WO2016078168A1 - 植酸酶突变体 - Google Patents

植酸酶突变体 Download PDF

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WO2016078168A1
WO2016078168A1 PCT/CN2014/093278 CN2014093278W WO2016078168A1 WO 2016078168 A1 WO2016078168 A1 WO 2016078168A1 CN 2014093278 W CN2014093278 W CN 2014093278W WO 2016078168 A1 WO2016078168 A1 WO 2016078168A1
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phytase
seq
amino acid
appa
mutant
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PCT/CN2014/093278
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French (fr)
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吴秀秀
王华明
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青岛蔚蓝生物集团有限公司
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Priority to ES14906425T priority Critical patent/ES2759083T3/es
Priority to EP14906425.5A priority patent/EP3222714B1/en
Priority to US15/524,390 priority patent/US11104908B2/en
Priority to DK14906425T priority patent/DK3222714T3/da
Publication of WO2016078168A1 publication Critical patent/WO2016078168A1/zh
Priority to US17/387,909 priority patent/US11739336B2/en

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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02006—Hydroxyacylglutathione hydrolase (3.1.2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03008—3-Phytase (3.1.3.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase

Definitions

  • the invention relates to the field of biotechnology, in particular to a phytase mutant, a preparation method and application thereof, a DNA molecule encoding the phytase mutant, a vector and a host cell.
  • Phytase is a phosphatase that hydrolyzes phytic acid. It degrades phytate phosphorus (inositol hexaphosphate) to inositol and inorganic phosphate. This enzyme is divided into two classes: 3-phytase (EC.3.1.3.8) and 6-phytase (EC.3.1.2.6).
  • Phytase is widely found in plants, animals and microorganisms, such as higher plants such as corn and wheat, prokaryotic microorganisms such as Bacillus subtilis, Pseudomonas, Lactobacillus, and Escherichia coli, and eukaryotic microorganisms such as yeast, Rhizopus, and Aspergillus. .
  • the basic storage form of phosphorus is phytate phosphorus, which is as high as 1% to 3%, which accounts for 60% to 80% of total phosphorus in plants.
  • the phosphorus in the form of phytate phosphorus is difficult to be utilized due to the lack of enzymes capable of decomposing phytic acid in monogastric animals, and its utilization rate is only 0% to 40%, which causes many problems: firstly, waste of phosphorus source On the one hand, the phosphorus source in the feed cannot be effectively utilized.
  • phytate phosphorus is also an anti-nutritional factor, which is chelated with various metal ions such as Zn 2+ , Ca 2+ , Cu 2+ , Fe 2+ , etc. during protein digestion and absorption in the gastrointestinal tract of animals. Corresponding insoluble complexes reduce the effective use of these nutrients by animals.
  • Phytase can be used as a feed additive for monogastric animals, and its feeding effect has been confirmed worldwide. It can increase the utilization of phosphorus in plant feed by 60%, and reduce the amount of phosphorus excretion in feces by 40%, while also reducing the anti-nutritional effect of phytic acid. Therefore, the addition of phytase to feed has important significance in improving the production efficiency of livestock and poultry industry and reducing the pollution of phytate phosphorus to the environment.
  • the phytase currently produced industrially mainly includes fungal phytase derived from Aspergillus niger and bacterial phytase derived from Escherichia coli.
  • the phytase APPA derived from Escherichia coli has high specific activity and good digestive tract stability. At present, it is mainly applied to the feed industry by spraying directly after powder feed or after spraying pellets.
  • the bacterial phytase APPA has poor thermal stability.
  • the residual enzyme activity of the aqueous solution is kept at 70 ° C for 5 minutes, and the residual enzyme activity is less than 30%. It is directly added to the animal feed to carry out the enzyme activity after granulation is generally less than 20%, so that APPA planting
  • the use of acid enzymes in pelleted feeds is limited.
  • the method of spraying phytase liquid onto the feed after granulation of feed not only increases the input of the equipment, but also can not guarantee the stability of the enzyme preparation and the uniformity of distribution in the feed. Therefore, improving the thermal stability of phytase is an important practical significance of phytase for feed.
  • the present invention provides a phytase mutant, obtains a mutant protein, and improves heat resistance, thereby facilitating the wide application of phytase in the field of feed.
  • the present invention provides the following technical solutions:
  • the present invention provides a phytase mutant having any one of the amino acid sequences shown in (I) and (II):
  • the amino acid sequence of the phytase mutant has a sequence that is at least 75% homologous to the amino acid sequence of the phytase.
  • the amino acid sequence of the phytase mutant has a sequence of at least 80% homology to the amino acid sequence of the phytase.
  • the amino acid sequence of the phytase mutant has a sequence that is at least 85% homologous to the amino acid sequence of the phytase.
  • the amino acid sequence of the phytase mutant has a sequence that is at least 90% homologous to the amino acid sequence of the phytase.
  • the amino acid sequence of the phytase mutant has a sequence that is at least 95% homologous to the amino acid sequence of the phytase.
  • the modification comprises amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation or carbonylation.
  • the substitution is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve , 13, 14, 15, 16 or 17 amino acids.
  • the substitution is a substitution of 12, 13, 16, or 17 amino acids.
  • the substitution is 25th, 46th, 62nd, 70th, 73rd, 75th, 114th, 137th, 142th, One or more amino acids in position 146, 159 or 255 are substituted.
  • the phytase has the amino acid sequence set forth in SEQ ID NO:1.
  • the amino acid at position 25 of the phytase having the amino acid sequence of SEQ ID NO: 1 is changed from Ala to Phe, and the amino acid at position 46 is changed from Trp to Glu, position 62.
  • the amino acid changes from Gln to Trp, the 70th amino acid changes from Gly to Glu, the 73rd amino acid changes from Ala to Pro, the 75th amino acid changes from Lys to Cys, and the 114th amino acid changes from Thr to His, the 137th position.
  • the amino acid changes from Asn to Val, the 142th amino acid changes from Asp to Arg, the 146th amino acid changes from Ser to Glu, the 159th amino acid changes from Arg to Tyr, and the 255th amino acid changes from Tyr to Asp.
  • the present invention also provides a DNA molecule encoding the above phytase mutant.
  • the phytase mutant has the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9.
  • the DNA molecule encoding the phytase mutant described above has a nucleoside as set forth in SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10. Acid sequence.
  • the present invention also discloses a vector having the above DNA molecule.
  • amino acid sequence of the above phytase mutant is SEQ ID NO: 3, and one of the nucleic acid sequences encoding the gene is SEQ ID NO: 4.
  • the present invention also encompasses a plasmid carrying a phytase mutant gene having the coding sequence of SEQ ID NO: 4.
  • the substitution further comprises the substitution of the amino acid at position 380.
  • the substituting further comprises changing the amino acid at position 380 of the phytase having the amino acid sequence SEQ ID NO: 3 from Ala to Pro.
  • amino acid sequence of the above mutant is SEQ ID NO: 5, and one of the nucleic acid sequences encoding the gene is SEQ ID NO: 6.
  • the present invention also encompasses a plasmid carrying a phytase mutant gene having the coding sequence of SEQ ID NO: 6.
  • the substitution further comprises the substitution of one or more amino acids at position 80, 176 or 187.
  • amino acid sequence of amino acid sequence SEQ ID NO: 5 is changed from Ser to Pro, the amino acid at position 176 is changed from Asn to Pro, and the amino acid at position 187 is changed from Ser to For Pro.
  • amino acid sequence of the above phytase mutant is SEQ ID NO: 7, and the nucleic acid sequence encoding one of the genes is SEQ ID NO: 8.
  • the present invention also encompasses a plasmid carrying a phytase mutant gene having the coding sequence of SEQ ID NO: 8.
  • the substitution further comprises the substitution of the amino acid at position 161.
  • amino acid sequence of amino acid sequence SEQ ID NO: 7 is changed from Thr to Pro.
  • amino acid sequence of the above phytase mutant is SEQ ID NO: 9, and the nucleic acid sequence encoding one of the genes is SEQ ID NO: 10.
  • the present invention also encompasses a plasmid carrying a phytase mutant gene having the coding sequence of SEQ ID NO: 10.
  • Step 1 Obtain a DNA molecule encoding any one of the amino acid sequences shown in (I), (II):
  • Step 2 merging the DNA molecule obtained in the step 1 with an expression vector to construct a recombinant expression vector, and transforming the host cell;
  • Step 3 Inducing a host cell containing the recombinant expression vector to express the fusion protein, and isolating and purifying the expressed fusion protein.
  • the modification in the method of preparation comprises amidation, phosphorylation, methylation, acetylation, ubiquitination, glycosylation or carbonylation.
  • the substitution in the preparation method is one, two, three, four, five, six, seven, eight, nine, ten, eleven , 12, 13, 14, 15, 16, or 17 amino acids.
  • the substitutions in the preparation method are 25th, 46th, 62nd, 70th, 73rd, 75th, 114th, 137th, and One or more amino acids in position 142, 146, 159 or 255 are substituted.
  • the substitution in the method of preparation further comprises the substitution of the amino acid at position 380.
  • the substitution in the method of preparation further comprises the substitution of one or more amino acids at position 80, 176 or 187.
  • the substitution in the method of preparation further comprises the substitution of the amino acid at position 161.
  • the step 1 is specifically using a cDNA encoding the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 as a template. Primer amplification.
  • the host cell in step 2 is Pichia pastoris.
  • the invention also provides the use of the above phytase mutants in feed.
  • 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 phytase mutant was remarkably improved.
  • the present invention provides a phytase mutant having any one of the amino acid sequences shown in (I) and (II):
  • the present invention is based on the phytase mutant APPA-M, and also provides A380P single point mutants PHY1, S80P, N176P, S187P and A380P four-point mutant PHY4, S80P, T161P, N176P, S187P and A380P five-point mutants.
  • PHY5. After 10 min treatment at 80 °C, the residual enzyme activities of mutant PHY1, PHY4 and PHY5 were increased by 33.85%, 53.11% and 75.86%, respectively, after APPA-M; after 5 min treatment at 85 °C, the residues of PHY1, PHY4 and PHY5 were mutated. The enzyme activity increased by 14.89%, 28.45% and 44.94%, respectively, compared with APPA-M. Its heat resistance is significantly higher than APPA-M, which is beneficial to the wide application of phytase in feed.
  • Figure 1 is a map of the recombinant plasmid pPIC9K-APPA
  • Figure 2 is a comparison of the heat resistance of APPA-M and PHY 1, PHY4 and PHY5.
  • the invention discloses a phytase mutant, a preparation method and application thereof, a DNA molecule encoding the phytase mutant, a carrier and a host cell, and those skilled in the art can learn from the contents of the present invention 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 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 changed without departing from the spirit, scope and scope of the invention. More in combination, to implement and apply the techniques of the present invention.
  • the present invention employs conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003). . These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can use other conventional methods, experimental solutions and reagents in the art based on the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention. For example, the following experimental materials and reagents can be used in the present invention:
  • 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.
  • Yeast screening medium 2% peptone, 2% agarose;
  • 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 amino acid sequence is SEQ ID) NO:1, which encodes a nucleotide sequence of SEQ ID NO: 2), and mutates 12 sites (A25F, W46E, Q62W, G70E, A73P, K75C, T114H, N137V, D142R, S146E, R159Y, Y255D).
  • the phytase mutant comprising the above 12 mutation sites was named APPA-M, and its amino acid sequence was SEQ ID NO: 3, and a coding nucleotide sequence was synthesized with reference to the sequence as SEQ ID NO: 4.
  • the sequence was optimized by Shanghai Jierui Bio-Bioengineering Co., Ltd. according to the cryptophilic preference of Pichia pastoris, and two restriction sites of EcoRI and NotI were added to the 5' and 3' ends of the synthetic sequence, respectively.
  • the nucleotide sequence encoding the wild type phytase APPA was synthesized in the same manner as SEQ ID NO: 2.
  • colony PCR (reaction system: monoclonal selected by template, rTaq DNA polymerase 0.5 ul, 10 ⁇ Buffer 2.0 ⁇ L, dNTPs (2.5 mM) 2.0 ⁇ L, 5′AOX primer (10 M): 0.5 ⁇ L, 3'AOX primer: 0.5 ⁇ L, ddH 2 O 14.5 ⁇ L, reaction procedure: pre-denaturation at 95 ° C for 5 min, 30 cycles: 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 2 min, 72 ° C for 10 min) to verify positive clones, after sequencing verification Finally, the correct recombinant expression plasmid was obtained.
  • the two recombinant expression plasmids were named pPIC9K-APPA (plasmid map shown in Figure 1) and pPIC9K-APPA-M.
  • Pichia pastoris GS115 strain was activated by YPD plate, cultured at 30 °C for 48h, inoculated with activated GS115 monoclonal in 6mL YPD liquid medium, cultured at 30°C, 220rpm, and cultured for about 12h, then transferred to 30mLYPD liquid medium.
  • the cell density was measured by UV spectrophotometer at 30 ° C and 220 rpm for about 5 h. After the OD600 value was in the range of 1.1 - 1.3, centrifuged at 9 ° C for 9 min at 4 ° C for 2 min to collect 4 mL of the cells to the sterilized EP tube.
  • the expression plasmids pPIC9K-APPA and pPIC9K-APPA-M were linearized with Sal I, linearized fragments were purified and recovered, and Pichia pastoris GS115 was transformed by electroporation, and the electroporation mixture was uniformly coated on MD plates in aseptic operation.
  • the table was dried in Taichung; the MD plate was inverted and cultured in a 30 °C incubator for 2-3 days, and the Pichia pastoris recombinant strains GS115/pPIC9K-APPA and GS115/pPIC9K-APPA-M were screened on MD plates, about 300 per plate.
  • the clones were washed with sterile water and plated on YPD (0.5 mg/mL-8 mg/mL) plates containing different concentrations of geneticin to screen multiple copies of the transformants.
  • Pichia pastoris APPA One transformant of Pichia pastoris recombinant strain GS115/pPIC9K-APPA was named Pichia pastoris APPA, and one transformant of recombinant strain GS115/pPIC9K-APPA-M was named Pichia pastoris APPA-M (Pichia) Pastoris APPA-M), transferred to BMGY medium, cultured at 30 ° C, 250 rpm for 1 d; then transferred to BMMY medium, shake culture at 30 ° C, 250 rpm; add 0.5% methanol per day, induce expression 4d; 9000 rpm The cells were removed by centrifugation for 10 min to remove the cells, and a fermentation supernatant containing phytase APPA and APPA-M was obtained.
  • the enzyme activities of the Pichia pastoris APPA and APPA-M fermentation supernatants were determined to be 166 U/mL and 195 U/mL, respectively, according to the above method.
  • Fermentation of Pichia pastoris APPA and Pichia pastoris APPA-M 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, ammonium dihydrogen phosphate. 55 g/L, magnesium sulfate 16.4 g/L, potassium sulfate 20.3 g/L, potassium hydroxide 1.65 g/L, and antifoaming agent 0.05%.
  • Fermentation production process pH value of 5.0, temperature of 30 ° C, stirring rate of 300 rpm, ventilation of 1.0-1.5 (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%, and the culture is carried out at 30 ° C for 24-26 h, which is marked by the completion of glucose; 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%, the stage ends, which lasts for about 30-60 minutes.
  • 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 150-180 h; after the fermentation is finished, the fermentation broth is processed through a plate and frame filter to obtain a crude enzyme solution.
  • the crude enzyme solution was detected by the phytase enzyme activity assay method described in 1.3.2, and the enzyme activity assay results are shown in Table 2.
  • the final fermentation enzyme activity of Pichia pastoris APPA recombinantly expressing wild-type phytase was 9800 U/mL, while the final fermentation enzyme activity of Pichia pastoris APPA-M recombinantly expressing phytase mutant was as high as 10257 U/mL.
  • the Pichia pastoris APPA and the above were determined at 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85 ° C, pH 5.5, respectively.
  • the enzyme activity of the crude enzyme solution obtained by fermentation of Pichia pastoris APPA-M was 100%, and the relative enzyme activity was calculated. The results showed that the optimal temperature of wild-type phytase APPA and mutant APPA-M was 75 °C.
  • Pichia pastoris APPA and Pichia pastoris APPA-M were fermented with 0.1 M acetic acid-sodium acetate buffer at pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, respectively.
  • the enzyme solution was diluted, and the enzyme activity was measured at 37 ° C.
  • the relative enzyme activity was calculated with the highest enzyme activity of 100%.
  • the experimental results showed that the optimum pH value of wild type phytase APPA and mutant APPA-M was obtained. Both are 5.0.
  • the crude enzyme solution obtained by fermentation of Pichia pastoris APPA and Pichia pastoris APPA-M was diluted 10 times with pH 0.25 M sodium acetate buffer preheated for 10 min, mixed uniformly, and treated at 75 ° C for 5 min. At the end, the sample was taken and cooled to room temperature. Then, the diluted enzyme activity was measured, and the residual enzyme activity was calculated by taking the enzyme activity of the untreated sample to 100%.
  • Residual enzyme activity (%) enzyme activity of the enzyme solution after treatment / enzyme activity of the untreated enzyme solution ⁇ 100%
  • the synthesized gene (sequence is SEQ ID NO: 4) was subjected to protein structure analysis, and the protein has two structures. Domain: The 134 amino acid residues at the N-terminus together with the 152 amino acid residues at the C-terminus constitute domain 1, the remaining 124 amino acid residues constitute the domain 2, and the conserved sequence and the active center are located in domain 1, without destroying the protein. Under the premise of secondary structure and active center, the site is further mutated.
  • XynII-F1 GGCGAATTC CAGTCAGAACCAGAGTTGAAGTT (underlined as restriction endonuclease EcoRI recognition site), as shown in SEQ ID NO: 11;
  • XynII-R1 ATAGCGGCCGC TTACAAGGAACAAGCAGGGAT (underlined as restriction endonuclease NotI recognition site), as shown in SEQ ID NO: 12;
  • the APPA-M gene (SEQ ID NO: 4) was used as a template, and the above primers were used for PCR amplification using the GeneMorph II random mutagenesis PCR kit (Stratagene).
  • the PCR product was recovered by gel, and EcoRI and NotI were digested with the same reaction.
  • the digested pET21a vector was ligated, transformed into E. coli BL21 (DE3), plated on LB+Amp plate, and cultured in inverted at 37 °C. After the transformants appeared, they were picked with a toothpick one by one to 96-well plates, each well.
  • A380P single point mutation S80P, N176P, S187P and A380P Four-point mutation, and five-point mutations of S80P, T161P, N176P, S187P and A380P.
  • the above phytase mutant containing A380P single point mutation was named PHY1, and its amino acid sequence was SEQ ID NO: 5, and a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 6 was obtained.
  • the above phytase mutant containing the four-point mutation of S80P, N176P, S187P and A380P was named PHY4, 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 phytase mutant containing the five-point mutation of S80P, T161P, N176P, S187P and A380P is named as PHY5, and the amino acid sequence thereof is SEQ ID NO: 9, and the nucleotide sequence is obtained by referring to the sequence as SEQ ID NO: 10.
  • the genetic sequences of the three mutants of SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 were optimized by Shanghai Jierui Bioengineering Co., Ltd. according to the preference bias of Pichia pastoris, and in the synthetic sequence 5' Two restriction enzyme sites, EcoRI and NotI, were added to the 3' ends.
  • the three gene sequences synthesized by 2.1 were digested with EcoRI and NotI, respectively, and then ligated with the same digested pPIC-9k vector at 16 ° C overnight, and transformed into E. coli DH5a, coated on LB + Amp plate, 37 ° C Inverted culture, after the emergence of the transformant, colony PCR (reaction system and procedure as in Example 1) verified positive clones, and finally confirmed the correct recombinant expression plasmid after sequencing, and the three recombinant expression plasmids were named pPIC9K-PHY1. , pPIC9K-PHY4 and pPIC9K-PHY5.
  • the expression plasmids pPIC9K-PHY1, pPIC9K-PHY4 and pPIC9K-PHY5 were linearized with Sac I, respectively.
  • the linearized fragments were purified and recovered, and then Pichia pastoris GS115 was transformed by electroporation, and the recombinant strain of Pichia pastoris was screened on MD plate.
  • GS115/ pPIC9K-PHY1, GS115/pPIC9K-PHY4 and GS115/pPIC9K-PHY5 were then screened for multiple copies of transformants on YPD plates (0.5 mg/mL-8 mg/mL) containing different concentrations of geneticin.
  • Pichia pastoris PHY1 One transformant of Pichia pastoris recombinant strain GS115/pPIC9K-PHY1 was named Pichia pastoris PHY1, and one transformant of recombinant strain GS115/pPIC9K-PHY4 was named Pichia pastoris PHY4.
  • One transformant of the recombinant strain GS115/pPIC9K-PHY5 was named Pichia pastoris PHY5.
  • Pichia pastoris PHY1, PHY4 and PHY5 were transferred to BMGY medium, cultured at 30 ° C, shaking at 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. 4d; the cells were removed by centrifugation at 9000 rpm for 10 min to obtain fermentation supernatants containing phytase mutants PHY1, PHY4 and PHY5, respectively.
  • the enzyme activities of the Pichia pastoris PHY1, PHY4 and PHY5 fermentation supernatants were 211 U/mL, 201 U/mL and 255 U/mL, respectively.
  • the fermentation of Pichia pastoris PHY1, Pichia pastoris PHY4 and Pichia pastoris PHY5 was carried out on a 10-liter fermenter.
  • the medium used for fermentation was: calcium sulfate 1.1g/L, potassium dihydrogen phosphate 5.5g/L, phosphoric acid. Ammonium dihydrogenate 55g / L, magnesium sulfate 16.4g / L, potassium sulfate 20.3g / L, potassium hydroxide 1.65g / L, defoamer 0.05%.
  • Fermentation production process pH 5.0, temperature 30 ° C, stirring rate 300 rpm, ventilation 1.0-1.5 (v/v), dissolved oxygen is controlled at 20% or more.
  • 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%, and the culture is carried out at 30 ° C for 24-26 h, which is marked by the completion of glucose; 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%, the stage ends, which lasts for about 30-60 minutes.
  • 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 150-180 h; after the fermentation is finished, the fermentation broth is processed through a plate and frame filter to obtain a crude enzyme solution.
  • the final fermentation enzyme activity of Pichia pastoris PHY1 recombinantly expressing phytase mutant PHY1 was 10317 U/mL
  • the final fermentation enzyme activity of Pichia pastoris PHY4 recombinantly expressing phytase mutant PHY4 was 10401 U/mL
  • the final fermentation enzyme activity of Pichia pastoris PHY5 of the acidase mutant PHY5 was as high as 10813 U/mL.
  • the Pichia pastoris PHY1 was determined at 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85 ° C, pH 5.5, respectively.
  • the enzyme activity of the crude enzyme solution obtained by fermentation of PHY4 and PHY5 was calculated to be relative enzyme activity at a maximum enzyme activity of 100%. The results showed that the optimum temperature of the mutants PHY1, PHY4 and PHY5 did not change, compared to the phytase mutant APPA-M, both at 75 °C.
  • the crude enzyme solution obtained by fermentation of Pichia pastoris PHY1, PHY4 and PHY5 was diluted 10 times with pH 0.25 M sodium acetate buffer preheated for 10 min, mixed uniformly, treated at 85 ° C for 5 min, treated at 80 ° C for 10 min, and sampled and cooled at the end. To the room temperature, the diluted enzyme activity was measured, and the residual enzyme activity was calculated by taking 100% of the enzyme activity of the untreated sample.
  • the present invention provides a phytase mutant PHY1 comprising a single point mutation of A380P, a phytase mutant PHY4 comprising four points mutations of S80P, N176P, S187P and A380P based on the phytase mutant APPA-M. And a phytase mutant PHY5 comprising a five point mutation of S80P, T161P, N176P, S187P and A380P.
  • the optimum temperature and pH of PHY1, PHY4 and PHY5 have not changed, but the heat resistance has been significantly improved, which is beneficial to the wide application of phytase in feed.

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Abstract

提供了野生型植酸酶APPA的突变体PHY1，PHY4和PHY5。80℃处理10min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高了33.85％、53.11％和75.86％；85℃处理5min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高了14.89％、28.45％和44.94％，其耐热性显著高于APPA-M。

Description

植酸酶突变体
本申请要求于2014年11月21日提交中国专利局、申请号为201410677220.8、发明名称为“植酸酶突变体”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
本发明涉及生物技术领域，特别涉及一种植酸酶突变体、其制备方法及应用、编码该植酸酶突变体的DNA分子、载体、宿主细胞。
背景技术
植酸酶是一种能水解植酸的磷酸酶类。它能将植酸磷(六磷酸肌醇)降解为肌醇和无机磷酸。此酶分为两类:3-植酸酶(EC.3.1.3.8)和6-植酸酶(EC.3.1.2.6)。植酸酶广泛存在于植物、动物和微生物中,如玉米、小麦等高等植物,枯草芽孢杆菌、假单孢杆菌、乳酸杆菌、大肠杆菌等原核微生物及酵母、根霉、曲霉等真核微生物中。
在谷物、豆类和油料等作物籽实中，磷的基本贮存形式是植酸磷，其含量高达1％～3％，它占植物中总磷的60％～80％。但是以植酸磷形式存在的磷却因单胃动物体内缺乏能分解植酸的酶而难以被利用，其利用率仅在0％～40％，从而造成了许多问题：首先是造成磷源浪费，一方面饲料中的磷源不能得到有效利用，另一方面为了满足动物对磷的需求，又必须在饲料中添加无机磷,提高了饲料成本；其次是形成高磷粪便污染环境。饲料中85％左右的植酸磷会被动物直接排出体外，粪便中大量的植酸磷使水和土壤受到严重污染。另外，植酸磷还是一种抗营养因子，它在动物胃肠道的消化吸收过程中会与多种金属离子如Zn2+、Ca2+、Cu2+、Fe2+等以及蛋白质螯合成相应的不溶性复合物，降低了动物对这些营养物质的有效利用。
植酸酶可作为一种单胃动物的饲料添加剂，它的饲喂效果已在世界范围内得到了确证。它可使植物性饲料中磷的利用率提60％，粪便中磷排泄量减少40％，同时还可降低植酸的抗营养作用。因此在饲料中添加植酸酶对提高畜禽业生产效益及降低植酸磷对环境的污染有重要意义。
现工业化生产的植酸酶主要有来源于黑曲霉的真菌植酸酶和来源于大肠杆菌的细菌植酸酶两种。其中来源于大肠杆菌的植酸酶APPA具有高比活性及良好的消化道稳定性等特点。目前主要通过在粉末饲料直接添加或颗粒饲料后喷涂的方法应用在饲料行业。
因为目前在颗粒饲料生产过程中有一个短暂的80-90℃的高温阶段。细菌植酸酶APPA热稳定性较差，其水溶液在70℃下保温5分钟剩余酶活性低于30％，直接添加到动物饲料中进行制粒后存留酶活一般低于20％，使APPA植酸酶在颗粒饲料的应用受到限制。采用饲料制粒后植酸酶液体喷涂到饲料上的方法不仅增加设备投入，而且对酶制剂的稳定性、饲料中分布均一性都无法很好的保证。因此，提高植酸酶热稳定性是目前饲料用植酸酶具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种植酸酶突变体，获得突变体蛋白，提高其耐热性，从而有利于植酸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：
本发明提供了一种植酸酶突变体，其具有(Ⅰ)、(Ⅱ)所示的氨基酸序列中任意一个：
(Ⅰ)与植酸酶的氨基酸序列具有至少70％同源性的序列；
(Ⅱ)具有所述植酸酶的至少一个免疫表位，且所述植酸酶的氨基酸序列经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列。
在本发明的一些实施例中，植酸酶突变体的氨基酸序列与植酸酶的氨基酸序列具有至少75％同源性的序列。
在本发明的另一些实施例中，植酸酶突变体的氨基酸序列与植酸酶的氨基酸序列具有至少80％同源性的序列。
在本发明的另一些实施例中，植酸酶突变体的氨基酸序列与植酸酶的氨基酸序列具有至少85％同源性的序列。
在本发明的另一些实施例中，植酸酶突变体的氨基酸序列与植酸酶的氨基酸序列具有至少90％同源性的序列。
在本发明的另一些实施例中，植酸酶突变体的氨基酸序列与植酸酶的氨基酸序列具有至少95％同源性的序列。
在本发明的一些实施例中，所述修饰包括酰胺化、磷酸化、甲基化、乙酰化、泛素化、糖基化或羰基化。
在本发明的另一些实施例中，所述取代为取代1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个或17个氨基酸。
在本发明的一些实施例中，所述取代为取代12个、13个、16个或17个氨基酸。
在本发明的另一些实施例中，所述取代为第25位、第46位、第62位、第70位、第73位、第75位、第114为、第137位、第142位、第146位、第159位或第255位中的一个或多个氨基酸被取代。
在本发明的一些实施例中，所述植酸酶具有如SEQ ID NO:1所示的氨基酸序列。
在本发明的另一些实施例中，所述取代为氨基酸序列为SEQ ID NO:1的植酸酶的第25位氨基酸由Ala变为Phe，第46位氨基酸由Trp变为Glu，第62位氨基酸由Gln变为Trp，第70位氨基酸由Gly变为Glu，第73位氨基酸由Ala变为Pro，第75位氨基酸由Lys变为Cys，第114位氨基酸由Thr变为His，第137位氨基酸由Asn变为Val，第142位氨基酸由Asp变为Arg，第146位氨基酸由Ser变为Glu，第159位氨基酸由Arg变为Tyr，第255位氨基酸由Tyr变为Asp。
本发明还提供了编码上述的植酸酶突变体的DNA分子。
在本发明的另一些实施例中，植酸酶突变体具有如SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7或SEQ ID NO:9所示的氨基酸序列。
在本发明的一些实施例中，编码上述的植酸酶突变体的DNA分子具有如SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8或SEQ ID NO:10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。
上述植酸酶突变体的氨基酸序列为SEQ ID NO:3，其一种编码基因的核酸序列为SEQ ID NO:4。
本发明还包括携带有编码序列为SEQ ID NO:4的植酸酶突变体基因的质粒。
在本发明的另一些实施例中，所述取代还包括第380位的氨基酸被取代。
在本发明的另一些实施例中，所述取代还包括将氨基酸序列为SEQ ID NO:3的植酸酶的第380位氨基酸由Ala变为Pro。
上述突变体的氨基酸序列为SEQ ID NO:5，其一种编码基因的核酸序列为SEQ ID NO:6。
本发明还包括携带有编码序列为SEQ ID NO:6的植酸酶突变体基因的质粒。
在本发明的另一些实施例中，所述取代还包括第80位、第176位或第187位中的一个或多个氨基酸被取代。
在本发明的另一些实施例中，氨基酸序列为SEQ ID NO:5的植酸酶的第80位氨基酸由Ser变为Pro，第176位氨基酸由Asn变为Pro，第187位氨基酸由Ser变为Pro。
上述植酸酶突变体的氨基酸序列为SEQ ID NO:7，其一种编码基因的核酸序列为SEQ ID NO:8。
本发明还包括携带有编码序列为SEQ ID NO:8的植酸酶突变体基因的质粒。
在本发明的另一些实施例中，所述取代还包括第161位的氨基酸被取代。
在本发明的另一些实施例中，氨基酸序列为SEQ ID NO:7的植酸酶的第第161位氨基酸由Thr变为Pro。
上述植酸酶突变体的氨基酸序列为SEQ ID NO:9，其一种编码基因的核酸序列为SEQ ID NO:10。
本发明还包括携带有编码序列为SEQ ID NO:10的植酸酶突变体基因的质粒。
本发明还提供了上述植酸酶突变体的制备方法，包括：
步骤1：获取编码具有(Ⅰ)、(Ⅱ)所示的氨基酸序列中任意一个的DNA分子：
(Ⅰ)与植酸酶的氨基酸序列具有至少70％同源性的序列；
(Ⅱ)具有植酸酶的至少一个免疫表位，且所述植酸酶的氨基酸序列经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列；
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。
在本发明的一些实施例中，制备方法中所述修饰包括酰胺化、磷酸化、甲基化、乙酰化、泛素化、糖基化或羰基化。
在本发明的另一些实施例中，制备方法中所述取代为取代1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个或17个氨基酸。
在本发明的另一些实施例中，制备方法中所述取代为第25位、第46位、第62位、第70位、第73位、第75位、第114为、第137位、第142位、第146位、第159位或第255位中的一个或多个氨基酸被取代。
在本发明的另一些实施例中，制备方法中所述取代还包括第380位的氨基酸被取代。
在本发明的另一些实施例中，制备方法中所述取代还包括第80位、第176位或第187位中的一个或多个氨基酸被取代。
在本发明的另一些实施例中，制备方法中所述取代还包括第161位的氨基酸被取代。
在本发明的一些实施例中，所述步骤1具体为以编码SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7或SEQ ID NO:9所示氨基酸序列的cDNA为模板，经引物扩增。
步骤2所述宿主细胞为毕赤酵母。
本发明还提供了上述植酸酶突变体在饲料中的应用。
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，重组表达的植酸酶突变体的耐热性得到显著提升。
本发明提供了一种植酸酶突变体，其具有(Ⅰ)、(Ⅱ)所示的氨基酸序列中任意一个：
(Ⅰ)与植酸酶的氨基酸序列具有至少70％同源性的序列；
(Ⅱ)具有所述植酸酶的至少一个免疫表位，且所述植酸酶的氨基酸序列经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列。本发明提供的4个植酸酶突变体的耐热性都比突变前得到显著提升。野生型植酸酶APPA经75℃处理5min后残留酶活不足10％，而植酸酶突变体APPA-M经75℃处理5min后残留酶活高于95％，其耐热性显著高于野生型APPA。本发明以植酸酶突变体APPA-M为基础，还提供了A380P单点突变体PHY1，S80P、N176P、S187P和A380P四点突变体PHY4，S80P、T161P、N176P、S187P和A380P五点突变体PHY5。80℃处理10min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高了33.85％、53.11％和75.86％；85℃处理5min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高了14.89％、28.45％和44.94％。，其耐热性显著高于APPA-M，从而有利于植酸酶在饲料中的广泛应用。
附图说明
图1为重组质粒pPIC9K-APPA图谱；
图2为APPA-M与PHY 1、PHY4和PHY5耐热性比较图。
具体实施方式
本发明公开了一种植酸酶突变体、其制备方法及应用、编码该植酸酶突变体的DNA分子、载体、宿主细胞，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。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法，例如MOLECULAR CLONING:A LABORATORY MANUAL,3nd Ed.(Sambrook,2001)和CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Ausubel,2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是，本领域的技术人员可以在本发明所记载的技术方案的基础上，采用本领域其它常规的方法、实验方案和试剂，而不限于本发明具体实施例的限定。例如，本发明可选用如下实验材料和试剂：
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％蛋白胨、2％琼脂糖；
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。
下面结合实施例，进一步阐述本发明：
实施例1植酸酶突变体
1.1野生型植酸酶APPA及突变体APPA-M基因的合成
为了提高大肠杆菌来源的植酸酶APPA(氨基酸序列为SEQ ID  NO:1，其编码核苷酸序列为SEQ ID NO:2)的耐热性，对12个位点进行突变(A25F，W46E，Q62W，G70E，A73P，K75C，T114H，N137V，D142R，S146E，R159Y，Y255D)。
将包含上述12个突变位点的植酸酶突变体命名为APPA-M，其氨基酸序列为SEQ ID NO:3，参照该序列合成一个编码核苷酸序列为SEQ ID NO:4。所述序列由上海捷瑞生物生物工程有限公司依照毕赤酵母的密码偏爱性优化合成，并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
采用同样的方法合成野生型植酸酶APPA的编码核苷酸序列SEQ ID NO:2。
1.2野生型及突变体基因表达载体的构建
将1.1合成的2条序列分别进行EcoRI和NotI双酶切后与经同样酶切后的pPIC-9k载体16℃过夜连接并转化大肠杆菌DH5a，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，菌落PCR(反应体系：模板挑取的单克隆，rTaqDNA聚合酶0.5ul，10×Buffer 2.0μL，dNTPs(2.5mM)2.0μL，5’AOX引物(10M)：0.5μL，3’AOX引物：0.5μL，ddH2O 14.5μL，反应程序：95℃预变性5min，30cycles：94℃30sec，55℃30sec，72℃2min，72℃10min)验证阳性克隆子，经测序验证后最后获得了正确的重组表达质粒，2个重组表达质粒分别命名为pPIC9K-APPA(质粒图谱见图1)和pPIC9K-APPA-M。
1.3毕赤酵母工程菌株的构建
1.3.1酵母感受态制备
将毕赤酵母GS115菌株进行YPD平板活化，30℃培养48h后接种活化的GS115单克隆于6mL YPD液体培养基中，30℃、220rpm，培养约12h后转接菌液于装有30mLYPD液体培养基的三角瓶中，30℃、220rpm培养约5h经紫外分光光度计检测其菌体密度，待其OD600值在1.1–1.3范围后，4℃9,000rpm离心2min分别收集4mL菌体至灭菌EP管中，轻轻弃上清，用灭菌的滤纸吸干残留的上清后用预冷的1mL灭菌水重悬菌体，4℃、9,000rpm离心2min，轻轻弃上清，重复用1mL灭菌水洗一遍 后4℃、9,000rpm离心2min，轻轻弃上清，预冷的1mL山梨醇(1mol/L)重悬菌体；4℃、9,000rpm离心2min，轻轻弃上清，预冷的100-150μl山梨醇(1mol/L)轻柔重悬菌体。
1.3.2转化和筛选
将表达质粒pPIC9K-APPA和pPIC9K-APPA-M用Sal I进行线性化，线性化片段纯化回收后通过电穿孔法转化毕赤酵母GS115，将电击混合物均匀涂布于MD平板上，在无菌操作台中晾干；将MD平板倒置培养在30℃培养箱2–3日，在MD平板上筛选得到毕赤酵母重组菌株GS115/pPIC9K-APPA和GS115/pPIC9K-APPA-M，每个平板约300个克隆子，将其分别用无菌水洗下来后涂布在含不同浓度遗传霉素的YPD(0.5mg/mL-8mg/mL)平板上筛选多拷贝的转化子。
将毕赤酵母重组菌株GS115/pPIC9K-APPA的一个转化子命名为毕赤酵母APPA(Pichia pastoris APPA)，重组菌株GS115/pPIC9K-APPA-M的一个转化子命名为毕赤酵母APPA-M(Pichia pastoris APPA-M)，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；9000rpm离心10min去除菌体离心去除菌体，得到含植酸酶APPA和APPA-M的发酵上清液。
(1)植酸酶酶活单位的定义
在温度为37℃、pH为5.0的条件下，每分钟从浓度为5.0mmol/L植酸钠中释放1μmol无机磷，即为一个植酸酶活性单位，以U表示。
(2)植酸酶酶活测定方法
取甲、乙两支25mL比色管，各加入1.8mL乙酸缓冲液(PH 5.0)、0.2mL样品反应液，混匀，37℃预热5min。在甲管中加入4mL底物溶液，乙管中加入4mL终止液，混匀，37℃反应30min，反应结束后甲管中加入4mL终止液，乙管中加入4mL底物溶液，混匀。静置10min，分别在415nm波长处测定吸光值。每种样品作3个平行，取吸光值的平均值，通过标准曲线用回归直线方程计算植酸酶活性。
酶活X＝F×C/(m×30)
其中:X——酶活力单位，U/g(mL)；
F——试样溶液反应前的总稀释倍数；
C——根据实际样液的吸光值由直线回归方程计算出的酶活性，U；
m——试样质量或体积，g/mL；
30——反应时间；
(3)酶活测定结果见表1。
表1 酶活检测结果
Figure PCTCN2014093278-appb-000001
按照上述方法测定毕赤酵母APPA和APPA-M发酵上清液的酶活分别为166U/mL和195U/mL。
1.4发酵验证
在10升发酵罐上分别进行毕赤酵母APPA和毕赤酵母APPA-M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、硫酸镁16.4g/L、硫酸钾20.3g/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1.3.2所述植酸酶酶活测定方法对上述粗酶液进行检测，酶活测定结果见表2。
表2 酶活测定结果
Figure PCTCN2014093278-appb-000002
重组表达野生型植酸酶的毕赤酵母APPA最终的发酵酶活为9800U/mL，而重组表达植酸酶突变体的毕赤酵母APPA-M最终的发酵酶活高达10257U/mL。
1.5酶学特性分析
1.5.1最适作用温度
分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃，pH5.5条件下测定上述毕赤酵母APPA和毕赤酵母APPA-M发酵所得粗酶液的酶活，以最高酶活力为100％，计算相对酶活。结果显示：野生型植酸酶APPA和突变体APPA-M的最适作用温度均为75℃。
1.5.2最适作用pH值
分别用pH2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0的0.1M乙酸-乙酸钠缓冲液对上述毕赤酵母APPA和毕赤酵母APPA-M发酵所得粗酶液进行稀释，在37℃条件下测定酶活，以最高酶活力为100％，计算相对酶活，实验结果显示：野生型植酸酶APPA和突变体APPA-M的的最适作用pH值均为5.0。
1.5.3温度稳定性
用预热10min的pH 0.25M乙酸钠缓冲液分别将上述毕赤酵母APPA和毕赤酵母APPA-M发酵所得粗酶液稀释10倍，混合均匀，75℃处理5min，结束时取样并冷却至室温，然后测定稀释后的酶活，以未处理样品的酶活为100％，计算残留酶活。
残余酶活(％)＝处理后酶液的酶活/未处理酶液的酶活×100％
结果显示：野生型植酸酶APPA经75℃处理5min后残余酶活不足10％，而植酸酶突变体APPA-M经75℃处理5min后残余酶活高于95％， 从而说明植酸酶突变体APPA-M的耐热性显著高于野生型植酸酶。
实施例2植酸酶突变体
为了进一步提高植酸酶突变体APPA-M(氨基酸序列为SEQ ID NO:3)的热稳定性，对合成的基因(序列为SEQ ID NO:4)进行蛋白结构分析，该蛋白有两个结构域：N端的134个氨基酸残基与C端的152个氨基酸残基共同组成结构域1，剩余中间124氨基酸残基组成结构域2，保守序列和活性中心均位于结构域1中，在不破坏蛋白二级结构与活性中心的前提下，进一步对位点进行突变。
设计PCR引物APPAM-F1、APPAM-R1：
XynII-F1：GGCGAATTC CAGTCAGAACCAGAGTTGAAGTT(下划线为限制性内切酶EcoRI识别位点)，如SEQ ID NO：11所示；
XynII-R1：ATAGCGGCCGC TTACAAGGAACAAGCAGGGAT(下划线为限制性内切酶NotI识别位点)，如SEQ ID NO：12所示；
以APPA-M基因(SEQ ID NO：4)为模板，以上述引物用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，胶回收PCR产物，EcoRI、NotI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，获得含有植酸酶的大肠杆菌细胞裂解液。
分别取出40ul裂解液至两块新的96孔板，其中一块板于80℃处理10min后，两块96孔板都加入80ul底物，于37℃反应30min后加入80ul终止液(钒酸铵：钼酸铵：硝酸＝1:1:2)测定生成的无机磷含量，不同的突变子高温处理后保持的活性不同。
实验结果表明，有些突变对APPA-M蛋白耐热性没有影响，有些突变甚至使其耐热性或酶活变得更差了，例如Q184E/Y289K/I405L三点突变体和去C端(CNZSMQTD)突变体在80℃处理5min后，残留酶活比APPA-M分别降低9％和17％，Q285Y和C178N这两个单点突变体基本 没有活性；另外还有些突变，虽然能提高蛋白对温度的耐受性，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。最终，得到既能显著提高APPA-M蛋白耐热性，又不会影响其酶活及原有酶学性质的突变位点及位点的组合：A380P单点突变，S80P、N176P、S187P和A380P四点突变，以及S80P、T161P、N176P、S187P和A380P五点突变。
将上述含A380P单点突变的植酸酶突变体命名为PHY1，其氨基酸序列为SEQ ID NO:5，参照该序列得到一个编码核苷酸序列为SEQ ID NO:6。
将上述含S80P、N176P、S187P和A380P四点突变的植酸酶突变体命名为PHY4，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S80P、T161P、N176P、S187P和A380P五点突变的植酸酶突变体命名为PHY5，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。
2.2突变体基因的合成及扩增
由上海捷瑞生物工程有限公司依照毕赤酵母的密码偏爱性分别优化合成SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10这三条突变体的基因序列，并且在合成序列5’和3’两端分别加上EcoRI和NotI两个酶切位点。
2.3突变体基因表达载体的构建
将2.1合成的3条基因序列分别进行EcoRI和NotI双酶切，然后与经同样酶切后的pPIC-9k载体16℃过夜连接，并转化大肠杆菌DH5a，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，菌落PCR(反应体系及程序同实施例1)验证阳性克隆子，经测序验证后最后获得了正确的重组表达质粒，3个重组表达质粒分别命名为pPIC9K-PHY1、pPIC9K-PHY4和pPIC9K-PHY5。
2.4毕赤酵母工程菌株的构建
分别将表达质粒pPIC9K-PHY1、pPIC9K-PHY4和pPIC9K-PHY5用Sac I进行线性化，线性化片段纯化回收后通过电穿孔法分别转化毕赤酵母GS115，在MD平板上筛选得到毕赤酵母重组菌株GS115/ pPIC9K-PHY1、GS115/pPIC9K-PHY4和GS115/pPIC9K-PHY5，然后在含不同浓度遗传霉素的YPD平板(0.5mg/mL-8mg/mL)上筛选多拷贝的转化子。
将毕赤酵母重组菌株GS115/pPIC9K-PHY1的一个转化子命名为毕赤酵母PHY1(Pichia pastoris PHY1)，重组菌株GS115/pPIC9K-PHY4的一个转化子命名为毕赤酵母PHY4(Pichia pastoris PHY4)，重组菌株GS115/pPIC9K-PHY5的一个转化子命名为毕赤酵母PHY5(Pichia pastoris PHY5)。将毕赤酵母PHY1、PHY4和PHY5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；9000rpm离心10min去除菌体，即得到分别含植酸酶突变体PHY1、PHY4和PHY5的发酵上清液。
采用实施例1中1.3.2所述植酸酶酶活测定方法对毕赤酵母PHY1、PHY4和PHY5发酵上清液进行酶活测定，检测结果见表3。
表3 酶活测定结果
Figure PCTCN2014093278-appb-000003
毕赤酵母PHY1、PHY4和PHY5发酵上清液的酶活分别为211U/mL、201U/mL和255U/mL。
2.5发酵验证
在10升发酵罐上分别进行毕赤酵母PHY1、毕赤酵母PHY4和毕赤酵母PHY5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、硫酸镁16.4g/L、硫酸钾20.3g/L、氢氧化钾1.65g/L、消泡剂0.05％。
发酵生产工艺：pH5.0、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1中1.3.2所述植酸酶酶活测定方法对上述粗酶液进行检测，结果见表4。
表4 酶活测定个结果
Figure PCTCN2014093278-appb-000004
重组表达植酸酶突变体PHY1的毕赤酵母PHY1最终的发酵酶活为10317U/mL，重组表达植酸酶突变体PHY4的毕赤酵母PHY4最终的发酵酶活为10401U/mL，而重组表达植酸酶突变体PHY5的毕赤酵母PHY5最终的发酵酶活高达10813U/mL。
2.6酶学特性分析
2.6.1最适作用温度
分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、80℃、85℃，pH5.5条件下测定上述毕赤酵母PHY1、PHY4和PHY5发酵所得粗酶液的酶活，以最高酶活力为100％，计算相对酶活。结果显示：与植酸酶突变体APPA-M相比，突变体PHY1、PHY4和PHY5的最适作用温度没有发生变化，均为75℃。
2.6.2最适作用pH值
分别用pH2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0的 0.1M乙酸-乙酸钠缓冲液对上述毕赤酵母PHY1、PHY4和PHY5发酵所得粗酶液进行稀释，在37℃条件下测定酶活，以最高酶活力为100％，计算相对酶活。结果显示：与植酸酶突变体APPA-M相比，突变体PHY1、PHY4和PHY5的最适作用pH均为5.0。
2.6.3耐热性分析
用预热10min的pH 0.25M乙酸钠缓冲液分别将上述毕赤酵母PHY1、PHY4和PHY5发酵所得粗酶液稀释10倍，混合均匀，85℃处理5min，80℃处理10min，结束时取样并冷却至室温，然后测定稀释后的酶活，以未处理样品的酶活计100％，计算残留酶活。
结果如图2所示：80℃处理10min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高33.85％、53.11％和75.86％；85℃处理5min后，突变体PHY1、PHY4和PHY5的残留酶活比APPA-M分别提高14.89％、28.45％和44.94％。
综上，本发明以植酸酶突变体APPA-M为基础，提供了包含A380P单点突变的植酸酶突变体PHY1，包含S80P、N176P、S187P和A380P四点突变的植酸酶突变体PHY4和包含S80P、T161P、N176P、S187P和A380P五点突变的植酸酶突变体PHY5。与APPA-M相比，PHY1、PHY4和PHY5的最适作用温度和pH没有发生改变，但其耐热性得到显著提升，从而有利于植酸酶在饲料中的广泛应用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
以上对本发明所提供的植酸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 PCTCN2014093278-appb-000005
Figure PCTCN2014093278-appb-000006
Figure PCTCN2014093278-appb-000007
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Figure PCTCN2014093278-appb-000009
Figure PCTCN2014093278-appb-000010
Figure PCTCN2014093278-appb-000011
Figure PCTCN2014093278-appb-000012
Figure PCTCN2014093278-appb-000013
Figure PCTCN2014093278-appb-000014
Figure PCTCN2014093278-appb-000015
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Figure PCTCN2014093278-appb-000020

Claims (10)

  1. 一种植酸酶突变体，其特征在于，其具有(Ⅰ)、(Ⅱ)所示的氨基酸序列中任意一个：
    (Ⅰ)与植酸酶的氨基酸序列SEQ ID NO:1具有至少70％同源性的序列；
    (Ⅱ)具有所述植酸酶的至少一个免疫表位，且所述植酸酶的氨基酸序列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；
    所述取代为取代1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个或17个氨基酸。
  2. 根据权利要求1所述的植酸酶突变体，其特征在于，所述取代为第25位、第46位、第62位、第70位、第73位、第75位、第114位、第137位、第142位、第146位、第159位或第255位中的一个或多个氨基酸被取代。
  3. 根据权利要求2所述的植酸酶突变体，其特征在于，所述取代还包括第380位的氨基酸被取代。
  4. 根据权利要求3所述的植酸酶突变体，其特征在于，所述取代还包括第80位、第176位或第187位中的一个或多个氨基酸被取代。
  5. 根据权利要求4所述的植酸酶突变体，其特征在于，所述取代还包括第161位的氨基酸被取代。
  6. 根据权利要求1至5任一项所述的植酸酶突变体，其特征在于，其具有如SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7或SEQ ID NO:9所示的氨基酸序列。
  7. 编码如权利要求1至6任一项所述的植酸酶突变体的DNA分子。
  8. 根据权利要求7所述的DNA分子，其特征在于，其具有如SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8或SEQ ID NO:10所示的核苷酸序列。
  9. 具有如权利要求7或8所述DNA分子的载体。
  10. 一种宿主细胞，其特征在于，包含如权利要求9所述的重组表达载体。
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