WO2019033775A1 - 木聚糖酶突变体 - Google Patents

木聚糖酶突变体 Download PDF

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WO2019033775A1
WO2019033775A1 PCT/CN2018/083635 CN2018083635W WO2019033775A1 WO 2019033775 A1 WO2019033775 A1 WO 2019033775A1 CN 2018083635 W CN2018083635 W CN 2018083635W WO 2019033775 A1 WO2019033775 A1 WO 2019033775A1
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seq
xylanase
mutant
amino acid
xyna1
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PCT/CN2018/083635
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French (fr)
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吴秀秀
邵弨
黄亦钧
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青岛蔚蓝生物集团有限公司
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Priority to EP18845993.7A priority Critical patent/EP3670654A4/en
Priority to US16/639,571 priority patent/US10968439B2/en
Publication of WO2019033775A1 publication Critical patent/WO2019033775A1/zh

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    • 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)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • the present invention relates to the field of protein engineering technology, and in particular to a xylanase mutant having improved heat resistance.
  • Xylan is a major component of plant hemicellulose and is widely found in corncob, bagasse, wheat bran, straw and other crop wastes. Because xylanase can decompose xylan into oligomeric wood of different lengths. Sugar and xylose, this product has important economic value. The xylanase makes full use of these available resources and exerts its potential application value. The research of xylanase has also received full attention.
  • Xylanases are glycosyl hydrolases that hydrolyze beta-1,4-linked xylopyranoside chains and have been shown to be present in at least hundreds of different organisms and can be produced economically on a large scale. Together with other glycosyl hydrolases, they form a superfamily comprising more than 40 different enzyme families. Trichoderma reesei is known to produce three different xylanases, of which xylanases I and II (XynI and XynII) have the best characteristics. XynI has a molecular weight of 19 kDa, and its isoelectric point and optimum pH are lower (pI 5.5, pH 3-4) compared to XynII. XynII has a molecular weight of 20 kDa, an isoelectric point of 9.0, and an optimum pH of 5.0-5.5.
  • Xylanase is now widely used in pulp bleaching, textile fiber modification, and in animal feed and human food production.
  • a common problem in all of these applications is the extreme conditions faced by xylanases.
  • the material from the alkaline wash has a higher temperature (> 80 ° C) and a pH (> 10). Most xylanases will be inactivated under such conditions, so the pulp must be cooled to neutralize the alkali, but this means an increase in cost.
  • xylanases are exposed to brief elevated temperatures during feed preparation (eg, 2-5 minutes at 90 °C). However, the catalytic activity of the xylanase requires a lower temperature (e.g., about 37 ° C). Thus, the xylanase will irreversibly deactivate at high temperatures.
  • thermostable xylanase suitable for industrial production.
  • the present invention provides a xylanase mutant, which obtains a mutant protein and improves its heat resistance, thereby facilitating the wide application of xylanase in the field of feed.
  • the present invention provides the following technical solutions:
  • the present invention provides a xylanase mutant comprising a disulfide bridge comprising one or more of T1C-T27C, Q33C-T187C or S109C-N153C (xylanase numbering) ;
  • (III) a nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2 or its complement or by the degeneracy of the genetic code and the nucleotide sequence shown in SEQ ID NO: 2 or its complement An amino acid sequence encoded by a sequence having a different nucleotide sequence;
  • One or more amino acids in which the substitution is at position 51, 143 or 161 are substituted.
  • the xylanase mutants provided herein have an amino acid sequence that has at least 96% homology to the amino acid sequence of the xylanase.
  • the xylanase mutants provided herein have an amino acid sequence that is at least 97% homologous to the amino acid sequence of the xylanase.
  • the xylanase mutants provided herein have an amino acid sequence that has at least 98% homology to the amino acid sequence of the xylanase.
  • the xylanase mutants provided herein have an amino acid sequence that has at least 99% homology to the amino acid sequence of the xylanase.
  • the xylanase mutants provided herein have one or more of the following disulfide bridges: T1C-T27C, Q33C-T187C, and S109C-N153C.
  • the mutants provided herein have two or three of the following disulfide bridges: T1C-T27C, Q33C-T187C and S109C-N153C.
  • the mutants of the invention have the following two disulfide bridges: Q33C-T187C and S109C-N153C.
  • the mutants of the invention have the following three disulfide bridges: T1C-T27C, Q33C-T187C and S109C-N153C.
  • the xylanase mutant has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.
  • the invention also provides DNA molecules encoding the xylanase mutants described above.
  • the DNA molecule encoding the xylanase mutant described above has the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6.
  • the xylanase mutant has at least one substituted amino acid residue at the 51, 143, 161 (xylanase numbering) position.
  • the xylanase mutant has at least 2 substituted amino acid residues at positions 51, 143, 161 (xylanase numbering).
  • the xylanase mutant has three substituted amino acid residues at positions 51, 143, 161 (xylanase numbering).
  • the substitution is at least one mutation selected from the group consisting of Q51N, H143K, Q161F.
  • the xylanase mutant has SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 or SEQ ID NO: 25.
  • the invention also provides DNA molecules encoding the xylanase mutants described above.
  • the DNA molecule encoding the xylanase mutant described above has SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO : 16 or the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 22 or SEQ ID NO: 24 or SEQ ID NO: 26.
  • the present invention also discloses a vector having the above DNA molecule.
  • the invention also provides a host cell comprising the above recombinant expression vector.
  • the host cell is Pichia pastoris.
  • the above plasmid was transferred into Pichia pastoris, and the heat resistance of the recombinantly expressed xylanase mutant was remarkably improved.
  • the present invention artificially introduces two or three unnatural disulfide bridges in xylanase by site-directed mutagenesis, and obtains heat-resistant mutants XynA1 and XynA2, especially mutants introducing three disulfide bridges.
  • XynA2 after 6 min treatment at 80 ° C, had an enzyme activity residual rate of 75%, which was 72% higher than that of the mutant XynA1.
  • the present invention further screens to obtain three mutation sites Q51N, H143K, Q161F which can significantly increase the heat tolerance of the mutant, and the mutation sites are introduced into the xylanase XynA1 by a single point or a combination of two points or a combination of three points.
  • the three-point combination mutant XynA1-H143K-Q51N-Q161F which has a residual activity of 61.28% after treatment at 80 °C for 5 min, and 26.89% of enzyme after treatment at 85 °C for 3 min. Residual, heat resistance is remarkable.
  • the mutant site was introduced into the mutant obtained by xylanase XynA2 at a single point. After treatment at 80 ° C for 5 min, the residual rate of enzyme activity generally increased by 9.26% -24.58%, and the residual rate of enzyme activity after treatment at 85 ° C for 3 min. It has generally increased by 26.63% to 46.61%.
  • thermostable mutation sites Q51N, H143K, and Q161F into the xylanase XynA2, respectively, and the combination of mutations having better heat resistance than the above single point mutant includes : H143K+Q51N, H143K+Q161F, Q51N+Q161F, H143K+Q51N+Q161F.
  • the residual activity of the combined mutants was 100% after treatment at 80 ° C for 5 min, and the residual rate of enzyme activity was over 95% after treatment at 85 ° C for 3 min, and the heat resistance was significantly improved.
  • the invention discloses a xylanase mutant, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • polypeptide refers to a compound consisting of a single strand of amino acid residues joined by peptide bonds.
  • protein as used herein may be synonymous with the term “polypeptide” or may additionally refer to a complex of two or more polypeptides.
  • expression refers to the process by which a gene-based nucleic acid sequence produces a polypeptide. This process includes transcription and translation.
  • the term "gene” means a DNA fragment involved in the production of a polypeptide chain, which may or may not contain a region before or after the coding region.
  • disulfide bridge or “disulphide bond” refers to a bond formed between a sulfur atom of a cysteine residue in a polypeptide or protein. .
  • the disulfide bridge or disulfide bond may be non-naturally occurring and may be introduced by point mutation.
  • enzyme refers to a protein or polypeptide that catalyzes a chemical reaction.
  • activity refers to a biological activity associated with a particular protein, such as an enzyme activity associated with a protease.
  • Biological activity refers to any activity that is generally attributed to the protein by those skilled in the art.
  • xylanase refers to a glycosyl hydrolase that hydrolyzes a beta-1,4-linked xylopyranoside chain.
  • wild-type refers to a natural or naturally occurring sequence or protein.
  • mutant mutations refers to changes in a single nucleotide in DNA, especially where this alteration will result in a change in the protein.
  • mutant refers to a type of organism or protein that differs from the wild type. Such alterations can be achieved by methods well known to those skilled in the art, for example, by point mutations, wherein the resulting protein can be referred to as a mutant.
  • modified refers to a sequence, such as an amino acid sequence comprising a polypeptide, which includes deletion, insertion, substitution or truncation of a naturally occurring sequence.
  • substituted refers to the replacement of naturally occurring residues.
  • thermostable refers to a property that remains stable in environments involving temperature.
  • a thermostable organism refers to a organism that is more stable than a non-thermally stable organism under certain temperature conditions.
  • thermally stable refers to the property of being thermally stable.
  • alpha-helix refers to a structure formed in this case: a single polypeptide chain is regularly rotated about itself to form a rigid cylinder in which each peptide bond is regularly And other peptide bonds on the nearby chain interact through hydrogen bonds.
  • the strain and vector Escherichia coli DH5 ⁇ , Pichia pastoris GS115, vector pPIC9K, Amp, G418 were purchased from Invitrogen.
  • Enzymes and kits PCR enzyme and ligase were purchased from Takara, restriction enzymes were purchased from Fermentas, plasmid extraction kits and gel purification kits were purchased from Omega, and GeneMorph II random mutagenesis kit was purchased from Beijing. Bomais Biotechnology Co., Ltd.
  • E. coli medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0);
  • LB-AMP medium LB medium plus 100 ⁇ g/mL ampicillin
  • Yeast medium 1% yeast extract, 2% peptone, 2% glucose;
  • Yeast screening medium 2% glucose, 2% agarose, 1.34% YNB, 4 x 10 -5 biotin;
  • BMGY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 x 10 -5 biotin, 1% glycerol;
  • BMMY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 x 10 -5 biotin, 0.5% methanol.
  • the materials and reagents used in the xylanase mutants provided by the present invention are commercially available.
  • Xyn-F1 5'-CGC GAATTC ACTATTCAACCTGGAACTGGATAC—3' (underlined as restriction endonuclease ECORI recognition site)
  • Xyn-R1 5'-CT CGCGGCCGC TTATGAGACTGTGATAGAGGCAG-3' (underlined as restriction endonuclease NOTI recognition site)
  • the above primers Xyn-F1 and Xyn-R1 were used for PCR amplification, and the PCR product was recovered by gel, linked to pEASY-T vector, transformed into E. coli DH5a, and picked correctly. Transformants were sequenced. The sequencing result showed that the nucleotide sequence of the xylanase in the transformant was SEQ ID NO: 2, and the encoded amino acid sequence was SEQ ID NO: 1. Applicants named the xylanase Xyn.
  • the disulfide bridge (also known as the Cys-Cys bridge) can stabilize the enzyme structure.
  • a certain amount of stable disulfide bridge is necessary to maintain proper stability of the enzyme.
  • the applicant modified the xylanase Xyn by analyzing its sequence and structure (crystal structure PDB ID: 2JIC).
  • the modification is to introduce one or more unnatural disulfide bridges by site-directed mutagenesis, for example, introducing an unnatural disulfide bridge T1C-T27C to stabilize the N-terminal region of the Xyn protein, and introducing a disulfide bridge S109C-N153C to The ⁇ -helix region was stabilized and a disulfide bridge Q33C-T187C was introduced to stabilize the C-terminal region.
  • the nucleotide sequences of the above xylanase mutants XynA1 and XynA2 were synthesized by Shanghai Jierui Biotechnology Co., Ltd.
  • the above two mutants were subjected to PCR amplification using the primers Xyn-F1 and Xyn-R1 described in Example 1, and EcoRI and Not I sites were introduced at both ends of the primer.
  • the PCR reaction conditions were: denaturation at 94 ° C for 5 min; then denaturation at 94 ° C for 30 s, renaturation at 56 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles, and incubation at 72 ° C for 10 min.
  • the results of agarose gel electrophoresis showed that the size of the two mutant gene fragments was about 600 bp.
  • the gene fragment of the wild-type xylanase Xyn was amplified by the same PCR method as described above.
  • the xylanase mutant gene XynA1 and XynA2 fragments obtained above were ligated to the expression vector pPIC9K via EcoRI and Not I sites to construct expression vectors pPIC9K-XynA1 and pPIC9K-XynA2.
  • the mutant expression plasmid was linearized with Sal I, and the expression plasmid linearized fragment was transformed into Pichia pastoris GS115 by electroporation, and the Pichia pastoris recombinant strains GS115/pPIC9K-XynA1 and GS115/pPIC9K-XynA2 were screened on MD plates, respectively. Then, multiple copies of the transformants were screened on YPD plates containing different concentrations of geneticin.
  • the positive transformants of the recombinant expression xylanase mutants XynA1 and XynA2 were named as Pichia pastoris XynA1 (Pichia pastoris XynA1) and Pichia pastoris XynA2 (Pichia pastoris XynA2), respectively, and then transferred to BMGY medium, respectively.
  • the medium was cultured at 30 ° C and 250 rpm for 1 d; then transferred to BMMY medium, shake culture at 30 ° C, 250 rpm; 0.5% methanol was added every day to induce expression for 4 days; the cells were removed by centrifugation to obtain xylanase-containing mutants.
  • the fermentation supernatants of XynA1 and XynA2 were analyzed by SDS-PAGE electrophoresis, and the results showed that the molecular weight of the xylanase mutant in the above fermentation supernatant was about 20.7 kDa.
  • Pichia pastoris Xyn The wild-type xylanase gene Xyn was cloned into Pichia pastoris GS115 host by the same restriction enzyme ligation method as described above, and a Pichia pastoris engineering strain recombinantly expressing wild-type xylanase Xyn was constructed, which was named Pichia pastoris Xyn. (Pichia pastoris Xyn).
  • the Pichia pastoris Xyn was fermented in a shake flask at 30 ° C and shaking at 250 rpm; 0.5% methanol was added every day to induce expression for 4 days; the cells were removed by centrifugation to obtain a fermentation supernatant containing wild-type xylanase Xyn.
  • the amount of enzyme required to release 1 ⁇ mol of reducing sugar per minute from a xylan solution having a concentration of 5 mg/ml at 37 ° C and a pH of 5.5 is an enzyme activity unit U.
  • X D is the activity of xylanase in the diluted enzyme solution, U/ml
  • a E is the absorbance of the enzyme reaction solution
  • a B is the absorbance of the enzyme blank
  • K is the slope of the standard curve
  • C 0 is the standard The intercept of the curve
  • M is the molar mass of xylose, 150.2 g/mol
  • t is the enzymatic reaction time, min
  • N is the dilution factor of the enzyme solution
  • the xylanase activity in the above-mentioned fermentation supernatants of Pichia pastoris Xyn, Pichia pastoris XynA1 and Pichia pastoris XynA2 was respectively measured according to the above method.
  • the results showed that the enzyme activity of Pichia pastoris Xyn fermentation supernatant was 115 U/ml, while the enzyme activities of Pichia pastoris XynA1 and Pichia pastoris XynA2 fermentation supernatant were 104 U/mL and 71 U/mL, respectively.
  • the fermentation of Pichia pastoris Xyn, Pichia pastoris XynA1 and Pichia pastoris XynA2 was carried out on a 10-liter fermenter.
  • the medium used for fermentation was: calcium sulfate 1.1 g/L, potassium dihydrogen phosphate 5.5 g/L, phosphoric acid. Ammonium dihydrogenate 55 g / L, potassium sulfate 20.3 g / L, magnesium sulfate 16.4 g / L, potassium hydroxide 1.65 g / L, defoaming agent 0.05%.
  • Fermentation production process pH value of 5.0, temperature of 30 ° C, stirring rate of 300 rpm, ventilation of 1.0 to 1.5 (v / v), dissolved oxygen control of more than 20%.
  • the whole fermentation process is divided into three stages: the first stage is the bacterial culture stage, the seed is inserted at a ratio of 7%, the culture is carried out at 30 °C for 24 to 26 hours, and the glucose is used as a marker; the second stage is the starvation stage, when the glucose is supplemented. After the completion, no carbon source is added. When the dissolved oxygen rises above 80%, it indicates that the stage ends, and the period is about 30-60 min.
  • the third stage is the induced expression stage, and methanol is added to induce, and the dissolved oxygen is kept above 20%.
  • the culture time is between 150 and 180 hours. After the fermentation is completed, the fermentation broth is treated by a plate and frame filter to obtain a crude enzyme solution.
  • the above crude enzyme solution was detected by the xylanase enzyme activity assay method described in Example 3.
  • the result showed that the final fermentation enzyme activity of Pichia pastoris Xyn recombinantly expressing wild-type xylanase was 7030 U/ml, and the recombination was carried out.
  • the final fermentation enzyme activities of Pichia pastoris XynA1 and Pichia pastoris XynA2 expressing xylanase mutants were 6954 U/ml and 5507 U/ml, respectively.
  • the crude enzyme solution obtained by fermentation in Example 4 was diluted with an acetic acid-sodium acetate buffer solution of pH 5.5 to about 20 U/ml, and treated at 65 ° C, 70 ° C, 75 ° C and 80 ° C for 5 min, respectively.
  • the residual enzyme activity was calculated by calculating the enzyme activity residual rate by 100% of the enzyme activity of the untreated sample. The specific results are shown in Table 1.
  • Enzyme activity residual rate (%) residual enzyme activity / enzyme activity of untreated sample ⁇ 100%
  • Table 1 shows the effect of introducing an unnatural disulfide bridge on the heat resistance of xylanase
  • the present invention provides a mutant XynA1 which introduces two unnatural disulfide bridges Q33C-T187C and S109C-N153C and introduces three unnatural disulfide bridges Q33C as compared with the wild-type xylanase.
  • the heat resistance of the mutant XynA2 of T187C, S109C-N153C and T1C-T27C was significantly or significantly improved, especially the mutant XynA2 which introduced three disulfide bridges, and the residual rate of enzyme activity after treatment at 80 ° C for 5 min Up to 75%, the enzyme activity residual rate of the mutant XynA1 was increased by 72%.
  • PCR amplification was performed using the GeneMorph II random mutagenesis PCR kit (Stratagene), and the PCR product was recovered by gel, and EcoRI and Not I were digested. After treatment, it was ligated with the same digested pET21a vector, transformed into E. coli BL21 (DE3), plated on LB+Amp plate, and cultured in inverted at 37 ° C. After the emergence of the transformant, the toothpick was picked one by one to the 96-well plate.
  • Some mutations have no effect on the heat tolerance of the xylanase mutant XynA1, such as S39D, K55A, E106T, T108F, etc. Some mutations even make their heat resistance or enzyme activity worse, such as N18A, N91K, S148I , A184V, etc.; there are also some mutations, although it can improve the temperature tolerance of the xylanase mutant XynA1, but the enzymatic properties of the mutations have changed significantly, which are not satisfactory.
  • the above xylanase mutant containing the Q51N single point mutation was named XynA1-Q51N, and the amino acid sequence thereof was SEQ ID NO: 7, and the coding sequence was obtained by referring to the sequence as SEQ ID NO: 8.
  • the above xylanase mutant containing H143K single point mutation was named XynA1-H143K, and its amino acid sequence was SEQ ID NO: 9, and the sequence was obtained by referring to the sequence of SEQ ID NO: 10.
  • the above xylanase mutant containing the Q161F single point mutation was named XynA1-Q161F, and its amino acid sequence was SEQ ID NO: 11, and a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 12 was obtained.
  • the nucleotide sequence of the above xylanase mutant was synthesized by Shanghai Jierui Biotechnology Co., Ltd.
  • the Pichia pastoris engineering strain recombinantly expressing the above mutant was constructed by the method described in Example 2-5, and the fermentation was verified and the heat resistance of the mutant was determined. The specific results are shown in Table 3.
  • Example 6 Using the method described in Example 6 for screening and obtaining mutants, Applicants screened for mutated combinations of xylanase thermostable sites Q51N, H143K, and Q161F. Further, the obtained mutants were each subjected to heat resistance measurement.
  • the xylanase mutant containing the above two-point mutation combination of H143K and Q51N was named XynA1-H143K-Q51N, and the amino acid sequence thereof was SEQ ID NO: 13, and the nucleotide sequence was obtained by referring to the sequence as SEQ ID NO: 14.
  • the xylanase mutant containing the above two-point mutation combination of H143K and Q161F was named XynA1-H143K-Q161F, and the amino acid sequence thereof was SEQ ID NO: 15, and the nucleotide sequence was obtained by referring to the sequence as SEQ ID NO: 16.
  • the above xylanase mutant containing the Q51N and Q161F two-point mutation combination was named XynA1-Q51N-Q161F, and the amino acid sequence thereof was SEQ ID NO: 17, and the nucleotide sequence was obtained by referring to the sequence as SEQ ID NO: 18.
  • the xylanase mutant containing the above-mentioned three-point mutation combination of H143K, Q51N and Q161F was named XynA1-H143K-Q51N-Q161F, and the amino acid sequence thereof was SEQ ID NO: 19, and the nucleotide sequence was obtained by referring to the sequence. SEQ ID NO:20.
  • the nucleotide sequence of the above xylanase mutant was synthesized by Shanghai Jierui Biotechnology Co., Ltd.
  • the Pichia pastoris engineering strain recombinantly expressing the above mutant was constructed by the method described in Example 2-5, and the fermentation was verified and the heat resistance of the mutant was determined. The specific results are shown in Table 4.
  • the applicants introduced the three single-point mutation sites Q51N, H143K and Q161F obtained in the screening of Example 6 into the xylanase XynA2 to obtain three new woods. Glycanase mutant protein.
  • the above xylanase mutant containing the Q51N single point mutation was named XynA2-Q51N, and its amino acid sequence was SEQ ID NO: 21, and the coding sequence was obtained by referring to the sequence as SEQ ID NO: 22.
  • the above xylanase mutant containing H143K single point mutation was named XynA2-H143K, and its amino acid sequence was SEQ ID NO: 23, and a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 24 was obtained.
  • the above xylanase mutant containing the Q161F single point mutation was named XynA2-Q161F, and its amino acid sequence was SEQ ID NO: 25, and a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 26 was obtained.
  • the nucleotide sequence of the above xylanase mutant was synthesized by Shanghai Jierui Biotechnology Co., Ltd.
  • the Pichia pastoris engineering strain recombinantly expressing the above mutant was constructed by the method described in Example 2-5, and the fermentation was verified and the heat resistance of the mutant was determined. The specific results are shown in Table 5.
  • the applicant introduces any two or three of the above-mentioned heat-resistant mutation sites Q51N, H143K, and Q161F into the xylanase XynA2, respectively, and heat-treats the obtained xylanase mutant protein. Determination.
  • the results showed that the combination of mutations with better heat resistance than the above single point mutants included: H143K+Q51N, H143K+Q161F, Q51N+Q161F, H143K+Q51N+Q161F.
  • the residual activity of the combined mutants was 100% after treatment at 80 ° C for 5 min, and the residual rate of enzyme activity was over 95% after treatment at 85 ° C for 3 min, and the heat resistance was significantly improved.

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Abstract

本发明涉及蛋白质工程改造技术领域，特别涉及耐热性得到提高的木聚糖酶突变体。本发明通过定点突变的方法在木聚糖酶中人为引入两个或三个非自然二硫桥，获得了耐热性提高的突变体XynA1和XynA2，尤其是引入三个二硫桥的突变体XynA2，其在80℃处理5min后酶活残留率高达75％，比突变体XynA1的酶活残留率提高了72％。本发明进一步筛选获得三个能显著提高突变体耐热性的突变位点Q51N，H143K，Q161F，所述突变位点以单点或两点组合或三点组合的方式引入木聚糖酶XynA1和XynA2，均能有效提高其耐热性。

Description

木聚糖酶突变体
本申请要求于2017年08月18日提交中国专利局、申请号为201710712383.9、发明名称为“木聚糖酶突变体”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及蛋白质工程改造技术领域，特别涉及耐热性得到提高的木聚糖酶突变体。
背景技术
木聚糖是植物半纤维素的主要组成部分，广泛存在于玉米芯、甘蔗渣、麦麸、秸秆等农作物废弃物中，由于木聚糖酶能够将木聚糖分解成不同长度的低聚木糖和木糖，该产物具有重要的经济价值，通过木聚糖酶将这些可利用资源充分利用，发挥其潜在的应用价值，木聚糖酶的研究也受到充分的重视。
木聚糖酶是水解β-1，4-连接的吡喃木糖苷链的糖基水解酶，已被证明存在于至少上百种不同生物体中，并可以经济地大规模生产。它们与其他糖基水解酶一起形成了包括超过40中不同酶家族的超家族。已知里氏木霉可以生产三种不同的木聚糖酶，其中木聚糖酶I和II(XynI和XynII)具有最好的特点。XynI分子量19kDa，与XynII相比，它的等电点和最适pH较低(pI 5.5，pH 3-4)。XynII分子量20kDa，它的等电点为9.0，最适pH 5.0-5.5。
现在木聚糖酶被广泛应用于纸浆漂白、纺织品纤维修饰以及在动物饲料和人类的食品生产中。所有这些应用中的共同问题是木聚糖酶面临的极端条件。在工业应用中的高温以及与许多木聚糖酶的最适pH与工业应用中的pH条件不同，降低了现有木聚糖酶在工业上的有效利用。
在应用于纸浆漂白过程中，来自于碱洗的物料具有较高的温度(>80℃)和pH(>10)。多数木聚糖酶在这样的条件下将会失活，因此必须将纸浆冷却，将碱性中和，但这意味着成本的增加。
在饲料应用中，木聚糖酶面临着饲料制备期间的短暂高温(如90℃下2-5分钟)。但是，木聚糖酶的催化活性需要的温度较低(如约37℃)。这样，木聚糖酶将在高温下不可逆地失活。
尽管已有大量研究改善了木聚糖酶的稳定性，但仍不能满足日益提高的工业生产需求，因此，提供一种适用于工业生产的耐高温木聚糖酶具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种木聚糖酶突变体，获得突变体蛋白，提高其耐热性，从而有利于木聚糖酶在饲料领域的广泛应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种木聚糖酶突变体，其包括二硫桥，所述二硫桥包括T1C-T27C、Q33C-T187C或S109C-N153C(木聚糖酶编号方式)中的一个或多个；
且其具有(Ⅰ)、(Ⅱ)或(Ⅲ)所示的氨基酸序列中任意一个：
(Ⅰ)与木聚糖酶酶的氨基酸序列SEQ ID NO:1具有至少95％同源性的序列；
(Ⅱ)具有所述木聚糖酶的至少一个免疫表位，且所述木聚糖酶的氨基酸序列SEQ ID NO:1经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列；
(Ⅲ)由如SEQ ID NO:2所示的核苷酸序列或其互补序列或因遗传密码的简并性而与如SEQ ID NO:2所示的核苷酸序列或其互补序列的核苷酸序列不同的序列编码的氨基酸序列；
所述取代为第51位、第143位或第161位的一个或多个氨基酸被取代。
在一些实施例中，本发明提供的木聚糖酶突变体的氨基酸序列与木聚糖酶的氨基酸序列具有至少96％同源性。
在一些实施例中，本发明提供的木聚糖酶突变体的氨基酸序列与木聚糖酶的氨基酸序列具有至少97％同源性。
在一些实施例中，本发明提供的木聚糖酶突变体的氨基酸序列与木聚糖酶的氨基酸序列具有至少98％同源性。
在一些实施例中，本发明提供的木聚糖酶突变体的氨基酸序列与木聚糖酶的氨基酸序列具有至少99％同源性。
在一些实施例中，本发明提供的木聚糖酶突变体具有下列二硫桥中的一个或更多个：T1C-T27C，Q33C-T187C和S109C-N153C。
在一些实施例中，本发明提供的突变体具有下列二硫桥中的两个或三个：T1C-T27C，Q33C-T187C和S109C-N153C。
在一些实施例中，本发明突变体具有下列两个二硫桥：Q33C-T187C和S109C-N153C。
在一些实施例中，本发明突变体具有下列三个二硫桥：T1C-T27C，Q33C-T187C和S109C-N153C。
在本发明的一些实施例中，木聚糖酶突变体具有如SEQ ID NO:3或SEQ ID NO:5所示的氨基酸序列。
本发明还提供了编码上述的木聚糖酶突变体的DNA分子。
在本发明的一些实施例中，编码上述的木聚糖酶突变体的DNA分子具有如SEQ ID NO:4或SEQ ID NO:6所示的核苷酸序列。
在本发明的另一些实施例中，所述木聚糖酶突变体在51，143，161(木聚糖酶编 号方式)位置上具有至少1个被取代的氨基酸残基。
在本发明的另一些实施例中，所述木聚糖酶突变体在51，143，161(木聚糖酶编号方式)位置上具有至少2个被取代的氨基酸残基。
在本发明的另一些实施例中，所述木聚糖酶突变体在51，143，161(木聚糖酶编号方式)位置上具有3个被取代的氨基酸残基。
在优选实施方案中，该取代为至少1个选自下面的突变：Q51N，H143K，Q161F。
在本发明的另一些实施例中，木聚糖酶突变体具有如SEQ ID NO:7或SEQ ID NO:9或SEQ ID NO:11或SEQ ID NO:13或SEQ ID NO:15或SEQ ID NO:17或SEQ ID NO:19或SEQ ID NO:21或SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列。
本发明还提供了编码上述的木聚糖酶突变体的DNA分子。
在本发明的一些实施例中，编码上述的木聚糖酶突变体的DNA分子具有如SEQ ID NO:8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14或SEQ ID NO:16或SEQ ID NO:18或SEQ ID NO:20或SEQ ID NO:22或SEQ ID NO:24或SEQ ID NO:26所示的核苷酸序列。
本发明还提拱了具有上述DNA分子的载体。
本发明还提供了一种宿主细胞，包含上述重组表达载体。
在本发明的一些实施例中，宿主细胞为毕赤酵母。
将上述的质粒转入毕赤酵母中，重组表达的木聚糖酶突变体的耐热性得到显著提升。
本发明通过定点突变的方法在木聚糖酶中人为引入两个或三个非自然二硫桥，获得了耐热性提高的突变体XynA1和XynA2，尤其是引入三个二硫桥的突变体XynA2，其在80℃处理5min后酶活残留率高达75％，比突变体XynA1的酶活残留率提高了72％。本发明进一步筛选获得三个能显著提高突变体耐热性的突变位点Q51N，H143K，Q161F，所述突变位点以单点或两点组合或三点组合的方式引入木聚糖酶XynA1，均能有效提高其耐热性，尤其是三点组合突变体XynA1-H143K-Q51N-Q161F，其在80℃处理5min后酶活残留率高达61.28％，85℃处理3min后仍有26.89％的酶活残留，耐热性显著。所述突变位点以单点的方式引入木聚糖酶XynA2获得的突变体，80℃处理5min后，酶活残留率普遍提高了9.26％-24.58％，85℃处理3min后，酶活残留率普遍提高了26.63％-46.61％。进一步地，申请人将上述耐热突变位点Q51N，H143K，Q161F中的任意两个或三个位点分别引入木聚糖酶XynA2，耐热性比上述单点突变体更好的突变组合包括：H143K+Q51N，H143K+Q161F，Q51N+Q161F，H143K+Q51N+Q161F。所述组合突变体80℃处理5min后酶活残留率均为100％，85℃处理3min后酶活残留率均超过95％，耐热性得到显著提升。
具体实施方式
本发明公开了一种木聚糖酶突变体，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明现在将通过参考的方式、仅仅使用下面给出的定义和实施例进行详细描述。除非在此处另外定义，所有此处使用的技术和科学术语都和本发明所属领域的普通技术人员所通常理解的术语意义相同。Singleton等所著的由纽约John Wiley and Sons于1994年出版的DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY第二版，以及由Hale和Marham所著，由纽约Harper Perennial于1991年出版的THE HARPER COLLINS DICTIONARY OF BIOLOGY，向技术人员提供用于本发明中的许多术语的综合词典。尽管与此处的描述相似或等价的任何材料和方法可以在本发明的实际应用和试验中使用，本发明还是描述了优选的方法和材料。数字范围包括了界定范围的数字。除非另外指出，分别地，核酸按照5′到3′的方向从左到右写出；氨基酸序列按照从氨基到羧基的方向从左到右写出。特别地，从业者可以参照Sambrook等，1989和Ausubel FM等，1993来理解本领域的定义和术语。应该理解，本发明不局限于所描述的具体方法学、方案和试剂，因为这些是可以改变的。
此处提供的标题并不是对本发明多个方面和实施方案的限制，通过参考作为整体的说明书可以知晓本发明各个方面和实施方案。因此，接下来定义的术语是通过参考作为整体的说明书而被更完整地定义出来。
正如此处所用，术语“多肽(polypeptide)”，指的是由氨基酸残基以肽键相连而成的单链构成的化合物。此处的术语“蛋白”可以与术语“多肽”同义，或者可以另外指两个或多个多肽的复合物。
正如此处所用，术语“表达(expression)”，指的是基于基因的核酸序列产生出多肽的过程。该过程包括转录和翻译。
正如此处所用，术语“基因(gene)”，意思是参与多肽链产生的DNA片段，它可以包含或者也可以不包含编码区域之前或者之后的区域。
如此处使用的，术语“二硫桥(disulphide bridge)”或者“二硫键(disulphide bond)”指的是，在多肽或蛋白质中，在半胱氨酸残基的硫原子之间形成的键。在本发明中，二硫桥或二硫键可以是非自然发生的以及可通过点突变方式引入的。
如此处使用的，“酶(enzyme)”指的是催化化学反应的蛋白质或多肽。
如此处使用的，术语“活性(activity)”指的是与特定蛋白相关的生物活性，比如与蛋白酶相关的酶活性。生物活性指的是本领域技术人员一般将其归属于该蛋白质的任何活性。
如此处使用的，术语“木聚糖酶(xylanase)”指的是水解β-1,4-连接的吡喃木糖苷链 的糖基水解酶。
如此处使用的，“野生型(wild-type)”指的是天然的或自然发生的序列或蛋白质。
如此处使用的，“点突变(poin mutations)”指的是DNA中单核苷酸的改变，尤其是在这个改变将会导致蛋白质的改变的情况下。
如此处使用的，“突变体(mutant)”指的是生物体或蛋白不同于野生型的类型。这种改变可通过本领域技术人员所熟知的方法加以实现，例如，通过点突变，其中得到的蛋白质就可以被称为突变体。
如此处使用的，“修饰的(modified)”，指的是一个序列，诸如包含多肽的氨基酸序列，其包括对自然发生序列的删除、插入、替换或截断。
如此处使用的，“取代的(substituted)”，指的将是对自然发生残基的替换。
如此处使用的，“热稳定的(thermostable)”指的是在涉及温度的环境下保持稳定的性质。例如，热稳定生物指的是特定温度条件下比非热稳定生物更稳定的生物。
如此处使用的，“热稳定性(thermostability)”指的是热稳定的性质。
如此处使用的，“α螺旋(α-helix)”指的是这种情况下所形成的结构：单条多肽链绕着自身规则地旋转，从而形成刚性圆柱体，其中每个肽键都规则地和附近链上的其他肽键通过氢键相互作用。
本发明优选实施例中所选用的实验材料和试剂：
菌株与载体：大肠杆菌DH5α、毕赤酵母GS115、载体pPIC9K、Amp、G418购自Invitrogen公司。
酶与试剂盒：PCR酶及连接酶购买自Takara公司，限制性内切酶购自Fermentas公司，质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司，GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方：
大肠杆菌培养基(LB培养基)：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH7.0)；
LB-AMP培养基：LB培养基加100μg/mL氨苄青霉素；
酵母培养基(YPD培养基)：1％酵母提取物，2％蛋白胨，2％葡萄糖；
酵母筛选培养基(MD培养基)：2％葡萄糖，2％琼脂糖，1.34％YNB，4×10 -5生物素；
BMGY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10 -5生物素，1％甘油；
BMMY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10 -5生物素，0.5％甲醇。
本发明提供的木聚糖酶突变体中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1木聚糖酶基因的扩增
Xyn-F1：5’—CGC GAATTCACTATTCAACCTGGAACTGGATAC—3’(下划线为 限制性内切酶ECORI识别位点)
Xyn-R1：5’—CT CGCGGCCGCTTATGAGACTGTGATAGAGGCAG—3’(下划线为限制性内切酶NOTI识别位点)
以里氏木霉(Trichoderma reesei)基因组为模板，利用上述引物Xyn-F1和Xyn-R1进行PCR扩增，胶回收PCR产物，连接pEASY-T载体，转化至大肠杆菌DH5a中，挑取正确的转化子进行测序。测序结果显示，转化子中木聚糖酶的核苷酸序列为SEQ ID NO：2，其编码的氨基酸序列为SEQ ID NO：1。申请人将该木聚糖酶命名为Xyn。
实施例2耐热突变体基因的筛选与获得
二硫桥(也就是Cys-Cys桥)对酶结构能起到稳定作用。一定量的稳定性二硫桥对保持该酶适当的稳定性来说是必需的。通过向蛋白质结构中人为引入一定数量的二硫桥，预期也能产生较稳定的，尤其是耐热的，但活性可能会有所降低的酶突变体。
为了提高木聚糖酶Xyn的耐热性，申请人通过分析木聚糖酶Xyn的序列及结构(晶体结构PDB ID:2JIC)，对其进行修饰。所述的修饰，为通过定点突变引入一个或多个非自然的二硫桥，例如，引入非自然二硫桥T1C-T27C以稳定Xyn蛋白的N-末端区域，引入二硫桥S109C-N153C以稳定α螺旋区域，引入二硫桥Q33C-T187C以稳定C末端区域。
申请人将上述引入Q33C-T187C和S109C-N153C两条二硫桥的突变体命名为XynA1，其氨基酸序列为SEQ ID NO：3，其编码核苷酸序列为SEQ ID NO：4；将上述引入Q33C-T187C、S109C-N153C和T1C-T27C三条二硫桥的突变体命名为XynA2，其氨基酸序列为SEQ ID NO：5，其编码核苷酸序列为SEQ ID NO：6。
上述木聚糖酶突变体XynA1和XynA2的核苷酸序列由上海捷瑞生物公司合成。用实施例1中所述引物Xyn-F1和Xyn-R1对上述两个突变体进行PCR扩增，引物两端引入EcoRI、Not I位点。PCR反应条件为：94℃变性5min；然后94℃变性30s，56℃复性30s，72℃延伸1min，30个循环后，72℃保温10min。琼脂糖凝胶电泳结果显示两个突变体基因片段大小均为600bp左右。
通过上述同样的PCR方法扩增得到野生型木聚糖酶Xyn的基因片段。
实施例3毕赤酵母工程菌株的构建
将上述克隆得到的木聚糖酶突变体基因XynA1和XynA2片段，通过EcoRI和Not I位点与表达载体pPIC9K相连接，构建表达载体pPIC9K-XynA1和pPIC9K-XynA2。
将突变体表达质粒用Sal I进行线性化，表达质粒线性化片段通过电穿孔法转化毕赤酵母GS115，在MD平板上分别筛选得到毕赤酵母重组菌株GS115/pPIC9K-XynA1和GS115/pPIC9K-XynA2，然后分别在含不同浓度遗传霉素的YPD平板上筛选多拷贝的转化子。
将筛选得到的重组表达木聚糖酶突变体XynA1和XynA2的阳性转化子分别命 名为毕赤酵母XynA1(Pichia pastoris XynA1)和毕赤酵母XynA2(Pichia pastoris XynA2)，然后分别转接于BMGY培养基中，30℃、250rpm振荡培养1d；再转入BMMY培养基中，30℃、250rpm振荡培养；每天添加0.5％的甲醇，诱导表达4d；离心去除菌体，分别得到含木聚糖酶突变体XynA1和XynA2的发酵上清液；将其进行SDS-PAGE电泳检测分析，结果显示上述发酵上清液中木聚糖酶突变体的分子量大小约为20.7kDa。
通过上述同样的酶切连接方法将野生型木聚糖酶基因Xyn克隆到毕赤酵母GS115宿主中，构建得到重组表达野生型木聚糖酶Xyn的毕赤酵母工程菌，命名为毕赤酵母Xyn(Pichia pastoris Xyn)。摇瓶水平发酵毕赤酵母Xyn，30℃、250rpm振荡培养；每天添加0.5％的甲醇，诱导表达4d；离心去除菌体，得到含野生型木聚糖酶Xyn的发酵上清液。
(1)木聚糖酶酶活单位的定义
在37℃、pH值为5.5的条件下，每分钟从浓度为5mg/ml的木聚糖溶液中释放1μmol还原糖所需要的酶量即为一个酶活力单位U。
(2)酶活测定方法
取2ml浓度为1％的木聚糖底物(pH5.5乙酸-乙酸钠缓冲液配制)，加入到比色管中，37℃平衡10min，再加入2ml经pH5.5乙酸-乙酸钠缓冲液适当稀释并经37℃平衡好的酸性木聚糖酶酶液，混匀于37℃精确保温反应30min。反应结束后，加入5ml DNS试剂，混匀以终止反应。然后沸水浴煮沸5min，用自来水冷却至室温，加蒸馏水定容至25ml，混匀后，以标准空白样为空白对照，在540nm处测定吸光值AE。
酶活计算公式：
Figure PCTCN2018083635-appb-000001
式中：X D为稀释酶液中木聚糖酶的活力，U/ml；A E为酶反应液的吸光度；A B为酶空白液的吸光度；K为标准曲线的斜率；C 0为标准曲线的截距；M为木糖的摩尔质量，150.2g/mol；t为酶解反应时间，min；N为酶液稀释倍数；1000为转化因子，1mmol＝1000μmol。
(3)酶活测定结果
按照上述方法分别检测上述毕赤酵母Xyn、毕赤酵母XynA1和毕赤酵母XynA2发酵上清液中木聚糖酶酶活。结果显示：毕赤酵母Xyn发酵上清液的酶活为115U/ml，而毕赤酵母XynA1和毕赤酵母XynA2发酵上清液的酶活分别为104U/mL和71U/mL。
实施例4发酵验证
在10升发酵罐上分别进行毕赤酵母Xyn、毕赤酵母XynA1和毕赤酵母XynA2的发酵，发酵使用的培养基配方为：硫酸钙1.1g/L、磷酸二氢钾5.5g/L、磷酸二氢铵55g/L、硫酸钾20.3g/L、硫酸镁16.4g/L、氢氧化钾1.65g/L、消泡剂0.05％。
发酵生产工艺：pH值5.0、温度30℃、搅拌速率300rpm、通风量1.0～1.5(v/v)、溶氧控制在20％以上。
整个发酵过程分为三个阶段：第一阶段为菌体培养阶段，按7％比例接入种子，30℃培养24～26h，以补完葡萄糖为标志；第二阶段为饥饿阶段，当葡萄糖补完之后，不流加任何碳源，当溶氧上升至80％以上表明该阶段结束，为期约30～60min；第三阶段为诱导表达阶段，流加甲醇诱导，并且保持溶氧在20％以上，培养时间在150～180h之间。发酵结束后，发酵液通过板框过滤机处理后获得粗酶液。
采用实施例3所述木聚糖酶酶活测定方法对上述粗酶液进行检测，结果显示，重组表达野生型木聚糖酶的毕赤酵母Xyn最终的发酵酶活为7030U/ml，而重组表达木聚糖酶突变体的毕赤酵母XynA1和毕赤酵母XynA2最终的发酵酶活分别为6954U/ml和5507U/ml。
实施例5木聚糖酶的耐热性质测定
将实施例4中发酵获得的粗酶液分别用pH5.5的乙酸-乙酸钠缓冲液稀释至约20U/ml，在65℃、70℃、75℃和80℃条件下分别处理5min后，测定其残留酶活，以未处理样品的酶活计100％，计算酶活残留率。具体结果见表1。
酶活残留率(％)＝残留酶活/未处理样品的酶活×100％
表1引入非自然二硫桥对木聚糖酶耐热性的影响
Figure PCTCN2018083635-appb-000002
注：与野生型相比， *示P＜0.05； #示P＜0.01.
从表1的结果可知，与野生型木聚糖酶相比，本发明提供的引入两个非自然二硫桥Q33C-T187C和S109C-N153C的突变体XynA1以及引入三个非自然二硫桥Q33C-T187C、S109C-N153C和T1C-T27C的突变体XynA2的耐热性均得到显著或极显著提高，尤其是引入三个二硫桥的突变体XynA2，其在80℃处理5min后酶活残留率高达75％，比突变体XynA1的酶活残留率提高了72％。从而说明，人为引入非自然二硫桥能大幅提高木聚糖酶的耐热性，但二硫桥的引入数量对木聚糖酶发酵酶活的影响也很明显。从实施例4最后的发酵数据可以看出，本发明引入两个非自然二硫桥Q33C-T187C和S109C-N153C的突变体XynA1的发酵酶活与野生型相当，但引入三个非自然二硫桥Q33C-T187C、S109C-N153C和T1C-T27C的突变体XynA2的发酵酶活下降了21.7％。
实施例6木聚糖酶突变体位点的获得及鉴定
为了进一步提高木聚糖酶突变体XynA1的耐热性，申请人通过定向进化技术对该酶进行了大量突变的筛选。
以XynA1基因为模板，利用实施例1中所述引物Xyn-F1、Xyn-R1，用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，胶回收PCR产物，EcoRI、Not I进行酶切处理后与经同样酶切后的pET21a载体连接，转化至大肠杆菌BL21(DE3)中，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，用牙签逐个挑至96孔板，每个孔中加入150ul含有0.1mM IPTG的LB+Amp培养基，37℃220rpm培养6h左右，离心弃上清，菌体用缓冲液重悬，反复冻融破壁，获得含有木聚糖酶的大肠杆菌细胞裂解液。
分别取出30ul裂解液至两块新的96孔板；将其中一块于75℃处理8min；然后将两块96孔板都加入30ul底物，于37℃反应30min后，DNS法测定生成的还原糖，计算不同突变子高温处理后的酶活水平。
有些突变对木聚糖酶突变体XynA1的耐热性没有影响，例如S39D，K55A，E106T，T108F等；有些突变甚至使其耐热性或酶活变得更差了，例如N18A，N91K，S148I，A184V等；另外还有些突变，虽然能提高木聚糖酶突变体XynA1对温度的耐受性，但突变后其酶学性质发生了显著的变化，这些均不符合要求。
所述突变体具体的耐热数据见表2。
表2木聚糖酶耐热性比较
突变体 75℃处理5min后酶活残留率
XynA1 32.12％
XynA1/S39D 27.99％
XynA1/K55A 29.21％
XynA1/E106T 29.73％
XynA1/T108F 28.07％
XynA1/N18A 19.83％
XynA1/N91K 22.57％
XynA1/S148I 25.63％
XynA1/A184V 19.13％
最终，申请人得到既能显著提高木聚糖酶突变体XynA1的耐热性，又不会显著影响其酶活及原有酶学性质的突变位点：Q51N，H143K，Q161F。
将上述含Q51N单点突变的木聚糖酶突变体命名为XynA1-Q51N，其氨基酸序列为SEQ ID NO:7，参照该序列得到一个编码核苷酸序列为SEQ ID NO:8。
将上述含H143K单点突变的木聚糖酶突变体命名为XynA1-H143K，其氨基酸序列为SEQ ID NO:9，参照该序列得到一个编码核苷酸序列为SEQ ID NO:10。
将上述含Q161F单点突变的木聚糖酶突变体命名为XynA1-Q161F，其氨基酸序列为SEQ ID NO:11，参照该序列得到一个编码核苷酸序列为SEQ ID NO:12。
上述木聚糖酶突变体的核苷酸序列由上海捷瑞生物公司合成。
采用实施例2-5所述方法，构建得到重组表达上述突变体的毕赤酵母工程菌株，并进行发酵验证以及突变体的耐热性质测定。具体结果见表3。
表3木聚糖酶突变体耐热性比较
Figure PCTCN2018083635-appb-000003
注：与XynA1相比， *示P＜0.05； #示P＜0.01.
从表3的数据可以看出，本发明筛选到的Q51N，H143K，Q161F三个单点突变，均能有效提高木聚糖酶突变体XynA1的耐热性，75℃处理5min后木聚糖酶残留酶活提高了43.72％-63.62％，其中XynA1-H143K突变体的残留酶活最高，达95.79％，取得了意料不到的技术效果。
实施例7木聚糖酶耐热组合突变体的获得
采用实施例6中所述筛选并获得突变体的方法，申请人对木聚糖酶耐热位点Q51N，H143K，Q161F进行突变组合筛选。进一步，对获得的突变体分别进行耐热性测定。
结果显示：耐热性比上述单点突变体更好的突变组合包括：H143K+Q51N，H143K+Q161F，Q51N+Q161F，H143K+Q51N+Q161F。
将上述含H143K和Q51N两点突变组合的木聚糖酶突变体命名为XynA1-H143K-Q51N，其氨基酸序列为SEQ ID NO:13，参照该序列得到一个编码核苷酸序列为SEQ ID NO:14。
将上述含H143K和Q161F两点突变组合的木聚糖酶突变体命名为XynA1-H143K-Q161F，其氨基酸序列为SEQ ID NO:15，参照该序列得到一个编码核苷酸序列为SEQ ID NO:16。
将上述含Q51N和Q161F两点突变组合的木聚糖酶突变体命名为XynA1-Q51N-Q161F，其氨基酸序列为SEQ ID NO:17，参照该序列得到一个编码核苷酸序列为SEQ ID NO:18。
将上述含H143K、Q51N和Q161F三点突变组合的木聚糖酶突变体命名为XynA1-H143K-Q51N-Q161F，其氨基酸序列为SEQ ID NO:19，参照该序列得到一个编码核苷酸序列为SEQ ID NO:20。
上述木聚糖酶突变体的核苷酸序列由上海捷瑞生物公司合成。
采用实施例2-5所述方法，构建得到重组表达上述突变体的毕赤酵母工程菌株，并进行发酵验证以及突变体的耐热性质测定。具体结果见表4。
表4木聚糖酶突变体耐热性比较
Figure PCTCN2018083635-appb-000004
注：分别与XynA1-Q51N、XynA1-H143K和XynA1-Q161F三个单点突变体相比， *示P＜0.05； #示P＜0.01.
从表4的数据可以看出，与XynA1-Q51N、XynA1-H143K和XynA1-Q161F三个单点突变体相比，其对应两点的组合突变体以及三点组合突变体的耐热性均得到显著提高，尤其是三点组合突变体XynA1-H143K-Q51N-Q161F，其在80℃处理5min后酶活残留率高达61.28％，85℃处理3min后仍有26.89％的酶活残留，耐热性显著。
实施例8耐热突变位点对木聚糖酶XynA2耐热性的影响
为了进一步提高木聚糖酶突变体XynA2的耐热性，申请人将实施例6筛选获得的3个单点突变位点Q51N，H143K，Q161F分别引入木聚糖酶XynA2，获得三个新的木聚糖酶突变体蛋白。
将上述含Q51N单点突变的木聚糖酶突变体命名为XynA2-Q51N，其氨基酸序列为SEQ ID NO:21，参照该序列得到一个编码核苷酸序列为SEQ ID NO:22。
将上述含H143K单点突变的木聚糖酶突变体命名为XynA2-H143K，其氨基酸序列为SEQ ID NO:23，参照该序列得到一个编码核苷酸序列为SEQ ID NO:24。
将上述含Q161F单点突变的木聚糖酶突变体命名为XynA2-Q161F，其氨基酸序列为SEQ ID NO:25，参照该序列得到一个编码核苷酸序列为SEQ ID NO:26。
上述木聚糖酶突变体的核苷酸序列由上海捷瑞生物公司合成。
采用实施例2-5所述方法，构建得到重组表达上述突变体的毕赤酵母工程菌株，并进行发酵验证以及突变体的耐热性质测定。具体结果见表5。
表5木聚糖酶突变体耐热性比较
Figure PCTCN2018083635-appb-000005
注：与XynA2相比， *示P＜0.05； #示P＜0.01.
从表5的结果可以看出，本发明将筛选到的三个突变位点引入木聚糖酶突变体XynA2后，同样能有效提升其耐热性，80℃处理5min后，引入上述突变位点的木聚糖酶突变体的酶活残留率普遍提高了9.26％-24.58％，85℃处理3min后，酶活残留率普遍提高了26.63％-46.61％，效果显著。所述三个突变位点中，H143K的引入对木聚糖酶耐热性的影响最明显。
进一步地，申请人将上述耐热突变位点Q51N，H143K，Q161F中的任意两个或三个位点分别引入木聚糖酶XynA2，并对获得的木聚糖酶突变体蛋白进行耐热性测定。结果显示：耐热性比上述单点突变体更好的突变组合包括：H143K+Q51N，H143K+Q161F，Q51N+Q161F，H143K+Q51N+Q161F。所述组合突变体80℃处理5min后酶活残留率均为100％，85℃处理3min后酶活残留率均超过95％，耐热性得到显著提升。
以上对本发明所提供的木聚糖酶突变体进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2018083635-appb-000006
Figure PCTCN2018083635-appb-000007
Figure PCTCN2018083635-appb-000008
Figure PCTCN2018083635-appb-000009
Figure PCTCN2018083635-appb-000010
Figure PCTCN2018083635-appb-000011
Figure PCTCN2018083635-appb-000012
Figure PCTCN2018083635-appb-000013
Figure PCTCN2018083635-appb-000014
Figure PCTCN2018083635-appb-000015
Figure PCTCN2018083635-appb-000016
Figure PCTCN2018083635-appb-000017
Figure PCTCN2018083635-appb-000018
Figure PCTCN2018083635-appb-000019
Figure PCTCN2018083635-appb-000020
Figure PCTCN2018083635-appb-000021

Claims (10)

  1. 一种木聚糖酶突变体，其特征在于，其包括二硫桥，所述二硫桥包括T1C-T27C、Q33C-T187C或S109C-N153C中的一个或多个；
    且其具有(Ⅰ)、(Ⅱ)或(Ⅲ)所示的氨基酸序列中任意一个：
    (Ⅰ)与木聚糖酶的氨基酸序列SEQ ID NO:1具有至少95％同源性的序列；
    (Ⅱ)具有所述木聚糖酶的至少一个免疫表位，且所述木聚糖酶的氨基酸序列SEQ ID NO:1经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列；
    (Ⅲ)由如SEQ ID NO:2所示的核苷酸序列或其互补序列或因遗传密码的简并性而与如SEQ ID NO:2所示的核苷酸序列或其互补序列的核苷酸序列不同的序列编码的氨基酸序列；
    所述取代为第51位、第143位或第161位的一个或多个氨基酸被取代。
  2. 根据权利要求1所述的木聚糖酶突变体，其特征在于，所述木聚糖酶突变体中的二硫桥为Q33C-T187C和S109C-N153C；所述木聚糖酶突变体的氨基酸序列如SEQ ID NO:3所示。
  3. 根据权利要求1所述的木聚糖酶突变体，其特征在于，所述木聚糖酶突变体中的二硫桥为T1C-T27C，Q33C-T187C和S109C-N153C；所述木聚糖酶突变体的氨基酸序列如SEQ ID NO:5所示。
  4. 根据权利要求1至3任一项所述的木聚糖酶突变体，其特征在于，所述取代为Q51N、H143K或Q161F。
  5. 根据权利要求4所述的木聚糖酶突变体，其特征在于，所述木聚糖酶突变体具有如SEQ ID NO:7或SEQ ID NO:9或SEQ ID NO:11或SEQ ID NO:13或SEQ ID NO:15或SEQ ID NO:17或SEQ ID NO:19或SEQ ID NO:21或SEQ ID NO:23或SEQ ID NO:25所示的氨基酸序列。
  6. 本发明还提供了编码如权利要求1至5任一项的木聚糖酶突变体的DNA分子。
  7. 根据权利要求6所述的DNA分子，其特征在于，其具有如SEQ ID NO:4或SEQ ID NO:6所示的核苷酸序列；或
    具有如SEQ ID NO:8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14或SEQ ID NO:16或SEQ ID NO:18或SEQ ID NO:20或SEQ ID NO:22或SEQ ID NO:24或SEQ ID NO:26所示的核苷酸序列。
  8. 具有如权利要求6或7所述DNA分子的载体。
  9. 一种宿主细胞，其特征在于，包含如权利要求8所述的载体。
  10. 根据权利要求1至5任一项所述木聚糖酶突变体的制备方法，其特征在于，包括如下步骤：
    步骤1：获取编码所述木聚糖酶突变体的DNA分子：
    步骤2：将步骤1获得的所述DNA分子与表达载体融合，构建重组表达载体，转化宿主细胞；
    步骤3：诱导含重组表达载体的宿主细胞表达融合蛋白，分离纯化表达的融合蛋 白。
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