WO2017166562A1 - 植酸酶突变体 - Google Patents

植酸酶突变体 Download PDF

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WO2017166562A1
WO2017166562A1 PCT/CN2016/093918 CN2016093918W WO2017166562A1 WO 2017166562 A1 WO2017166562 A1 WO 2017166562A1 CN 2016093918 W CN2016093918 W CN 2016093918W WO 2017166562 A1 WO2017166562 A1 WO 2017166562A1
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phytase
seq
amino acid
acid sequence
sequence
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PCT/CN2016/093918
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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 ES16896334T priority Critical patent/ES2879292T3/es
Priority to DK16896334.6T priority patent/DK3438253T3/da
Priority to US16/089,044 priority patent/US11214776B2/en
Priority to EP16896334.6A priority patent/EP3438253B1/en
Publication of WO2017166562A1 publication Critical patent/WO2017166562A1/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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • 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
    • 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
    • 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)
    • C12Y301/03002—Acid phosphatase (3.1.3.2)

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 thermal stability has important practical significance for the current 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), (II) or (III):
  • (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;
  • the substitution is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve , 13, 14, 15, 16, 17, or 18 amino acids.
  • 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 a substitution of 16, 17, or 18 amino acids.
  • the substitution is 46th, 62nd, 70th, 73rd, 75th, 80th, 114th, 137th, 142th, One or more amino acids in positions 146, 159, 161, 176, 187, 255 or 380 are substituted.
  • the substitution is 46th, 62nd, 70th, 73rd, 75th, 80th, 114th, 137th, 142th, Amino acids at positions 146, 159, 161, 176, 187, 255 and/or 380 are substituted.
  • the phytase has the amino acid sequence set forth in SEQ ID NO: 1, and one of the nucleotide sequences encoding the phytase is set forth in SEQ ID NO: 2.
  • 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 present invention also provides a DNA molecule encoding the above phytase mutant.
  • 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.
  • the amino acid at position 46 of the phytase having the amino acid sequence of SEQ ID NO: 1 is changed from Trp to Glu, and the amino acid at position 62 is changed from Gln to Trp, and position 70.
  • amino acid sequence of the above phytase mutant is shown in SEQ ID NO: 3, and one of the encoded nucleotide sequences is shown in 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 126th and/or 211th amino acids.
  • the substitution further comprises the substitution of the amino acid at position 126.
  • the substituting further comprises changing the amino acid at position 126 of the phytase having the amino acid sequence SEQ ID NO: 3 from Asn to Asp.
  • 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 the amino acid at position 211.
  • amino acid sequence 211 of the phytase of SEQ ID NO: 3 is changed from Vla to Trp.
  • 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 simultaneous substitution of amino acids at positions 126 and 211.
  • amino acid sequence of amino acid sequence SEQ ID NO: 3 is changed from Asn to Asp and the amino acid at position 211 is changed from Vla to Trp.
  • 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.
  • the invention also provides a preparation method of the above phytase mutant, comprising:
  • Step 1 Obtain a DNA molecule encoding any one of the amino acid sequences shown in (I), (II) or (III):
  • (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;
  • substitution is substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, or 18 amino acids;
  • 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 substitutions in the preparation method are 46th, 62nd, 70th, 73rd, 75th, 80th, 114th, 137th, and One or more amino acids of position 142, 146, 159, 161, 176, 187, 255 or 380 are substituted.
  • the substitution is 46th, 62nd, 70th, 73rd, 75th, 80th, 114th, 137th, 142th, Amino acids at positions 146, 159, 161, 176, 187, 255 and/or 380 are substituted.
  • the substitution in the method of preparation further comprises the substitution of the amino acid at position 126 and/or position 211.
  • 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), (II) or (III):
  • (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;
  • substitution is substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, or 18 amino acids.
  • the present invention provides a phytase mutant Phy7.1 comprising a single point mutation of N126D, a phytase mutant Phy7.2 comprising a single point mutation of V211W, comprising a two-point mutation of N126D and V211W, based on phytase PHY6.
  • mutants Phy7.1, Phy7.2 and Phy8 were increased by 12.48%, 15.50% and 20.90%, respectively; after treatment at 85 °C for 5 min, mutant Phy7
  • the residual enzyme activities of Phy7.2 and Phy8 were increased by 13.05%, 18.50% and 27.56%, respectively, and their heat resistance was significantly higher than that of phytase PHY6, which was beneficial to the wide application of phytase in feed.
  • Figure 1 is a comparison of PHY6, Phy7.1, Phy7.2 and Phy8 heat resistance.
  • 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 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.
  • 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.
  • those skilled in the art can adopt other conventional methods, experimental solutions, and other methods in the art based on the technical solutions described in the present invention.
  • Reagents are not limited to the definition of specific embodiments of the 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.
  • 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% peptone, 2% agarose;
  • 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.
  • SEQ ID NO: 4 has two domains: 134 amino acid residues at the N-terminus and 152 amino acid residues at the C-terminus. Co-constituting domain 1, the remaining intermediate 124 amino acid residues constitute domain 2, the conserved sequence and the active center are located in domain 1, and the gene is further mutated without destroying the secondary structure and active center of the protein.
  • PHY6-F1 GGC GAATTC CAGTCAGAACCAGAGTTGAAGTT (underlined as restriction endonuclease EcoRI recognition site), as shown in SEQ ID NO:11;
  • PHY6-R1 ATA GCGGCCGC TTACAAGGAACAAGCAGGGAT (underlined as restriction endonuclease NotI recognition site), as shown in SEQ ID NO: 12;
  • the PHY6 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 enzyme.
  • the pET21a vector was ligated, transformed into E. coli BL21 (DE3), plated on LB+Amp plate, and cultured in inverted at 37 °C. After the transformation appeared, pick up the 96-well plate with a toothpick, and add 150 ul to each well.
  • the LB+Amp medium containing 0.1 mM IPTG was cultured at 37 ° C for 220 hours at 220 ° C. The supernatant was centrifuged, the cells were resuspended in a buffer, and the cells were repeatedly frozen and thawed to obtain a phytase-containing Escherichia coli cell lysate
  • the phytase mutant containing the N126D single point mutation is named Phy7.1, and the amino acid sequence thereof is SEQ ID NO: 5, and the nucleotide sequence of the coding sequence is SEQ ID NO: 6;
  • Phy7.2 The above phytase mutant containing V211W single point mutation is named Phy7.2, the amino acid sequence of which is SEQ ID NO: 7, and the sequence of the nucleotide sequence is SEQ ID NO: 8;
  • the above phytase mutant containing the N126D and V211W two-point mutation was named Phy8, and the amino acid sequence thereof was SEQ ID NO: 9, and the coding sequence was obtained by referring to the sequence as SEQ ID NO: 10.
  • the genetic sequence of PHY6 was synthesized by Shanghai Jierui Bioengineering Co., Ltd. according to the preference bias of Pichia pastoris, and the gene sequences of the above three mutants were SEQ ID NO: 6, SEQ ID NO: 8. SEQ ID NO: 10, and two restriction sites for EcoRI and NotI were added to the 5' and 3' ends of the synthetic sequence, respectively.
  • the four gene sequences synthesized in 1.2 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, and plated on LB + Amp plate, 37 °C inverted culture, after the emergence of transformants, colony PCR (reaction system: template picked monoclonal, rTaq DNA polymerase 0.5ul, 10 ⁇ Buffer 2.0 ⁇ L, dNTPs (2.5mM) 2.0 ⁇ L, 5'AOX primer (10M) : 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).
  • the positive clone was verified and the correct recombinant expression plasmid was obtained after sequencing.
  • the recombinant expression plasmid carrying the PHY6 gene sequence SEQ ID NO: 4 was named pPIC9K-PHY6 and will carry the above three mutant genes SEQ ID NO: 6.
  • the recombinant expression plasmids of SEQ ID NO: 8, SEQ ID NO: 10 were designated as pPIC9K-Phy7.1, pPIC9K-Phy7.2, and pPIC9K-Phy8, respectively.
  • 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 flask was cultured at 30 ° C and 220 rpm for about 5 hours.
  • the cell density was measured by UV spectrophotometer. After the OD600 value was in the range of 1.1–1.3, centrifuged at 9000 rpm for 2 min at 4 ° C to collect 4 mL of the cells to the sterilized EP tube.
  • the expression plasmids pPIC9K-PHY6, pPIC9K-Phy7.1, pPIC9K-Phy7.2 and pPIC9K-Phy8 were linearized with Sac I, respectively. The linearized fragments were purified and recovered, and then Pichia pastoris GS115 was transformed by electroporation.
  • the Pichia pastoris recombinant strains GS115/pPIC9K-PHY6, GS115/pPIC9K-Phy7.1, GS115/pPIC9K-Phy7.2 and GS115/pPIC9K-Phy8 were screened, and then YPD plates containing different concentrations of geneticin (0.5mg) Multiple copies of the transformants were screened on /mL-8 mg/mL.
  • Pichia pastoris recombinant strain GS115/pPIC9K-PHY6 was named Pichia pastoris PHY6, and one transformant of recombinant strain GS115/pPIC9K-Phy7.1 was named Pichia Phy7.1 (Pichia).
  • Pichia Phy7.1 Pichia Phy7.1
  • Pichia pastoris Phy7.2 Pichia pastoris Phy7.2
  • Pichia Yeast Phy8 Pichia Yeast Phy8 (Pichia pastoris Phy8).
  • Pichia pastoris PHY6 Pichia pastoris Phy7.1, Pichia pastoris Phy7.2 and Pichia pastoris Phy8 were transferred to BMGY medium, shaken at 30 ° C, 250 rpm for 1 d; then transferred to BMMY medium, 30 Incubation culture was carried out at °C and 250 rpm; 0.5% methanol was added every day to induce expression for 4 days; and the cells were removed by centrifugation at 9000 rpm for 10 minutes to obtain fermentation supernatants containing phytase mutants PHY6, Phy7.1, Phy7.2 and Phy8, respectively.
  • Pichia pastoris PHY6, Pichia pastoris Phy7.1, Pichia pastoris Phy7.2, and Pichia pastoris Phy8 fermentation supernatants were 241 U/mL, 223 U/mL, 205 U/mL, and 237 U/mL, respectively.
  • Pichia pastoris PHY6, Pichia pastoris Phy7.1, Pichia pastoris Phy7.2 and Pichia pastoris Phy8 was carried out on a 10-liter fermenter.
  • the medium used for fermentation was: calcium sulfate 1.1 g/L, phosphoric acid Potassium dihydrogenate 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, defoaming agent 0.05%.
  • Fermentation production process pH 5.0, temperature 30 ° C, stirring rate 300 rpm, ventilation 1.0-1.5 (v / v), dissolved oxygen control above 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 final fermentation enzyme activity of Pichia pastoris PHY6 expressing recombinant phytase mutant PHY6 was 11403 U/mL, and the final fermentation enzyme activity of Pichia pastoris Phy7.1 recombinantly expressing phytase mutant Phy7.1 was 10807 U/mL.
  • the final fermentation enzyme activity of Pichia pastoris Phy7.2 expressing recombinant phytase mutant Phy7.2 was as high as 10,713 U/mL, and the final fermentation enzyme activity of Pichia pastoris Phy8 recombinantly expressing phytase mutant Phy8 was as high as 11133 U/mL.
  • the Pichia pastoris PHY6 was measured 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 Phy7.1, Phy7.2 and Phy8 was calculated to be relative enzyme activity with the highest enzyme activity of 100%. The results showed that the optimum temperature of the mutants Phy7.1, Phy7.2 and Phy8 provided by the present invention did not change compared to the phytase mutant PHY6, both of which were 75 °C.
  • the above Pichia pastoris PHY6, Phy7.1, Phy7.2 and Phy8 were respectively treated 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.
  • the crude enzyme solution obtained by fermentation was diluted, and the enzyme activity was measured at 37 ° C, and the relative enzyme activity was calculated with the highest enzyme activity of 100%.
  • the results showed that compared with the phytase mutant PHY6, the optimum pH of the mutant Phy7.2 provided by the present invention did not change, which was 5.0, while the optimum pH of Phy7.1 and Phy8 was down-regulated by 0.5 units, which was 4.5. .
  • the crude enzyme solution obtained by fermenting the above Pichia pastoris PHY6, Phy7.1, Phy7.2 and Phy8 was diluted 10 times with 0.25 M sodium acetate buffer preheated for 10 min and pH 5.0, mixed uniformly, and treated at 85 ° C for 5 min, 80 After treatment at ° C for 10 min, samples were taken at the end and cooled to room temperature, and then the diluted enzyme activity was measured, and the residual enzyme activity was calculated by taking 100% of the enzyme activity of the untreated sample.
  • Fig. 1 Compared with phytase PHY6, the residual enzyme activities of the mutants Phy7.1, Phy7.2 and Phy8 provided by the present invention were increased by 12.48%, 15.50% and 20.90, respectively, after treatment at 80 °C for 10 min. After 5 min treatment at 85 ° C, the residual enzyme activities of the mutants Phy7.1, Phy7.2 and Phy8 provided by the present invention were increased by 13.05%, 18.50% and 27.56%, respectively, and were significantly different from phytase PHY6. (P ⁇ 0.01).
  • the present invention provides a phytase mutant Phy7.1 comprising a single point mutation of N126D, a phytase mutant Phy7.2 comprising a V211W single point mutation, comprising N126D and V211W, based on phytase PHY6.
  • Point mutation of the phytase mutant Phy8 Compared with phytase PHY6, the optimum temperature of mutants Phy7.1, Phy7.2 and Phy8 did not change, the optimum pH of Phy7.2 did not change, and the optimum pH of Phy7.1 and Phy8 did not change.

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Abstract

提供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、含有所述DNA分子的载体、以及含有所述载体的宿主细胞。

Description

植酸酶突变体
本申请要求于2016年03月28日提交中国专利局、申请号为2016101843371、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，提高热稳定性对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)或(Ⅲ)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；
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、17个或18个氨基酸。
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。
在本发明的一些实施例中，所述取代为取代16个、17个或18个氨基酸。
在本发明的另一些实施例中，所述取代为第46位、第62位、第70位、第73位、第75位、第80位、第114位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第255位或第380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，所述取代为第46位、第62位、第70位、第73位、第75位、第80位、第114位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第255位和/或第380位的氨基酸被取代。
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，编码所述植酸酶的核苷酸序列之一如SEQ ID NO:2所示。
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。
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。
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80位氨基酸由Ser变为Pro，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161位氨基酸由Thr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，第255位氨基酸由Tyr变为Asp，第380位氨基酸由Ala变为Pro。
上述植酸酶突变体的氨基酸序列如SEQ ID NO:3所示，编码的核甘酸序列之一如SEQ ID NO:4所示。
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126位和/或第211位氨基酸被取代。
在本发明的另一些实施例中，所述取代还包括第126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126位氨基酸由Asn变为Asp。
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211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:3的植酸酶的第211位氨基酸由Vla变为Trp。
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126位和第211位的氨基酸同时被取代。
在本发明的另一些实施例中，氨基酸序列为SEQ ID NO:3的植酸酶的第126位氨基酸由Asn变为Asp，第211位氨基酸由Vla变为Trp。
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)或(Ⅲ)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；
(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、17个或18个氨基酸；
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第46位、第62位、第70位、第73位、第75位、第80位、第114位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第255位或第380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，所述取代为第46位、第62位、第70位、第73位、第75位、第80位、第114位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第255位和/或第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126位和/或第211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)或(Ⅲ)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；
(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、17个或18个氨基酸。
本发明以植酸酶PHY6为基础，提供了包含N126D单点突变的植酸酶突变体Phy7.1，包含V211W单点突变的植酸酶突变体Phy7.2，包含N126D和V211W两点突变的植酸酶突变体Phy8。与植酸酶PHY6相比，80℃处理10min后，突变体Phy7.1、Phy7.2和Phy8的残留酶活分别提高了12.48％、15.50％和20.90％；85℃处理5min后，突变体Phy7.1、Phy7.2和Phy8的残留酶活分别提高了13.05％、18.50％和27.56％，其耐热性显著高于植酸酶PHY6，从而有利于植酸酶在饲料中的广泛应用。
附图说明
图1为PHY6，Phy7.1，Phy7.2和Phy8耐热性比较图。
具体实施方式
为了提高植酸酶的耐热性，申请人在野生型植酸酶APPA((氨基酸序列为SEQ ID NO:1，其编码核苷酸序列为SEQ ID NO:2)基础上进行了16个位点突变(W46E，Q62W，G70E，A73P，K75C，S80P，T114H，N137V，D142R，S146E，R159Y，T161P，N176P，S187P，Y255D，A380P)，获得了植酸酶突变体PHY6，其氨基酸序列为SEQ ID NO:3，其编码核苷酸序列为SEQ ID NO:4。所述植酸酶突变体PHY6的耐热性得到显著提升。(此部分内容已经在2015年8月26日申请的申请号为201510532520.1、专利名称为“植酸酶突变体”的国内发明专利中详细阐述)。
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耐热突变体的筛选
为了进一步提高植酸酶突变体PHY6的耐热性，对PHY6基因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，进一步对该基因进行突变。
1.1设计PCR引物PHY6-F1、PHY6-R1：
PHY6-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；
PHY6-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PHY6基因(SEQ ID NO：4)为模板，利用上述引物用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，将其中一块96孔板于80℃处理10min；然后向两块96孔板中各加入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PHY6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，另外还有些突变虽然能提高PHY6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，申请人得到既能显著提高PHY6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：N126D单点突变、V211W单点突变、N126D和V211W两点突变。
将上述含N126D单点突变的植酸酶突变体命名为Phy7.1，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V211W单点突变的植酸酶突变体命名为Phy7.2，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N126D和V211W两点突变的植酸酶突变体命名为Phy8，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。
1.2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PHY6的基因序列SEQ ID NO:4，以及上述3条突变体的基因序列SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10，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。
1.3表达载体的构建
将1.2中合成的4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：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，经测序验证后获得了正确的重组表达质粒，将携带PHY6基因序列SEQ ID NO:4的重组表达质粒命名为pPIC9K-PHY6，将携带上述3个突变体基因SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10的重组表达质粒依次分别命名为pPIC9K-Phy7.1、pPIC9K-Phy7.2、pPIC9K-Phy8。
1.4毕赤酵母工程菌株的构建
1.4.1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℃9000rpm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℃、9000rpm离心2min，轻轻弃上清，重复用1mL灭菌水洗一遍后，4℃、9000rpm离心2min，轻轻弃上清，预冷的1mL山梨醇(1mol/L)重悬菌体；4℃、9000rpm离心2min，轻轻弃上清，预冷的100-150μl山梨醇(1mol/L)轻柔重悬菌体。
1.4.2转化和筛选
分别将表达质粒pPIC9K-PHY6、pPIC9K-Phy7.1、pPIC9K-Phy7.2和pPIC9K-Phy8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-PHY6、GS115/pPIC9K-Phy7.1、GS115/pPIC9K-Phy7.2和GS115/pPIC9K-Phy8,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-PHY6的一个转化子命名为毕赤酵母PHY6(Pichia pastoris PHY6)，重组菌株GS115/pPIC9K-Phy7.1的一个转化子命名为毕赤酵母Phy7.1(Pichia pastoris Phy7.1)，重组菌株GS115/pPIC9K-Phy7.2的一个转化子命名为毕赤酵母Phy7.2(Pichia pastoris Phy7.2)，重组菌株GS115/pPIC9K-Phy8的一个转化子命名为毕赤酵母Phy8(Pichia pastoris Phy8)。将毕赤酵母PHY6、毕赤酵母Phy7.1、毕赤酵母Phy7.2和毕赤酵母Phy8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PHY6、Phy7.1、Phy7.2和Phy8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；
采用上述植酸酶酶活测定方法对毕赤酵母PHY6、Phy7.1、Phy7.2和Phy8发酵上清液进行酶活测定，检测结果见表1。
表1 酶活测定结果
样品 吸光值1 吸光值2 吸光值3 平均值 酶活(U/mL)
PHY6 0.491 0.487 0.490 0.489 241
Phy7.1 0.472 0.467 0.470 0.470 223
Phy7.2 0.470 0.463 0.466 0.466 205
Phy8 0.485 0.479 0.483 0.482 237
毕赤酵母PHY6、毕赤酵母Phy7.1、毕赤酵母Phy7.2和毕赤酵母Phy8发酵上清液的酶活分别为241U/mL、223U/mL、205U/mL和237U/mL。1.5发酵验证
在10升发酵罐上分别进行毕赤酵母PHY6、毕赤酵母Phy7.1、毕赤酵母Phy7.2和毕赤酵母Phy8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1.4.2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2。
表2 酶活测定结果
样品 吸光值1 吸光值2 吸光值3 平均值 酶活(U/mL)
PHY6 0.488 0.487 0.490 0.488 11403
Phy7.1 0.475 0.478 0.480 0.478 10807
Phy7.2 0.469 0.473 0.470 0.471 10713
Phy8 0.483 0.480 0.481 0.481 11133
重组表达植酸酶突变体PHY6的毕赤酵母PHY6最终的发酵酶活为11403U/mL，重组表达植酸酶突变体Phy7.1的毕赤酵母Phy7.1最终的发酵酶活为10807U/mL，重组表达植酸酶突变体Phy7.2的毕赤酵母Phy7.2最终的发酵酶活高达10713U/mL，重组表达植酸酶突变体Phy8的毕赤酵母Phy8最终的发酵酶活高达11133U/mL。
1.6酶学特性分析
1.6.1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PHY6、Phy7.1、Phy7.2和Phy8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PHY6相比，本发明提供的突变体Phy7.1、Phy7.2和Phy8的最适作用温度没有发生变化，均为75℃。
1.6.2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PHY6、Phy7.1、Phy7.2和Phy8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PHY6相比，本发明提供的突变体Phy7.2的最适pH没有变化，为5.0，而Phy7.1和Phy8最适作用pH均下调0.5个单位，为4.5。
1.6.3耐热性分析
用预热10min、pH5.0的0.25M乙酸钠缓冲液分别将上述毕赤酵母PHY6、Phy7.1、Phy7.2和Phy8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1所示：与植酸酶PHY6相比，80℃处理10min后，本发明提供的突变体Phy7.1、Phy7.2和Phy8的残留酶活分别提高了12.48％、15.50％和20.90％；85℃处理5min后，本发明提供的突变体Phy7.1、Phy7.2和Phy8的残留酶活分别提高了13.05％、18.50％和27.56％，与植酸酶PHY6相比具有极显著差异(P＜0.01)。
综上，本发明以植酸酶PHY6为基础，提供了包含N126D单点突变的植酸酶突变体Phy7.1，包含V211W单点突变的植酸酶突变体Phy7.2，包含N126D和V211W两点突变的植酸酶突变体Phy8。与植酸酶PHY6相比，突变体Phy7.1、Phy7.2和Phy8的最适作用温度没有发生改变，Phy7.2的最适pH没有发生变化，Phy7.1和Phy8的最适作用pH均下调0.5个单位，但Phy7.1、Phy7.2和Phy8的耐热性都得到极显著(P＜0.01)提升，从而有利于其在饲料中的广泛应用。
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 PCTCN2016093918-appb-000001
Figure PCTCN2016093918-appb-000002
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Figure PCTCN2016093918-appb-000004
Figure PCTCN2016093918-appb-000005
Figure PCTCN2016093918-appb-000006
Figure PCTCN2016093918-appb-000007
Figure PCTCN2016093918-appb-000008
Figure PCTCN2016093918-appb-000009
Figure PCTCN2016093918-appb-000010
Figure PCTCN2016093918-appb-000011
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Figure PCTCN2016093918-appb-000013
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Figure PCTCN2016093918-appb-000015

Claims (11)

  1. 一种植酸酶突变体，其特征在于，其具有(Ⅰ)、(Ⅱ)或(Ⅲ)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；
    (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、17或18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46位、第62位、第70位、第73位、第75位、第80位、第114位、第126位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第211位、第255位或第380位中的一个或多个氨基酸被取代。
  3. 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46位、第62位、第70位、第73位、第75位、第80位、第114位、第137位、第142位、第146位、第159位、第161位、第176位、第187位、第255位和第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，所述植酸酶突变体的氨基酸序列如SEQ ID NO:3所示，编码该氨基酸的核苷酸序列如SEQ ID NO:4所示。
  5. 根据权利要求2至4任一项所述的植酸酶突变体，其特征在于，所述取代还包括第126位和/或第211位的氨基酸被取代。
  6. 根据权利要求5所述的植酸酶突变体，其特征在于，其具有如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6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: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所述的载体。
  11. 根据权利要求1至6任一项所述植酸酶突变体的制备方法，其特征在于，包括如下步骤：
    步骤1：获取编码具有(Ⅰ)、(Ⅱ)或(Ⅲ)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SEQ ID NO:1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；
    (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；
    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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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114807087A (zh) * 2022-06-28 2022-07-29 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体和应用
EP4144840A4 (en) * 2020-04-29 2024-06-05 Nanjing Bestzyme Bio Eng Co Ltd PARENT PHYTASE VARIANT

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108251439B (zh) * 2018-01-11 2021-03-30 山西大学 一种人工改造的耐胰蛋白酶的植酸酶及其制备方法和应用
EP3805381A4 (en) * 2018-05-30 2022-04-20 Nanjing Bestzyme Bio-engineering Co. Ltd. MUTATED PHYTASE
WO2020063268A1 (zh) * 2018-09-28 2020-04-02 青岛蔚蓝生物集团有限公司 植酸酶突变体
WO2020063267A1 (zh) * 2018-09-28 2020-04-02 青岛蔚蓝生物集团有限公司 植酸酶突变体
CN111218436B (zh) * 2018-11-27 2022-08-30 青岛蔚蓝生物集团有限公司 一种植酸酶突变体
US10995325B2 (en) 2019-03-21 2021-05-04 Fornia Biosolutions, Inc. Additional phytase variants and methods
CN110724676B (zh) * 2019-10-21 2021-09-07 广东溢多利生物科技股份有限公司 一种植酸酶突变体及其载体与应用
CN110698557B (zh) * 2019-11-06 2021-06-18 郑州伊美诺生物技术有限公司 Acth突变体、重组蛋白及其应用，以及含有该acth重组蛋白的试剂盒
CN113717959B (zh) * 2020-05-22 2024-04-02 青岛蔚蓝生物集团有限公司 植酸酶突变体
CN113717958B (zh) * 2020-05-22 2024-04-12 青岛蔚蓝生物集团有限公司 比活力提高的植酸酶突变体
CN113699134B (zh) * 2020-05-22 2024-04-05 潍坊康地恩生物科技有限公司 植酸酶突变体

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR0215406A (pt) * 2001-12-28 2004-11-09 Syngenta Participations Ag Fitase termotolerante para ração animal
CN101724611A (zh) * 2008-10-24 2010-06-09 福建福大百特科技发展有限公司 酸性植酸酶appa,其突变体及其制备
CN102392002A (zh) * 2011-11-03 2012-03-28 青岛蔚蓝生物集团有限公司 一种改进的大肠杆菌植酸酶htp6m及其基因和应用
CN102943083A (zh) * 2012-11-27 2013-02-27 青岛根源生物技术集团有限公司 一种定点突变的耐高温植酸酶基因tp及其表达载体和应用
CN104404012A (zh) * 2014-12-19 2015-03-11 青岛蔚蓝生物集团有限公司 一种新型的植酸酶
CN104450643A (zh) * 2014-12-19 2015-03-25 青岛蔚蓝生物集团有限公司 植酸酶突变体及其应用
CN104911160A (zh) * 2015-07-10 2015-09-16 青岛玛斯特生物技术有限公司 一种中性植酸酶突变体及其应用
CN105219749A (zh) * 2015-11-04 2016-01-06 广东溢多利生物科技股份有限公司 优化改良的植酸酶突变体及其编码基因和应用
CN105624131A (zh) * 2014-11-21 2016-06-01 青岛蔚蓝生物集团有限公司 植酸酶突变体

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4516358B2 (ja) * 2004-05-26 2010-08-04 富士通株式会社 無線基地局装置および無線通信方法
AU2011231572B2 (en) * 2010-03-26 2015-10-22 Novozymes A/S Thermostable phytase variants
US9765313B2 (en) * 2013-11-12 2017-09-19 Feed Research Institute, Chinese Academy Of Agricultural Sciences Method for producing a phytase variant with improved thermal stability, and a phytase variant and the use thereof
US10351832B2 (en) * 2016-06-30 2019-07-16 Fornia Biosolutions, Inc. Phytases and uses thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR0215406A (pt) * 2001-12-28 2004-11-09 Syngenta Participations Ag Fitase termotolerante para ração animal
US7081563B2 (en) * 2001-12-28 2006-07-25 Syngenta Participations Ag Thermotolerant phytase for animal feed
CN101724611A (zh) * 2008-10-24 2010-06-09 福建福大百特科技发展有限公司 酸性植酸酶appa,其突变体及其制备
CN102392002A (zh) * 2011-11-03 2012-03-28 青岛蔚蓝生物集团有限公司 一种改进的大肠杆菌植酸酶htp6m及其基因和应用
CN102943083A (zh) * 2012-11-27 2013-02-27 青岛根源生物技术集团有限公司 一种定点突变的耐高温植酸酶基因tp及其表达载体和应用
CN105624131A (zh) * 2014-11-21 2016-06-01 青岛蔚蓝生物集团有限公司 植酸酶突变体
CN104404012A (zh) * 2014-12-19 2015-03-11 青岛蔚蓝生物集团有限公司 一种新型的植酸酶
CN104450643A (zh) * 2014-12-19 2015-03-25 青岛蔚蓝生物集团有限公司 植酸酶突变体及其应用
CN104911160A (zh) * 2015-07-10 2015-09-16 青岛玛斯特生物技术有限公司 一种中性植酸酶突变体及其应用
CN105219749A (zh) * 2015-11-04 2016-01-06 广东溢多利生物科技股份有限公司 优化改良的植酸酶突变体及其编码基因和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4144840A4 (en) * 2020-04-29 2024-06-05 Nanjing Bestzyme Bio Eng Co Ltd PARENT PHYTASE VARIANT
CN114807087A (zh) * 2022-06-28 2022-07-29 中国农业科学院北京畜牧兽医研究所 一种提高植酸酶热稳定性的方法及突变体和应用

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