WO2015007033A1 - Mutant of xylanase xynas9-m with improved thermal stability and gene and use thereof - Google Patents
Mutant of xylanase xynas9-m with improved thermal stability and gene and use thereof Download PDFInfo
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- WO2015007033A1 WO2015007033A1 PCT/CN2013/086988 CN2013086988W WO2015007033A1 WO 2015007033 A1 WO2015007033 A1 WO 2015007033A1 CN 2013086988 W CN2013086988 W CN 2013086988W WO 2015007033 A1 WO2015007033 A1 WO 2015007033A1
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- xylanase
- xynas9
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- mutated
- gene
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- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 25
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 17
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 10
- 239000004220 glutamic acid Substances 0.000 claims abstract description 10
- 150000001413 amino acids Chemical group 0.000 claims abstract description 9
- 239000004471 Glycine Substances 0.000 claims abstract description 7
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 235000003704 aspartic acid Nutrition 0.000 claims abstract description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 5
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims abstract description 5
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- 239000004474 valine Substances 0.000 claims abstract 3
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims abstract 3
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- 238000000034 method Methods 0.000 claims description 6
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- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 38
- 108090000790 Enzymes Proteins 0.000 abstract description 38
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 abstract 2
- 229960002989 glutamic acid Drugs 0.000 abstract 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 26
- 102200039236 rs121908522 Human genes 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 12
- 239000000872 buffer Substances 0.000 description 10
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- 102000004169 proteins and genes Human genes 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
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- 239000012634 fragment Substances 0.000 description 5
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- 238000002741 site-directed mutagenesis Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
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- 239000000203 mixture Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 229920001221 xylan Polymers 0.000 description 4
- 150000004823 xylans Chemical class 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
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- 238000006460 hydrolysis reaction Methods 0.000 description 3
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- 239000000600 sorbitol Substances 0.000 description 3
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- 239000000758 substrate Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 101150102573 PCR1 gene Proteins 0.000 description 2
- 238000013494 PH determination Methods 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 230000006698 induction Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 101100519159 Arabidopsis thaliana PCR3 gene Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 239000000026 Pentaerythritol tetranitrate Substances 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 150000001450 anions Chemical class 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
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- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010026195 glycanase Proteins 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
Definitions
- 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- ⁇ -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 ⁇ -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.
- xylanase 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.
- xylanase has been widely used in feed, pulp and paper, food, energy and other industries, especially in industrial papermaking.
- 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.
- the artificial evolution and transformation of enzyme molecules has become a hot topic in the field of enzyme engineering. So far, many researchers 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.
- 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.
- 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.
- a xylanase XynAS9-m mutant having improved thermostability 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.
- the aspartic acid at position 185 is mutated to guanidine.
- the acid, more preferably, the serine at position 186 is mutated to glutamic acid.
- 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.
- 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.
- 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
- the serine at position 186 is mutated to glutamic acid
- amino acid sequence thereof is as shown in SEQ ID N0.4:
- 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.
- the Tm values increased by 6.84 °C and 6.99 °C, respectively, showing potential applications in pulp brewing, bioenergy and other industries.
- Figure 1 Schematic diagram of Overlap-PCR of xylanase XynAS9-m mutation
- Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitragen.
- E. coli medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.
- the primer sequences are as follows:
- V81P/G82E-R 5- ACTTC ATGGTGTTttcgggGGTG ATCTGGC-3 '
- D185P/S186E-R 5 '-CGATGTActcgggGCCGATCTTCT-3 '
- 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.
- 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.
- the PCR1 and PCR2 reaction products are mixed and used as a template.
- 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.
- 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:
- 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:
- Pichia pastoris GS115 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.
- Electroporation conversion The electrorotator was pre-warmed in advance, and the linearized plasmid was purified and dissolved in 10 ⁇ L 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.
- 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.
- 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.
- 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.
- 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%.
- the mutant enzyme 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. .
- 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.
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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
一种热稳定性改良的木聚糖酶 XynAS9-m突变体及其基因和应用 技术领域 Thermodynamically improved xylanase XynAS9-m mutant and gene and application thereof
本发明属于基因工程和酶工程技术领域, 具体内容涉及一种热稳定性改良 的木聚糖酶 XynAS9-m突变体及其基因和应用。 背景技术 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
木聚糖酶(endo-l,4- β -xylanases, EC 3.2.1.8)是一类重要的工业用酶, 降解 木聚糖成低聚糖和木糖的一类酶的总称。 主要以内切方式水解木聚糖分子中的 β -1,4-糖苷键, 生成低聚木糖和木糖, 是半纤维素水解酶系中最关键的水解酶 之一, 目前所克隆的木聚糖酶大多属于 F/10和 G/11家族, 且第十家族的木聚 糖酶较第十一家族的木聚糖酶具有底物特异性低、 水解速度快、 水解产物聚合 度低等优势, 因此在工业上具有重要的应用价值。 近几年来木聚糖酶已经在饲 料、 制浆造纸、 食品、 能源等行业中得到广泛应用, 尤其是在工业造纸上的应 用。 但是目前大多木聚糖酶的最适温度为 45-55°C, 且热稳定性差, 不能满足纸 浆酿造中的要求, 并且高温木聚糖酶或耐热酶具有降低酶制剂的成本, 提高反 应催化效率, 降低反应能耗, 降低了杂菌的污染及对化学变性剂及相关金属利 息具有较高的耐受力。 基于此寻找有效途径和手段, 提高木聚糖酶在高温环境 中的稳定性及催化活性已成为国内木聚糖酶工业的当务之急。 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.
随着蛋白质工程技术和分子生物学的发展, 运用定向进化和理性设计的手 段对 With the development of protein engineering technology and molecular biology, the use of directed evolution and rational design
酶分子进行人工进化和改造已成为当前酶工程领域研究的热点。 到目前为止, 已有许多学者运用这项技术成功对蛋白的热稳定性进行了改造, 本发明应用定 点突变的方法改造木聚糖酶, 分别获得木聚糖酶 XynAS9-m突变体 V81P/G82E 和木聚糖酶 XynAS9-m突变体 V81P/G82E/D185P/S186E, 将木聚糖酶 XynAS9- m的最适温度提高了 20°C, Tm值提高了 7°C, 扩大了其在工业上的应用。 发明内容 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
本发明的目的是通过定点突变的方法对木聚糖酶进行改造, 使改造后的木 聚糖酶 XynAS9-m突变体在耐受性上性质更加优良。 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.
本发明的另一目的是提供编码上述木聚糖酶 XynAS9-m突变体的基因。
本发明的另一目的是提供包含上述木聚糖酶 XynAS9-m突变体基因的重组 载体。 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.
本发明还提供了一种宿主细胞, 其含有如前所述的木聚糖酶 XynAS9-m突 变体的基因或如前所述的重组载体。 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.
本发明对链霉菌来源的木聚糖酶 XynAS9-m基因进行定点突变, 该木聚糖 酶的成熟蛋白具有如 SEQ ID NO. 1所示的氨基酸序列, 该成熟蛋白是由如 SEQ 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.
ID NO. 2所示的核苷酸序列编码的。 The nucleotide sequence shown by ID NO. 2 is encoded.
SEQ ID NO. 1 SEQ ID NO. 1
I FRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK 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 I T VGNDHTAP I T GWTVTWTLS S 401 GQS I SHM丽 G NLTVNGQDVT VRDVGYNGTL GGNGS TTFGF QGEGVADTPA 451 DVTCTPGRP S GT SA 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 GGCAGCGTTASEQ ID NO. 2 i ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA
61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC 2丄 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC 2丄 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC
1 S1 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC1 S1 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC
241 GTCGGCAACA CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC241 GTCGGCAACA CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC
301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC
361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC
421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC
48] TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG48] TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG
541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG
60i GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC60i GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC
66i GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG66i GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG
721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC
7S1 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC7S1 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC
841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG
901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC
961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG
1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC
1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC
1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT
1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC
1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC
1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC
13S1 GGGACTTCGG CGTAG 13S1 GGGACTTCGG CGTAG
根据本发明的热稳定性提高的木聚糖酶 XynAS9-m突变体, 氨基酸序列如
SEQ ID NO. 1所示的木聚糖酶的第 81位缬氨酸突变为脯氨酸且第 82位甘氨酸 突变为谷氨酸, 优选进一步, 第 185位的天冬氨酸突变为脯氨酸, 更优选, 第 186位的丝氨酸突变为谷氨酸。 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.
根据本发明的具体实施方式, 采用定点突变的方法, 获得了 2个热稳定性 提高的木聚糖酶 XynAS9-m 突变体, 分别命名为 V81P/G82E 、 V81P/G82E/D185P/S186E, BP: V81P/G82E为第 81位缬氨酸突变为脯氨酸且第 82位甘氨酸突变为谷氨酸; V81P/G82E/D185P/S186E为第 81位缬氨酸突变为 脯氨酸, 第 82位甘氨酸突变为谷氨酸, 第 185位的天冬氨酸突变为脯氨酸且第 186位的丝氨酸突变为谷氨酸。 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.
因此根据本发明的热稳定性改良的木聚糖酶 XynAS9-m 突变体 Therefore, the thermostability-improved xylanase XynAS9-m mutant according to the present invention
V81P/G82E, 其中第 81 位缬氨酸突变为脯氨酸, 第 82位甘氨酸突变为谷氨 酸, 其氨基酸序列如 SEQ ID NO.3所示 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 SEQ ID NO.3
1 FRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK 1 FRHHPTRGR RTAGLLAAAL ATLSAGLTAV APAHPARADT ATLGELAEAK
51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT PENT KWQYT EPSRGRFDYT51 GRYFGSATDN PELPDTQYTQ ILGSEFSQIT PENT KWQYT EPSRGRFDYT
101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GV RDHITHE101 AAEEIVDLAE SNGQSVRGHT LVWHNQLPSW VDDVPAGELL GV RDHITHE
151 VDHFKGRLIH WDWNEAFEE DGSRRQSVFQ QKIGDSYIAE AFKAARAADP151 VDHFKGRLIH WDWNEAFEE DGSRRQSVFQ QKIGDSYIAE AFKAARAADP
201 DVKLYYNDYN IEGIGPKSDA VYE VKSFKA QGIPIDGVG QAHLIAGQVP201 DVKLYYNDYN IEGIGPKSDA VYE VKSFKA QGIPIDGVG QAHLIAGQVP
251 ASLQENIRRF ADLGVDVALT ELDIR TLPR TAAKDAQQAT DYGAWEACL251 ASLQENIRRF ADLGVDVALT ELDIR TLPR TAAKDAQQAT DYGAWEACL
301 WSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL301 WSRCVGITV WDYTDKYSWV PSVFPGQGAA LPWDEDFAKK PAYHAIAAAL
351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS351 NGGSPAPGGN CTATYRVTSQ WQGGFTAEIT VGNDHTAPIT GWTVTWTLSS
401 GQSISHM丽 G NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA 451 DVTCTPGRPS GTSA 因此根据本发明的热稳定性改良的木聚糖酶 XynAS9-m 突变体 V81P/G82E/D185P/S186E, 其中第 81位缬氨酸突变为脯氨酸, 第 82位甘氨酸 突变为谷氨酸, 第 185位的天冬氨酸突变为脯氨酸且第 186位的丝氨酸突变为 谷氨酸, 其氨基酸序列如 SEQ ID N0.4所示: 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 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丽 G NLTVNGQDVT VRDVGYNGTL GGNGSTTFGF QGEGVADTPA 451 DVTCTPGRPS GTSA 本发明还提供了编码上述热稳定性改良的木聚糖酶 XynAS9-m突变体基因 序列, 其核苷酸序列如 SEQ ID NO.5、 6所示
SEQ ID NO. 5 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 GGCAGCGTTA1 ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA
61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC
121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC
181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC
241 CCCGAAKKCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC241 CCCGAAKKCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC
301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC
361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC
421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC
481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG
541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG541 CAGAAGATCG GCGACAGTTA CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG
601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC
661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG
721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC
781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC
841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG
901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC
961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG
1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC
1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC
1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT
1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC
1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC
1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC
1381 GGGACTTCGG CGTAG 1381 GGGACTTCGG CGTAG
SEQ ID NO. 6 SEQ ID NO. 6
1 ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA 1 ATGTTCCGCC ACCACCCGAC CCGAGGCCGC CGCACGGCCG GCCTCCTCGC GGCAGCGTTA
61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC61 GCAACCCTGT CGGCCGGCCT GACCGCGGTT GCGCCCGCTC ATCCGGCCCG CGCCGACACC
121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC121 GCCACCCTGG GCGAACTGGC CGAGGCCAAG GGCCGTTACT TCGGCTCCGC CACGGACAAC
181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC181 CCCGAACTGC CCGACACTCA GTACACGCAG ATCCTGGGCA GCGAGTTCAG CCAGATCACC
241 CCCGAAkkCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC241 CCCGAAkkCk CCATGAAGTG GCAGTACACC GAGCCGTCTC GGGGCCGGTT CGACTACACC
301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC301 GCCGCCGAGG AGATAGTCGA CCTGGCCGAG TCCAACGGCC AGTCGGTGCG CGGACACACC
361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC361 CTGGTGTGGC ACAACCAGCT GCCGAGCTGG GTCGACGACG TGCCGGCCGG TGAGCTCCTC
421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC421 GGGGTCATGC GCGACCACAT CACCCACGAG GTCGACCACT TCAAGGGGCG ACTGATCCAC
481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG481 TGGGACGTGG TCAACGAGGC GTTCGAGGAG GACGGCAGCC GCCGGCAGTC GGTCTTCCAG
541 CAGAAGATCG QCCCCGAGlk CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG541 CAGAAGATCG QCCCCGAGlk CATCGCCGAG GCATTCAAGG CCGCCCGCGC CGCCGATCCG
601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC601 GACGTCAAGC TCTACTACAA CGACTACAAC ATCGAAGGCA TCGGCCCCAA GAGCGATGCC
661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG661 GTCTACGAGA TGGTGAAGTC CTTCAAGGCC CAGGGCATCC CCATCGACGG CGTCGGCATG
721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC721 CAGGCACATC TGATCGCCGG CCAGGTCCCG GCAAGCCTGC AGGAGAACAT CCGGCGCTTC
781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC781 GCCGACCTGG GCGTCGACGT CGCCCTCACC GAACTCGACA TCCGCATGAC CCTGCCGCGC
841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG841 ACCGCTGCCA AGGATGCCCA GCAGGCCACC GACTACGGTG CCGTGGTCGA GGCATGCCTG
901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC901 GTGGTCTCCC GGTGCGTCGG CATCACCGTC TGGGACTACA CCGACAAGTA CTCCTGGGTC
961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG961 CCCTCCGTCT TCCCGGGCCA GGGTGCCGCC CTGCCATGGG ACGAGGACTT CGCCAAGAAG
1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC1021 CCCGCCTATC ACGCCATCGC CGCCGCGCTC AACGGCGGCA GCCCCGCCCC CGGTGGCAAC
1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC1081 TGCACCGCTA CCTACCGCGT CACCAGCCAG TGGCAGGGCG GCTTCACCGC CGAGATCACC
1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT1141 GTCGGGAACG ACCACACCGC GCCGATTACC GGCTGGACCG TCACCTGGAC GCTGTCCAGT
1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC1201 GGCCAGTCCA TCAGCCACAT GTGGAACGGA AACCTCACCG TCAACGGACA GGACGTCACC
1261 GTCCGCGACG TCGGCTACAA CGGCACCCTC GGCGGCAACG GAAGCACCAC CTTCGGCTTC
1321 CAGGGCGAAG GCGTGGCCGA CACTCCGGCG GACGTGACCT GTACCCCCGG CCGGCCGTCC 1381 GGGACTTCGG CGTAG 将上述编码热稳定性改良的木聚糖酶 XynAS9-m突变体的 cDNA分子以合 适的取向和正确的读码框***到所述载体的限制性酶切位点之间, 使其核苷酸 序列可操作的与表达调控序列相连接。 本发明优选的载体为 pPIC9, 使改造的 木聚糖酶基因***到质粒 pPIC9上的 EcoRI和 Not I限制性酶切位点之间, 使该 核苷酸序列位于 AOX1 启动子的下游并受其调控, 得到各突变体的重组酵母表 达质粒。 本发明优选的宿主菌为毕赤酵母 GS115。 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.
相比于野生型, 本发明的两个木聚糖酶 XynAS9-m突变体 V81P/G82E和 V81P/G82E/D185P/S186E的热稳定性明显提高, 最适温度较 70°C分别提高了 20 °〇和 20°C, Tm值分别提高了 6.84 °C和 6.99°C, 显示出了在纸浆酿造、 生物能 源等工业上潜在的应用价值。 附图说明 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
图 1木聚糖酶 XynAS9-m突变的 Overlap-PCR示意图; Figure 1. Schematic diagram of Overlap-PCR of xylanase XynAS9-m mutation;
图 2为 pPIC9载体图谱; Figure 2 is a map of pPIC9 vector;
图 3为 pPIC9-XynAS9-m重组载体图谱 Figure 3 shows the map of pPIC9-XynAS9-m recombinant vector
图 4为改造前、 后的木聚糖酶在不同温度下的酶活曲线图; Figure 4 is a graph showing the activity of xylanase at different temperatures before and after the transformation;
图 5为改造前、 后的木聚糖酶在不同温度处理不同时间后酶活曲线图; 图 6为改造前、 后的木聚糖酶在不同 pH下的酶活曲线图; 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;
图 7为改造前、 后的木聚糖酶对不同 pH的耐受性曲线图。 具体实施方式 试验材料和试剂 Figure 7 is a graph showing the tolerance of xylanase to different pH before and after modification. DETAILED DESCRIPTION Test Materials and Reagents
1、 菌株及载体: 毕赤酵母表达载体 pPIC9及菌株 GS115购自于 Invitragen 公司。 1. Strains and vectors: Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitragen.
2、 酶类及其它生化试剂: 内切酶购自 TaKaRa 公司, 连接酶购自 Invitragen公司。 购自 Sigma公司, 其它都为国产试剂 (均可从普通生化试剂公 司购买得到)。 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、 培养基:
( 1 ) i¾fltop/wra sp.培养基为马铃薯培养基: 1000mL 200g土豆煮汁, 10g 葡萄糖, 25g琼脂, pH5.0。 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 ) 大肠杆菌培养基 LB (1%蛋白胨、 0.5%酵母提取物、 1% NaCl, pH7.0)。 (2) E. coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).
( 3 ) BMGY培养基: 1%酵母提取物, 2%蛋白胨, 1.34%YNB, 0.00004% Biotin, 1%甘油 (V/V)。 (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).
(4) BMMY培养基: 除以 0.5%甲醇代替甘油,其余成份均与 BMGY相 同, H4.0 o (4) BMMY medium: Divided by 0.5% methanol instead of glycerol, the rest of the ingredients are the same as BMGY, H4.0 o
说明: 以下实施例中未作具体说明的分子生物学实验方法, 均参照 《分子 克隆实验指南》 (第三版) J.萨姆布鲁克一书中所列的具体方法进行, 或者按 照试剂盒和产品说明书进行。 实施例 1 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、 突变基因的获得- 以来源于链霉菌 Streptomyces sp. S9的木聚糖酶 XynAS9-m的基因序列 1. Obtaining the mutated gene - the gene sequence of the xylanase XynAS9-m derived from Streptomyces sp. S9
(SEQ ID NO. 2) 进行改造, 通过 Overlap PCR的方式引入突变, 并对其进行 测序, 获得突变基因。 (SEQ ID NO. 2) was modified, and the mutation was introduced by Overlap PCR and sequenced to obtain a mutant gene.
突变包括六条 PCR引物: S9 BF、 S9 BR、 V81P/G82E-F、 V81P/G82E-R、 D185P/S186E-F、 D185P/S186E-R。 The mutation includes six PCR primers: S9 BF, S9 BR, V81P/G82E-F, V81P/G82E-R, D185P/S186E-F, D185P/S186E-R.
引物序列如下: The primer sequences are as follows:
S9BF:5 '- TAGAATTCGACACCGCCACCCTGGGCGAACT-3 ' S9BF: 5 '- TAGAATTCGACACCGCCACCCTGGGCGAACT-3 '
S9 BR: 5 '-TATGCGGCCGCCTACGCCGAAGTCCCGGACGGC-3 ' S9 BR: 5 '-TATGCGGCCGCCTACGCCGAAGTCCCGGACGGC-3 '
V81P/G82E-F: 5'-GCCAGATCACCcccgaaAACACCATGAAGT-3 ' V81P/G82E-F: 5'-GCCAGATCACCcccgaaAACACCATGAAGT-3 '
V81P/G82E-R: 5- ACTTC ATGGTGTTttcgggGGTG ATCTGGC-3 ' V81P/G82E-R: 5- ACTTC ATGGTGTTttcgggGGTG ATCTGGC-3 '
D185P/S186E-F: 5 '-AGAAGATCGGCcccgagTACATCG-3 '; D185P/S186E-F: 5 '-AGAAGATCGGCcccgagTACATCG-3 ';
D185P/S186E-R: 5 '-CGATGTActcgggGCCGATCTTCT-3 ' D185P/S186E-R: 5 '-CGATGTActcgggGCCGATCTTCT-3 '
下划线代表限制性酶切位点 EcoRI和 NotI, 小写字母表示的为突变碱基 重叠延伸 PCR法是通过 3个 PCR反应来完成的。 以 ,《A¾>-m- ρ/Υί^质粒为 模板, 以突变体 V81P/G82E为例: 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反应体系: PCR reaction system:
PCR1
2*Pfu mix 25.0 μΐ PCR1 2*Pfu mix 25.0 μΐ
S9BF 0.5 ul S9BF 0.5 ul
V81P/G82E-R 0.5 ul V81P/G82E-R 0.5 ul
ddH20 22.0 μΐ ddH 2 0 22.0 μΐ
Template 2単 Template 2単
总体积 50.0 μΐ Total volume 50.0 μΐ
PCR1程序设定: PCR1 program settings:
95。C, 5 min; 95. C, 5 min;
94°C, 30 sec; 60°C, 30 sec; 72°C, 1 min, 30个循环; 94 ° C, 30 sec; 60 ° C, 30 sec; 72 ° C, 1 min, 30 cycles;
72°C, 10 min; 10°C, hold。 72 ° C, 10 min; 10 ° C, hold.
PCR扩增产物经 1. 2%的琼脂糖凝胶电泳后, 用 DNA回收试剂盒进行胶回收, 浴 于 2(^LddH20, 命名为 V81P/G82E_R。 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 PCR 2
2*PfU mix PCR2 : 25.0 μΐ 2*Pf U mix PCR2 : 25.0 μΐ
S9BR 0.5 ul S9BR 0.5 ul
V81P/G82E-F 0.5 ul V81P/G82E-F 0.5 ul
ddH20 22.0 μΐ ddH 2 0 22.0 μΐ
Template 2単 Template 2単
总体积 50.0 μΐ Total volume 50.0 μΐ
PCR2程序设定: PCR2 program settings:
95。C, 5 min; 95. C, 5 min;
94。C, 30 sec; 60。C, 30 sec; 72。C, 1 min, 30个循环; 94. C, 30 sec; 60. C, 30 sec; 72. C, 1 min, 30 cycles;
72°C, 10 min; 10°C, hold。 72 ° C, 10 min; 10 ° C, hold.
PCR扩增产物经 1. 2%的琼脂糖凝胶电泳后, 用 DNA回收试剂盒进行胶回收, 浴 于 2(^LddH20,命名为 V81P/G82E_F。 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 PCR3
以 PCR1和 PCR2反应产物混合后为模板 The PCR1 and PCR2 reaction products are mixed and used as a template.
2*Pfu mix 25.0 μΐ 2*Pfu mix 25.0 μΐ
S9BF 0.5 ul S9BF 0.5 ul
S9BR 0.5 ul ddH20 22.0 μΐ S9BR 0.5 ul ddH 2 0 22.0 μΐ
Template 2単 Template 2単
总体积 50.0 μΐ
PCR程序设定: Total volume 50.0 μΐ PCR program settings:
95。C, 5 min; 95. C, 5 min;
94。C, 30 sec; 60。C, 30 sec,; 72。C, 2 min,30个循环; 94. C, 30 sec; 60. C, 30 sec,; 72. C, 2 min, 30 cycles;
72°C, 10 min; 10°C, hold。 72 ° C, 10 min; 10 ° C, hold.
PCR扩增产物经 1.2%的琼脂糖凝胶电泳后, 切去 1.2kb作用的条带, 用 DNA回收试剂盒进行胶回收, 溶于 2(VL ddH20。 通过 DNA测序确定为突变的链 霉菌的木聚糖酶基因。 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、 木聚糖酶基因 XynAS9-m与表达载体的连接 2. The connection of the xylanase gene XynAS9-m to the expression vector
将上述获得突变的 , -m与表达载体 pPIC9分别进行限制性内切酶 EcoR I/Not I双酶切, 酶切条件如下: 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片段酶切体系 (50μυ 质粒 pPIC9酶切体系 (50μυ PCR fragment digestion system (50μυ plasmid pPIC9 digestion system (50μυ)
PCR片段 20 pPIC9 20 PCR fragment 20 pPIC9 20
10*H 5μί 10*Buffer 5μί 10*H 5μί 10*Buffer 5μί
BSA 5μί BSA 5μί BSA 5μί BSA 5μί
Triton 5μί Triton 5μί Triton 5μί Triton 5μί
EcoR I 1辈 EcoR I 1辈 EcoR I 1 Generation EcoR I 1 Generation
Not I 1辈 Not I 1辈 Not I 1 generation Not I 1 generation
DdH20 13辈 DdH20 13辈 DdH 2 0 13 generation DdH 2 0 13 generation
37°C水浴酶切处理 2h, 电泳后分别回收两个目的片段, 溶于 20nLddH2O。 用 T4 DNA连接酶进行连接, 连接体系如下: 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片段 5μΙ pPIC9 2 μΐPCR fragment 5μΙ pPIC9 2 μΐ
T4 DNA ligase Ιμί T4 DNA ligase Ιμί
10*Buffer 1.5 L 10*Buffer 1.5 L
DdH2Q 5.5 DdH 2 Q 5.5
总体积 15.0 室温连接 20-30min, 电泳检测结果显示有大约 9kb的片段, 并且用 EcoR I/Not I 双酶切后发现有 1.2kb和 8kb左右两条带, 说明连接成功, 构建得到分别包含 V81P/G82E 、 V81P/G82E/D185P/S186E 突变体的 pPIC9-XynAS9-m 载体 (XynAS9-m为定点突变改造后的各木聚糖酶基因的代号) 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、 XynAS9-m表达载体转化毕赤酵母 GS115及工程菌的筛选
受体感受态细胞的制备: 将毕赤酵母 GS 115菌株在 YPD平板上划线, 30°C 培养 48 h, 挑取生长茁壮的单菌落于 20 mL YPD液体培养基中, 30°C、 200 rpm 摇床培养 48 h; 将活化的新鲜 GS 115菌液按 0.1%的比例接种于 200 mL的 YPD 中, 200 rpm, 30°C培养至 OD6(K)约为 1.0-1.3, 放置冰上预冷; 将菌液转移至预 冷的离心管中, 4°C、 5000rpm离心 5 min, 弃上清, 用 200 mL预冷的无菌水重 悬菌体; 将重悬菌体在 4。C, 5000rpm离心 5 min, 弃上清后沉淀再用 100 mL预 冷的无菌水重悬; 将重悬菌体在 4°C, 5000 rpm离心 5 min, 用 20 mL预冷的 1 mol/L山梨醇溶液重悬沉淀; 将重悬的菌体沉淀在 4。C, 5000 rpm离心 5 min, 用 60(^L预冷的 1 mol/L的山梨醇溶液重悬; 对重悬细胞悬浮液分别以 80 分 装到 1.5 mL的预冷的 EP管中, 并保存在 -70°C, 备用。 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.
电击转化: 将电转仪提前开启预热, 将线性化的质粒纯化后溶于 10 μL无 菌水, 取 80 已制备好的 GS 115感受态细胞与之混匀, 后将其转至预冷的电 转杯中; 调整电转仪的参数为电压 2 kV, 电击; 电击完成后立即加入。 0.5-1 mL预冷的 1 mol/L山梨醇, 混匀后转移至 1.5ml的 EP管中, 在 30°C恒温箱中 静置 30min后, 5000rpm离心 3min, 弃去部分上清, 剩余液体混匀后涂布于 MD平板上, 30°C倒置培养培养 2-3天; 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;
酵母转化子的筛选: 将在 MD平板上长出的单克隆用灭菌牙签按编号挑至 MM上, 再点到相应编号的 MD平板上; 将两平板置于 30°C培养箱中培养 2 天。 按编号挑取正常生长的转化子接种于装有 3 mL BMGY培养基的酵母管 中, 此酵母管需要提前湿热灭菌且八层纱布包裹, 将其置于 30°C、 220 rpm摇 床培养 48 h; 将摇床培养 48 h的菌液置于 4400rpm离心 10 min, 去上清, 向离 心管中加入 1 mL含有 0.5%甲醇的 BMMY培养基, 在 30°C、 220 rpm诱导培 养。 诱导培养 48 h后, 将菌液置于 12000 rpm离心 3 min, 取上清检测活性, 从 中筛选出具有木聚糖酶活性的转化子; 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;
目的基因在毕赤酵母中摇瓶水平的表达: 将含有较高酶活力的阳性菌株接 种于 400 mL BMGY培养基的 1 L三角瓶中, 置于 30。C, 220 rpm摇床培养 48 h; 后将培养液 5000rpm离心 5 min, 弃上清, 沉淀用 200 mL含有 0.5%甲醇的 BMMY培养基重悬, 并再次置于 30°C, 220 rpm条件下诱导培养。 每隔 12 h补 加 1 mL甲醇, 使菌液中的甲醇浓度保持在 0.5%, 诱导液经离心后上清为突变 XynAS9-m的粗酶液, 此粗酶液经 6KDa中空纤维柱及 lOKDa超滤膜浓缩后, 进一步用丙酮沉淀进行浓缩, 再经脱盐柱及阴离子柱纯化后获得与野生型一致
的蛋白分子量。 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、 本发明所述的突变木聚糖酶活力的测定 4. Determination of mutant xylanase activity according to the present invention
1个木聚糖酶的活性单位 (U)定义: 在一定的条件下, 每分钟分解木聚糖释 放出 1 μηιοΐ ϋ-木糖的还原糖所需要的酶量。 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.
重组酶反应最适 pH和 pH稳定性的测定: Determination of pH and pH stability of the recombinant enzyme reaction:
将纯化好的重组突变木聚糖酶在 60°C下, 不同 pH的底物中进行酶学反 应, 以测定其最适 pH。 所用缓冲液为: pH 2.0-7.0的 Mcllvaine缓冲液 (0.2 M 磷酸氢二钠 /0.1 M柠檬酸), pH 8.0-9.0的 0.1 mol/L的 Tris-HCl缓冲液, 以及 pH 10.0-12.0的 Gly-NaOH缓冲液。 结果 (图 6) 表明各突变酶的最适 pH没有 太大改变, 且作用范围基本一致, 只是在缓冲液换取中酶活力有差异。 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.
pH稳定性测定: 将浓缩后的纯酶液在不同 pH值的缓冲液中置于 37°C下 处理 1 h, 用各自最适 pH的缓冲液作适当稀释, 在最适条件下测定剩余酶活 性。 结果 (图 7) 显示, 突变酶的 pH耐受性几乎与野生型一致, 只是突变酶 V81P/G82E与 V81P/G82E/D185P/S186E在 pH3.0时稳定。 氨基酸的取代不影响 其最适 pH和 pH耐受性。 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.
重组酶反应最适温度和热稳定性的测定: Determination of the optimum temperature and thermal stability of the recombinant enzyme reaction:
在最适 pH的缓冲液中及不同温度下 (40-90°C)测定活性以确定最适温度。 耐热性测定为在不同温度下处理不同时间后, 再在各自最适条件下测定其 剩余酶活力。 结果 (图 4,5 ) 显示, 突变酶较原酶的最适温度分别提高了 10-20 , 并且在 65 °C处理 60min后, 野生型仅剩余 9.3%的酶活, 而突变酶 V81P/G82E与 V81P/G82E/D185P/S186E剩余将近 83%的酶活, D185P/S186E剩 余 47%的酶活, 70°C处理 60min后, V81P/G82E与 V81P/G82E/D185P/S186E剩 余酶活力是野生型的 12倍, 即突变酶为 64%而野生型为 5.9%而已, 此外在其 他高温条件下处理后, 突变酶始终保持高于野生型的剩余酶活力, 实验结果显 示热稳定性确有显著提高。 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. .
重组酶比活、 Km值及 Vmax的测定重组酶 Recombinase specific activity, Km value and Vmax determination of recombinase
用不同浓度的木聚糖 (0.5, 0.75, 1.0, 2.0, 4.0, 5.0, 6.0、 8.0、 10.0mg/ml) 为底物, 在最适条件下测定酶活性, 利用双倒数作图法计算出相应的反应速 度, m值及 Vmi«。 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«.
按照 Bio-Rad试剂盒的方法, 绘制标准曲线。 选用蛋白浓度分写为 2.0、 1.5、 1.0、 0.75、 0.5、 0.25和 0.125mg/ml, 采用 5uL (蛋白) 和 250 uL (显色
液) 的反应体系, 室温反应 10-60min, 在 OD595下测定其吸收值绘制标准曲线。 比活力的测定方法: 首先通过标准曲线计算目的蛋白的含量, 其次是在最适条 件下测得重组酶的酶活, 用酶活数与蛋白浓度的比值即为酶的比活。 比活力的 定义为: 每毫克酶蛋白所具有的酶活力单位数。 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.
表 1为改造前、 后的木聚糖酶的动力学参数及比活力的比较 Table 1 compares the kinetic parameters and specific activities of xylanase before and after transformation.
不同突变位点 Specific activity Km ^cat kcat/ Km V¾iax Different mutation sites Specific activity Km ^cat kcat/ Km V3⁄4iax
-1 -1
的酶 U.mg- 1 s ml.s '.mg"1 U.mg"1 野生型的酶 630.27 1.03 790.20 760.33 950.82 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 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 206.93 1.05 212.46 202.51 255.64
V81P/G82E/ V81P/G82E/
160.37 2.21 235.59 106.48 283.47 160.37 2.21 235.59 106.48 283.47
D 185P/S 186E
D 185P/S 186E
Claims
1、 一种木聚糖酶 XynAS9-m突变体, 其特征在于, 将氨基酸序列如 SEQ ID NO. 1所示的木聚糖酶的第 81位缬氨酸突变为脯氨酸, 且第 82位甘氨酸突 变为谷氨酸。 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、 根据权利要求 1所述的木聚糖酶 XynAS9-m突变体, 其特征在于, 氨基 酸序列如 SEQ ID NO. 1所示的木聚糖酶的第 185位的天冬氨酸突变为脯氨酸。 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、 根据权利要求 1或 2所述的木聚糖酶 XynAS9-m突变体, 其特征在于, 氨基酸序列如 SEQ ID NO. 1所示的木聚糖酶的第 186位的丝氨酸突变为谷氨 酸。 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、 一种木聚糖酶突变体基因, 其特征在于, 其编码权利要求 1、 2或 3所 述的木聚糖酶 XynAS9-m突变体。 4. A xylanase mutant gene, characterized in that it encodes the xylanase XynAS9-m mutant described in claim 1, 2 or 3.
5、 根据权利要求 4所述的木聚糖酶突变体基因, 其特征在于, 所述基因的 核苷酸序列如 SEQ ID NO. 5或 6所示。 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、 包含权利要求 4所述木聚糖酶突变体基因的重组载体。 6. A recombinant vector comprising the xylanase mutant gene of claim 4.
7、 包含权利要求 4所述木聚糖酶突变体基因的重组菌株。 7. A recombinant strain comprising the xylanase mutant gene of claim 4.
8、 一种制备稳定性改良的木聚糖酶 XynAS9-m的方法, 其特征在于, 包括 以下步骤: 8. A method for preparing xylanase XynAS9-m with improved stability, characterized by comprising the following steps:
1 ) 用权利要求 6的重组载体转化宿主细胞, 得重组菌株; 1) Transform host cells with the recombinant vector of claim 6 to obtain a recombinant strain;
2) 培养重组菌株, 诱导重组定点突变的木聚糖酶 XynAS9-m的表达; 以及 2) Cultivate the recombinant strain and induce the expression of the recombinant site-directed mutated xylanase XynAS9-m; and
3) 回收并纯化所表达的定点突变的木聚糖酶 XynAS9-m。 3) Recover and purify the expressed site-directed mutated xylanase XynAS9-m.
9、 权利要求 1、 2或 3所述的木聚糖酶 XynAS9-m突变体的应用。
9. Application of the xylanase XynAS9-m mutant described in claim 1, 2 or 3.
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