic acid.

2. an xylanase gene XynAS9-m for rite-directed mutagenesis, is characterized in that, the zytase XynAS9-m mutant D185P/S186E of its rite-directed mutagenesis according to claim 1 of encoding.

3. the xylanase gene XynAS9-m of rite-directed mutagenesis according to claim 2, is characterized in that, the nucleotide sequence of described gene is as SEQ ID NO.5.

4. comprise the recombinant vectors of the xylanase gene XynAS9-m of rite-directed mutagenesis described in claim 2.

5. comprise the recombinant bacterial strain of the xylanase gene XynAS9-m of rite-directed mutagenesis described in claim 2.

6. recombinant bacterial strain according to claim 5, is characterized in that, described recombinant bacterial strain is pichia spp.

7. prepare a method of the zytase XynAS9-m of improved stability, it is characterized in that, comprise the following steps:

1) with the recombinant vectors transformed host cell of claim 4, recombinant bacterial strain is obtained;

2) recombinant bacterial strain is cultivated, the expression of the zytase XynAS9-m of induction restructuring rite-directed mutagenesis; And

3) the zytase XynAS9-m of the rite-directed mutagenesis also expressed by purifying is reclaimed.

8. the zytase XynAS9-m mutant D185P/S186E of rite-directed mutagenesis according to claim 1 is used for the application of hydrolyzed xylan.