WO2020168943A1 - 植酸酶突变体 - Google Patents

植酸酶突变体 Download PDF

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WO2020168943A1
WO2020168943A1 PCT/CN2020/074735 CN2020074735W WO2020168943A1 WO 2020168943 A1 WO2020168943 A1 WO 2020168943A1 CN 2020074735 W CN2020074735 W CN 2020074735W WO 2020168943 A1 WO2020168943 A1 WO 2020168943A1
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mutant
phytase
seq
amino acid
present
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PCT/CN2020/074735
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English (en)
French (fr)
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吴秀秀
黄亦钧
刘扬
张霞
程斯达
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青岛蔚蓝生物集团有限公司
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Priority to US17/431,185 priority Critical patent/US20220154154A1/en
Priority to EP20759529.9A priority patent/EP3929284A4/en
Publication of WO2020168943A1 publication Critical patent/WO2020168943A1/zh

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    • 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
    • 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
    • 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)

Definitions

  • the present invention relates to the field of biotechnology, in particular to a phytase mutant, its preparation method and application, and DNA molecules, vectors and host cells encoding the phytase mutant.
  • Phytase is a phosphatase that can hydrolyze phytate. It can degrade phytate phosphorus (inositol hexaphosphate) into inositol and inorganic phosphate. This enzyme is divided into two categories: 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 corn, wheat and other higher plants, Bacillus subtilis, Pseudomonas, Lactobacillus, Escherichia coli and other prokaryotic microorganisms and yeast, Rhizopus, Aspergillus and other eukaryotic microorganisms .
  • the basic storage form of phosphorus is phytate phosphorus, and its content is as high as 1% to 3%, which accounts for 60% to 80% of the total phosphorus in plants.
  • phosphorus in the form of phytate phosphorus is difficult to use due to the lack of enzymes that can decompose phytate in monogastric animals, and its utilization rate is only 0%-40%, which causes many problems: firstly, waste of phosphorus source On the one hand, the phosphorus source in the feed cannot be effectively used.
  • inorganic phosphorus inorganic phosphorus must be added to the feed, which increases the cost of the feed; secondly, the formation of high-phosphorus manure pollutes the environment. About 85% of the phytate phosphorus in the feed is directly excreted by the animal, and the large amount of phytate phosphorus in the feces causes serious pollution of water and soil.
  • phytate phosphorus is also an anti-nutritional factor, it will chelate with a variety of metal ions such as Zn 2+ , Ca 2+ , Cu 2+ , Fe 2+, etc. and protein during the digestion and absorption process of the animal’s gastrointestinal tract. The corresponding insoluble compound reduces the effective utilization 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 rate of phosphorus in plant feeds by 60%, reduce phosphorus excretion in feces by 40%, and at the same time reduce the anti-nutritional effect of phytic acid. Therefore, adding phytase to feed is of great significance to improve the production efficiency of livestock and poultry industry and reduce the pollution of phytate phosphorus to the environment.
  • the industrial phytases are mainly fungal phytases derived from Aspergillus niger and bacterial phytases derived from Escherichia coli.
  • the phytase APPA from E. coli has the characteristics of high specific activity and good digestive tract stability. At present, it is mainly applied in the feed industry by directly adding powder feed or spraying after pellet feed.
  • Bacterial phytase APPA has poor thermal stability. Its aqueous solution is kept at 70°C for 5 minutes and the remaining enzyme activity is less than 30%. It is directly added to animal feed for pelleting and the remaining enzyme activity is generally less than 20%, so that APPA plant
  • the application of acid enzymes in pellet feed is restricted.
  • the method of spraying the phytase liquid onto the feed after pelleting not only increases the equipment investment, but also cannot guarantee the stability of the enzyme preparation and the uniformity of the feed. Therefore, improving the thermal stability has important practical significance for the current feed phytase.
  • the present invention provides a mutant of phytase to obtain mutant protein and improve its heat resistance, thereby facilitating the wide application of phytase in the feed field.
  • the present invention relates to a phytase mutant, which comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1, and comprises an amino acid in at least one position selected from the group as compared with SEQ ID NO:1 Replaced by: 36, 69, 89, 91, 111, 202, 213, 225, 238, 243, 253, 258, 266.
  • the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, compared with SEQ ID NO:1. Or at least 99% identity.
  • the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% compared with SEQ ID NO:1, Or at least 99.9% identity.
  • the mutant comprises a substitution of at least one amino acid in the following group: A36P, D69F, D69Q, V89T, E91Q, T111P, A202P, L213F, L213W, Q225Y, T238R, W243V, W243L, Q253Y , Q258E, S266P.
  • substitution or combination of substitutions contained in the mutant is selected from the following substitutions and combinations of substitutions:
  • the mutant further comprises a substitution of at least one amino acid in the following group: A25F, D35Y, W46E, Q62W, G70E, A73P, K75C, S80P, T114H, N126D, N137V, D142R, S146E , R159Y, T161P, N176P, K180N, S187P, V211W, Q253V, Y255D, T327Y, A380P.
  • the substitution is selected from the combination of substitutions of the amino acids in the following group: W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D;
  • the present invention also relates to DNA molecules encoding the aforementioned phytase mutants.
  • the present invention also relates to a recombinant expression vector containing the above-mentioned DNA molecule.
  • the present invention also relates to a host cell comprising the above-mentioned recombinant expression vector.
  • the host cell is Pichiapastoris.
  • the host cell is Trichoderma reesei.
  • the present invention also provides a method for preparing the above phytase mutant, including:
  • Step 1 Obtain a DNA molecule encoding a phytase mutant, the phytase mutant comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1 and is selected from SEQ ID NO:1 At least one position in the following group contains at least one amino acid substitution: 36, 69, 89, 91, 111, 202, 213, 225, 238, 243, 253, 258, 266;
  • Step 2 Fusion the DNA molecule obtained in step 1 with an expression vector, construct a recombinant expression vector, and transform a host cell;
  • Step 3 Induce the host cell containing the recombinant expression vector to express the fusion protein, and separate and purify the expressed fusion protein.
  • the phytase mutant described in step 1 contains at least one amino acid substitution in the following group: A36P, D69F, D69Q, V89T, E91Q, T111P, A202P, L213F, L213W, Q225Y, T238R , W243V, W243L, Q253Y, Q258E, S266P.
  • the host cell described in step 2 is Pichia pastoris.
  • the host cell described in step 2 is Trichodermareesei.
  • the invention also provides the application of the phytase mutant in feed.
  • the present invention is based on wild-type phytase APPA, and provides a phytase mutant containing a single mutation site of Q258E and S266P and a combination of two mutation sites of Q258E/S266P. After treatment at 65°C for 3 minutes, the enzyme activity The residue rate is 17.62%-31.80%, which is 59.9%-188.6% higher than that of the wild-type phytase APPA, and the heat resistance is significantly improved, which is beneficial to the wide application of phytase in feed.
  • the present invention discloses a phytase mutant, its preparation method and application, and DNA molecules, vectors, and host cells encoding the phytase mutant. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the nomenclature used to define amino acid positions is based on the amino acid sequence of E. coli phytase deposited in Genbank under the number ABF60232, which is given in the sequence table as SEQ ID NO:1 (SEQ ID NO:1 Amino acids 1-410). Therefore, in this context, the base SEQ ID NO:1 for position numbering starts at Q1 (Gln1) and ends at L410 (Leu410). SEQ ID NO: 1 serves as the standard for position numbering, and therefore serves as the basis for naming.
  • the present invention uses conventional techniques and methods used in the fields of genetic engineering and molecular biology, such as 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 use other conventional methods, experimental schemes, and reagents in the field on the basis of the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention.
  • the present invention can choose the following experimental materials and reagents:
  • Escherichia coli DH5 ⁇ , Pichia pastoris GS115, vectors pPIC9k, Amp, and G418 were purchased from Invitrogen.
  • Enzymes and kits PCR enzymes and ligases were purchased from Takara, restriction enzymes were purchased from Fermentas, plasmid extraction kits and gel purification recovery kits were purchased from Omega, GeneMorphII random mutagenesis kits were purchased from Beijing Bo Maisi Biological Technology Co., Ltd.
  • E. coli culture medium (LB medium): 0.5% yeast extract, 1% peptone, 1% NaCL, pH 7.0);
  • Yeast medium 1% yeast extract, 2% peptone, 2% glucose;
  • Yeast selection medium 2% peptone, 2% agarose;
  • BMGY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10-5 biotin, 1% glycerol;
  • BMMY medium 2% peptone, 1% yeast extract, 100 mM potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 ⁇ 10-5 biotin, 0.5% methanol;
  • LB-AMP medium 0.5% yeast extract, 1% peptone, 1% NaCL, 100 ⁇ g/mL ampicillin, pH 7.0;
  • LB-AMP plate 0.5% yeast extract, 1% peptone, 1% NaCL, 1.5% agar, 100 ⁇ g/mL ampicillin, pH7.0;
  • Lower medium plate 2% glucose, 0.5% (NH4)2SO4, 1.5% KH2PO4, 0.06% MgSO4, 0.06% CaCl2, 1.5% agar.
  • the amino acid sequence of wild-type phytase APPA derived from Escherichia coli is SEQ ID NO: 1, and its coding nucleotide sequence is SEQ ID NO: 2.
  • the applicant conducted a protein structure analysis of its gene.
  • the protein has two domains: the N-terminal 134 amino acid residues and the C-terminal 152 amino acid residues together form domain 1.
  • the remaining 124 amino acid residues in the middle constitute domain 2, and the conserved sequence and active center are located in domain 1.
  • the gene is further mutated without destroying the secondary structure and active center of the protein.
  • APPA-F1 GGCGAATTC CAGTCAGAACCAGAGTTGAAGTT (underlined is the restriction enzyme EcoRI recognition site), as shown in SEQ ID NO: 3;
  • APPA-R1 ATAGCGGCCGC TTACAAGGAACAAGCAGGGAT (underline is the recognition site of restriction enzyme NotI), as shown in SEQ ID NO: 4;
  • APPA gene (SEQ ID NO: 2) as template, use the above primers to perform PCR amplification with GeneMorph II Random Mutation PCR Kit (Stratagene), gel to recover the PCR product, and digest with EcoRI and NotI after digestion with the same digestion After ligating the pET21a vector, it was transformed into E.
  • the present invention provides at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 phytase mutants: A36P, D69F, D69Q, V89T, E91Q, T111P, A202P, L213F, L213W, Q225Y, T238R, W243V, W243L, Q253Y, Q258E, S266P.
  • the mutant further comprises at least one mutation site in the following group: A25F, D35Y, W46E, Q62W, G70E, A73P, K75C, S80P, T114H, N126D, N137V, D142R, S146E, R159Y, T161P, N176P, K180N, S187P , V211W, Q253V, Y255D, T327Y, A380P.
  • the mutant further comprises a combination of mutation sites selected from the following group:
  • the present invention provides a phytase mutant further comprising a combination of W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D mutation sites , Respectively named M4 and M5.
  • the present invention further provides W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P Phytase mutants with a combination of mutation sites were named M6 and M7, respectively.
  • amino acid sequences of the mutants M1, M2, M3, M4, M5, M6, and M7 are SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13, respectively. Or SEQ IDNO: 15 or SEQ ID NO: 17.
  • the coding nucleotide sequences of the mutants M1, M2, M3, M4, M5, M6, and M7 are SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ, respectively.
  • the gene sequence SEQ ID NO: 2 of APPA and the gene sequence of the mutant were optimized and synthesized, and two enzymes EcoRI and NotI were added to the 5'and 3'ends of the synthetic sequence. Cut site.
  • the synthesized APPA and mutant gene sequences were digested with EcoRI and NotI respectively, and then ligated with the pPIC-9K vector after the same digestion at 16°C overnight, and transformed into E. coli DH5a, and spread on the LB+Amp plate.
  • colony PCR reaction system: template picked monoclonal, rTaqDNA 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, ddH2O 14.5 ⁇ L, reaction procedure: 95°C pre-denaturation 5min, 30cycles: 94°C30sec, 55°C30sec, 72°C2min, 72°C10min). The positive clones were verified, and the correct recombinant expression plasmid was obtained after sequencing verification.
  • the Pichia pastoris GS115 strain was activated on a YPD plate. After culturing at 30°C for 48 hours, the activated GS115 monoclonal was inoculated in 6mLYPD liquid medium at 30°C and 220 rpm for about 12 hours. Then the bacterial solution was transferred to a 30mLYPD liquid medium. Incubate in a triangular flask at 30°C and 220rpm for about 5h, and detect the cell density by UV spectrophotometer.
  • centrifuge at 4°C for 2min at 9000rpm to collect 4mL cells respectively into the sterilized EP tube Gently discard the supernatant, blot the remaining supernatant with sterile filter paper, resuspend the bacteria in pre-chilled 1mL sterile water, centrifuge at 4°C, 9000rpm for 2min, gently discard the supernatant, and repeat sterilization with 1mL
  • centrifuge at 4°C and 9000rpm for 2min gently discard the supernatant, and resuspend the bacteria in pre-cooled 1mL sorbitol (1mol/L); centrifuge at 4°C, 9000rpm for 2min, gently discard the supernatant, pre-cooled 100 -150 ⁇ l sorbitol (1mol/L) gently resuspend the bacteria.
  • the expression plasmids constructed in 2.1 were linearized with Sac I. After the linearized fragments were purified and recovered, they were transformed into Pichia pastoris GS115 by electroporation. The recombinant strains of Pichia pastoris were screened on MD plates, and then genetically modified at different concentrations. Multiple copies of transformants were screened on YPD plates (0.5 mg/mL-8 mg/mL) with mycin.
  • the obtained transformants were transferred to BMGY medium and cultured with shaking at 30°C and 250rpm for 1d; then transferred to BMMY medium and cultured with shaking at 30°C and 250rpm; 0.5% methanol was added every day to induce expression for 4d; centrifuged at 9000rpm After removing the bacteria in 10 minutes, fermentation supernatants containing wild-type phytase APPA and phytase mutants were obtained.
  • X enzyme activity unit, U/g(mL);
  • the phytase activity of the constructed Pichia pastoris recombinant strain fermentation supernatant was determined by the above method.
  • the gene sequence SEQ ID NO: 2 of APPA and the gene sequence of the mutant were optimized and synthesized, and KpnI and MluI were added to the 5'and 3'ends of the synthetic sequence. Restriction sites.
  • the synthesized phytase gene fragment and pSC1G vector were digested with restriction enzymes KpnI and MluI (Fermentas) respectively, and the digested products were purified using a gel purification kit, and the digested product was purified with T4 DNA ligase (Fermentas).
  • the phytase gene was ligated with the digested product of the pSC1G vector and transformed into E. coli Trans5 ⁇ (Transgen), selected with ampicillin, and the clone was sequenced (Invitrogen) for verification. After the sequencing is correct, a recombinant plasmid containing the phytase gene is obtained.
  • Trichodermareesei UE spore suspension Take the host bacterium Trichodermareesei UE spore suspension, inoculate it on a PDA plate, and culture it for 6 days at 30°C. After the spores are abundant, cut a colony of about 1cm ⁇ 1cm and place it in a spore containing 120mLYEG+U (0.5% Yeast powder, 1% glucose, 0.1% uridine) in a liquid medium, 30°C, 220rpm shaking culture for 14-16h;
  • the applicant separately constructed and obtained the engineered strains of Trichoderma reesei that recombinantly express APPA and the above phytase mutant.
  • the engineered strains of Trichoderma reesei obtained by the above construction were respectively inoculated to PDA solid plates, and cultured in a constant temperature incubator at 30°C for 6-7 days. After the spores were enriched, two hypha blocks with a diameter of 1 cm were respectively inoculated with 50 mL 250mL of fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH4)2SO4, 0.09% MgSO4, 2% KH2PO4, 0.04% CaCl2, 0.018% Tween-80, 0.018% trace elements) In an Erlenmeyer flask, incubate at 30°C for 48 hours, and then at 25°C for 48 hours. Centrifuge the fermentation broth to obtain fermentation supernatants containing phytase APPA and the above-mentioned phytase mutant respectively.
  • Example 2 The method described in Example 2 was used to determine the phytase activity of the constructed fermentation supernatant of the recombinant Trichoderma reesei strain.
  • Enzyme activity residual rate (%) enzyme activity of untreated sample/enzyme activity of heat-treated sample ⁇ 100%.
  • the phytase mutants M1, M2, and M3 containing Q258E and/or S266P mutation sites provided by the present invention have a residual enzyme activity rate of 17.62% after being treated at 65°C for 3 minutes. 31.80%, which is 59.9%-188.6% higher than the residual rate of phytase APPA.
  • the present invention provides a phytase mutant containing a combination of W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D mutation sites on the basis of phytase APPA, and its amino acid sequence It is SEQ ID NO: 19. It can be seen from the data in Table 2 that, compared with the control mutant, the mutants M4 and M5 provided by the present invention further comprising a single mutation site of Q258E and S266P, after being treated at 80°C for 4 minutes, the enzyme activity residue rate increased By 122.2%-147.4%, the heat resistance has been significantly improved.
  • the present invention provides a combination of W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P based on phytase APPA.
  • the amino acid sequence of the mutant phytase is SEQ ID NO: 20. It can be seen from the data in Table 3 that, compared with the control mutant, the mutants M6 and M7 further containing single mutation sites of Q258E and S266P provided by the present invention have an increased residual rate of enzyme activity after being treated at 85°C for 5 minutes The heat resistance is significantly improved by 45.6%-82.0%.
  • the Q258E and S266P mutation sites provided by the present invention can significantly improve the heat resistance of phytase, thereby facilitating the wide application of phytase in feed.

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Abstract

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。本发明提供的突变体在选自下组中的至少一个位置上包含氨基酸的取代：36，69，89，91，111，202，213，225，238，243，253，258，266。所述突变体的耐热性均得到显著提高，从而有利于植酸酶在饲料中的广泛应用。

Description

植酸酶突变体
本申请要求于2019年02月18日提交中国专利局、申请号为2019101193080、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Zn 2+、Ca 2+、Cu 2+、Fe 2+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：
本发明涉及一种植酸酶突变体，其包含与SEQ ID NO:1具有至少90％同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代：36，69，89，91，111，202，213，225，238，243，253，258，266。
在本发明的一些实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91％，92％，93％，94％，95％，96％，97％，98％，或至少99％的同一性。
在一些更具体的实施例中，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，或至少99.9％的同一性。
在本发明的一些实施例中，所述突变体包含下组中至少一个氨基酸的取代：A36P，D69F，D69Q，V89T，E91Q，T111P，A202P，L213F，L213W，Q225Y，T238R，W243V，W243L，Q253Y，Q258E，S266P。
在本发明的一些实施例中，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：
Q258E；
Q258E/S266P；
V89T/Q258E；
E91Q/Q258E；
Q225Y/Q258E；
Q253Y/Q258E；
V89T/E91Q/Q258E；
V89T/Q225Y/Q258E；
V89T/Q253Y/Q258E；
V89T/Q258E/S266P；
E91Q/Q225Y/Q258E；
E91Q/Q253Y/Q258E；
E91Q/Q258E/S266P；
Q225Y/Q253Y/Q258E；
Q225Y/Q258E/S266P；
V89T/E91Q/Q225Y/Q258E；
V89T/E91Q/Q253Y/Q258E；
V89T/E91Q/Q258E/S266P；
E91Q/Q225Y/Q253Y/Q258E；
E91Q/Q225Y/Q258E/S266P；
V89T/Q225Y/Q253Y/Q258E；
V89T/Q225Y/Q258E/S266P；
E91Q/Q225Y/Q253Y/Q258E；
E91Q/Q225Y/Q258E/S266P；
E91Q/Q253Y/Q258E/S266P；
V89T/Q253Y/Q258E/S266P；
V89T/E91Q/Q225Y/Q253Y/Q258E；
V89T/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/Q253Y/Q258E/S266P；
V89T/E91Q/Q225Y/Q258E/S266P；
E91Q/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
S266P；
V89T/S266P；
E91Q/S266P；
Q225Y/S266P；
Q253Y/S266P；
V89T/E91Q/S266P；
V89T/Q225Y/S266P；
V89T/Q253Y/S266P；
E91Q/Q225Y/S266P；
E91Q/Q253Y/S266P；
Q225Y/Q253Y/S266P；
V89T/E91Q/Q225Y/S266P；
V89T/E91Q/Q253Y/S266P；
V89T/Q225Y/Q253Y/S266P；
V89T/E91Q/Q225Y/Q253Y/S266P；
V89T/E91Q；
V89T/Q253Y；
E91Q/Q253Y；
V89T/E91Q/Q253Y；
V89T/E91Q/Q225Y；
V89T/Q225Y/Q253Y；
E91Q/Q225Y/Q253Y；
V89T/E91Q/Q225Y/Q253Y；
V89T/E91Q/A202P/Q253Y；
V89T/E91Q/L213F/Q253Y；
V89T/E91Q/L213W/Q253Y；
V89T/E91Q/W243V/Q253Y；
V89T/E91Q/W243L/Q253Y；
D69F/V89T/E91Q/Q253Y；
D69Q/V89T/E91Q/Q253Y；
V89T/E91Q/T111P/Q253Y；
V89T/E91Q/T238R/Q253Y；
A36P/V89T/E91Q/Q253Y；
V89T/E91Q/A202P/L213F/Q253Y；
V89T/E91Q/A202P/L213W/Q253Y；
V89T/E91Q/A202P/W243V/Q253Y；
V89T/E91Q/A202P/W243L/Q253Y；
V89T/E91Q/L213F/W243V/Q253Y；
V89T/E91Q/L213W/W243L/Q253Y；
V89T/E91Q/L213F/W243L/Q253Y；
V89T/E91Q/L213W/W243V/Q253Y；
V89T/E91Q/A202P/L213F/W243V/Q253Y；
V89T/E91Q/A202P/L213W/W243L/Q253Y；
V89T/E91Q/A202P/L213F/W243L/Q253Y；
V89T/E91Q/A202P/L213W/W243V/Q253Y；
V89T/E91Q/A202P/Q225Y/Q253Y；
V89T/E91Q/L213F/Q225Y/Q253Y；
V89T/E91Q/L213W/Q225Y/Q253Y；
V89T/E91Q/W243V/Q225Y/Q253Y；
V89T/E91Q/W243L/Q225Y/Q253Y；
D69F/V89T/E91Q/Q225Y/Q253Y；
D69Q/V89T/E91Q/Q225Y/Q253Y；
V89T/E91Q/T111P/Q225Y/Q253Y；
V89T/E91Q/T238R/Q225Y/Q253Y；
A36P/V89T/E91Q/Q225Y/Q253Y；
V89T/E91Q/A202P/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/L213F/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/L213W/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/W243V/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/W243L/Q225Y/Q253Y/Q258E/S266P；
D69F/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
D69Q/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/T111P/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/T238R/Q225Y/Q253Y/Q258E/S266P；
A36P/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213F/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213W/Q225Y/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/Q225Y/W243V/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/Q225Y/W243L/Q253Y/Q258E/S266P；
V89T/E91Q/L213F/Q225Y/W243V/Q253Y/Q258E/S266P；
V89T/E91Q/L213W/Q225Y/W243L/Q253Y/Q258E/S266P；
V89T/E91Q/L213F/Q225Y/W243L/Q253Y/Q258E/S266P；
V89T/E91Q/L213W/Q225Y/W243V/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213F/Q225Y/W243V/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213W/Q225Y/W243L/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213F/Q225Y/W243L/Q253Y/Q258E/S266P；
V89T/E91Q/A202P/L213W/Q225Y/W243V/Q253Y/Q258E/S266P；
A202P；
L213F；
L213W；
W243V；
W243L；
D69Q；
D69F；
T111P；
T238R；
A36P；
A202P/L213F；
A202P/L213W；
A202P/W243L；
A202P/W243V；
A202P/L213F/W243L；
A202P/L213F/W243V；
A202P/L213W/W243V；
A202P/L213W/W243L。
在本发明的一些实施例中，所述突变体还进一步包含下组中至少一个氨基酸的取代：A25F，D35Y，W46E，Q62W，G70E，A73P，K75C，S80P，T114H，N126D，N137V，D142R，S146E，R159Y，T161P，N176P，K180N，S187P，V211W，Q253V，Y255D，T327Y，A380P。
在本发明的优选实施例中，所述取代选自下组中所述氨基酸的取代组合：W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P。
本发明还涉及编码上述植酸酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达载体。
本发明还涉及一种宿主细胞，包含上述重组表达载体。
将上述的质粒转入宿主细胞中，重组表达的植酸酶突变体的耐热性得到显著提升。
在本发明的一些实施例中，宿主细胞为毕赤酵母(Pichiapastoris)。
在本发明的一些实施例中，宿主细胞为里氏木霉(Trichoderma reesei)。
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：获取编码植酸酶突变体的DNA分子，所述植酸酶突变体包含与SEQ ID NO:1具有至少90％同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含至少一种氨基酸的取代：36，69，89，91，111，202，213，225，238，243，253，258，266；
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，步骤1所述的植酸酶突变体包含下组中至少一个氨基酸的取代：A36P，D69F，D69Q，V89T，E91Q，T111P，A202P，L213F，L213W，Q225Y，T238R，W243V，W243L，Q253Y，Q258E，S266P。
在本发明的一些实施例中，步骤2所述的宿主细胞为毕赤酵母(Pichia pastoris)。
在本发明的一些实施例中，步骤2所述的宿主细胞为里氏木霉(Trichodermareesei)。
本发明还提供了上述植酸酶突变体在饲料中的应用。
本发明以野生型植酸酶APPA为基础，提供了包含Q258E、S266P单个突变位点以及Q258E/S266P两个突变位点组合的植酸酶突变体，在65℃条件下处理3min后，酶活残留率为17.62％-31.80％，比野生型植酸酶APPA的酶活残留率提高了59.9％-188.6％，耐热性得到显著提高，从而有利于植酸酶在饲料中的广泛应用。
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。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。
本发明中，用于限定氨基酸位置的命名法基于以编号ABF60232保藏于Genbank的大肠杆菌的植酸酶的氨基酸序列，其作为SEQ ID NO:1在序列表中给出(SEQ ID NO:1的氨基酸1-410)。因此，在本上下文中，用于位置编号的基础SEQ ID NO:1，始于Q1(Gln1)并且止于L410(Leu410)。SEQ ID NO:1作为位置编号的标准，并因此作为命名的基础。
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，GeneMorphII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)；
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；
LB-AMP培养基：0.5％酵母提取物，1％蛋白胨，1％NaCL，100μg/mL氨苄青霉素，pH7.0；
LB-AMP平板：0.5％酵母提取物，1％蛋白胨，1％NaCL，1.5％琼脂，100μg/mL氨苄青霉素，pH7.0；
上层培养基：0.1％MgSO4，1％KH2PO4，0.6％(NH4)2SO4，1％葡萄糖，18.3％山梨醇，0.35％琼脂糖；
下层培养基平板：2％葡萄糖，0.5％(NH4)2SO4，1.5％KH2PO4，0.06％MgSO4，0.06％CaCl2，1.5％琼脂。
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
来源于大肠杆菌的野生型植酸酶APPA的氨基酸序列为SEQ ID NO:1，其编码核苷酸序列为SEQ ID NO:2。为了提高植酸酶APPA的耐热性，申请人对其基因进行蛋白结构分析，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APPA-F1、APPA-R1：
APPA-F1：GGCGAATTC CAGTCAGAACCAGAGTTGAAGTT(下划线为限制性内切酶EcoRI识别位点)，如SEQ ID NO：3所示；
APPA-R1：ATAGCGGCCGC TTACAAGGAACAAGCAGGGAT(下划线为限制性内切酶NotI识别位点)，如SEQ ID NO：4所示；
以APPA基因(SEQ ID NO：2)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75℃处理5min；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，有些突变对植酸酶APPA的耐热性没有影响，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APPA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APPA耐热性，又不会影响其酶活及原有酶学性质的突变位点：A36P，D69F，D69Q，V89T，E91Q，T111P，A202P，L213F，L213W，Q225Y，T238R，W243V，W243L，Q253Y，Q258E，S266P。
在植酸酶APPA的基础上，本发明提供了包含下组中至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，至少11个，至少12个，至少13个突变位点的植酸酶突变体：A36P，D69F，D69Q，V89T，E91Q，T111P，A202P，L213F，L213W，Q225Y，T238R，W243V，W243L，Q253Y，Q258E，S266P。
所述突变体进一步包含下组中至少一个突变位点：A25F，D35Y， W46E，Q62W，G70E，A73P，K75C，S80P，T114H，N126D，N137V，D142R，S146E，R159Y，T161P，N176P，K180N，S187P，V211W，Q253V，Y255D，T327Y，A380P。
所述突变体进一步包含选自下组中的突变位点组合：
W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P；
A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P。
其中，本发明在植酸酶APPA的基础上，提供的包含Q258E或S266P单个突变位点，以及Q258E/S266P突变位点组合的突变体，分别命名为M1，M2，M3。
本发明在植酸酶突变体M1和M2的基础上，提供的进一步包含W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D突变位点组合的植酸酶突变体，分别命名为M4和M5。
本发明在植酸酶突变体M1和M2的基础上，提供的进一步包含W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P突变位点组合的植酸酶突变体，分别 命名为M6和M7。
所述突变体M1，M2，M3，M4，M5，M6，M7的氨基酸序列分别为SEQ ID NO:5或SEQ ID NO:7或SEQ ID NO:9或SEQ ID NO:11或SEQ IDNO:13或SEQ IDNO:15或SEQ ID NO:17。
所述突变体M1，M2，M3，M4，M5，M6，M7的编码核苷酸序列分别为SEQ ID NO:6或SEQ ID NO:8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14或SEQ ID NO:16或SEQ ID NO:18。
实施例2植酸酶突变体在毕赤酵母中的表达
依照毕赤酵母的密码偏爱性分别对APPA的基因序列SEQ ID NO:2，以及突变体的基因序列进行优化合成，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。
2.1表达载体的构建
将合成的APPA和突变体的基因序列分别进行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×Buffer2.0μL，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。
2.2毕赤酵母工程菌株的构建
2.2.1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6mLYPD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。
2.2.2转化和筛选
分别将2.1构建得到的表达质粒用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，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。
将获得的转化子分别转接于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，即得到分别含野生型植酸酶APPA和植酸酶突变体的发酵上清液。
(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。
采用上述方法分别对构建得到的毕赤酵母重组菌株发酵上清液进行 植酸酶酶活测定。
实施例3植酸酶突变体在里氏木霉中的表达
依照木霉的密码子偏爱性，分别对APPA的基因序列SEQ ID NO:2，以及突变体的基因序列进行优化合成，并且在合成序列5’和3’两端分别加上KpnI和MluI两个酶切位点。
3.1表达载体的构建
将合成后的植酸酶基因片段与pSC1G载体分别用限制性内切酶KpnI和MluI(Fermentas)进行酶切，使用凝胶纯化试剂盒将酶切产物纯化，并用T4DNA连接酶(Fermentas)分别将上述植酸酶基因与pSC1G载体的酶切产物连接并转化大肠杆菌Trans5α(Transgen)，用氨苄青霉素进行选择，并对克隆进行测序(Invitrogen)验证。测序正确后，即得到含有植酸酶基因的重组质粒。
3.2里氏木霉重组菌株的构建
(1)原生质体制备
取宿主菌里氏木霉(Trichodermareesei)UE孢子悬液，接种于PDA平板上，30℃培养6天；待其产孢丰富后，切取约1cm×1cm的菌落置于含120mLYEG+U(0.5％酵母粉、1％葡萄糖、0.1％尿苷)的液体培养基中，30℃，220rpm振荡培养14～16h；
用无菌纱布过滤收集菌丝体，并用无菌水清洗一次；将菌丝体置于含有20mL 10mg/mL裂解酶液(Sigma L1412)的三角瓶中，30℃，90rpm作用1-2h；用显微镜观察检测原生质体转化进展；
将预冷的20mL 1.2M山梨醇(1.2M山梨醇，50mM Tris-Cl，50mM CaCl2)加入上述三角瓶中，轻轻摇匀，用无菌Miracloth滤布过滤收集滤液，3000rpm，4℃离心10min；弃上清，加入预冷的5mL 1.2M山梨醇溶液悬浮菌体，3000rpm，4℃离心10min；弃上清，加入适量预冷的1.2M山梨醇悬浮分装(200μL/管，原生质体浓度为108个/mL)。
(2)表达载体转化
以下操作均在冰上进行，分别取10μg上述构建的到的重组质粒加入到含有200μL原生质体溶液的7mL无菌离心管中，然后加入50μL25％ PEG(25％PEG，50mM Tris-Cl，50mM CaCl2)，轻弹管底混匀，冰上放置20min；加入2mL 25％PEG，混匀后室温放置5min；加入4mL 1.2M山梨醇，轻轻混匀后倒入熔化并保持在55℃的上层培养基中；轻轻混匀后铺在制备好的下层培养基平板上，30℃培养5～7d至有转化子长出，将生长出的转化子挑至下层培养基平板进行复筛，菌落边缘形态较光滑的菌株为阳性转化子。
按照上述方法，申请人分别构建得到重组表达APPA和上述植酸酶突变体的里氏木霉工程菌株。
(3)发酵验证和酶活测定
将上述构建得到的里氏木霉工程菌株分别接种至PDA固体平板，在30℃恒温培养箱倒置培养6-7天，待孢子丰富后，分别取两块直径1cm的菌丝块接种于含有50mL发酵培养基(1.5％葡萄糖，1.7％乳糖，2.5％玉米浆，0.44％(NH4)2SO4，0.09％MgSO4，2％KH2PO4，0.04％CaCl2，0.018％吐温-80，0.018％微量元素)的250mL三角瓶中，30℃培养48小时，然后25℃培养48小时。将发酵液离心，即得到分别含植酸酶APPA和上述植酸酶突变体的发酵上清液。
采用实施例2所述方法分别对构建得到的里氏木霉重组菌株发酵上清液进行植酸酶酶活测定。
实施例4热稳定性分析
用预热10min、pH5.0的0.25M乙酸钠缓冲液将上述获得的表达植酸酶突变体的重组菌株发酵上清液各稀释10倍；然后将稀释后的样品分别进行如下处理：65℃处理3min，80℃处理4min，85℃处理5min，结束时取样并冷却至室温；分别测定热处理后样品的植酸酶酶活，以未处理样品的酶活计100％，计算酶活残留率，具体结果见表1-3。
酶活残留率(％)＝未处理样品的酶活/热处理后样品的酶活×100％。
表1 植酸酶残留酶活比较
植酸酶 65℃处理3min酶活残留率
APPA 11.02％
M1 31.80％
M2 17.62％
M3 23.60％
从表1的数据可以看出，本发明提供的包含Q258E和/或S266P突变位点的植酸酶突变体M1、M2、M3在65℃条件下处理3min后，酶活残留率为17.62％-31.80％，比植酸酶APPA的酶活残留率提高了59.9％-188.6％。从而说明，本发明在植酸酶APPA基础上提供的Q258E、S266P突变位点能显著提高其耐热性。
表2 植酸酶残留酶活比较
Figure PCTCN2020074735-appb-000001
作为对照，本发明在植酸酶APPA基础上提供了包含W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D突变位点组合的植酸酶突变体，其氨基酸序列为SEQ ID NO:19。从表2的数据可以看出，与该对照突变体相比，本发明提供的进一步包含Q258E、S266P单个突变位点的突变体M4和M5在80℃条件下处理4min后，酶活残留率提高了122.2％-147.4％，耐热性得到显著提高。
表3 植酸酶残留酶活比较
Figure PCTCN2020074735-appb-000002
作为对照，本发明在植酸酶APPA基础上提供了包含W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y /T161P/N176P/S187P/Y255D/A380P突变位点组合的植酸酶突变体，其氨基酸序列为SEQ ID NO:20。从表3的数据可以看出，与该对照突变体相比，本发明提供的进一步包含Q258E、S266P单个突变位点的突变体M6和M7在85℃条件下处理5min后，酶活残留率提高了45.6％-82.0％，耐热性得到显著提高。
综上所述，本发明提供的Q258E、S266P突变位点能显著提高植酸酶的耐热性，从而有利于促进植酸酶在饲料中的广泛应用。

Claims (10)

  1. 一种植酸酶突变体，其特征在于，所述的突变体包含与SEQ ID NO:1具有至少90％同一性的氨基酸序列，且与SEQ ID NO:1相比在选自下组中的至少一个位置上包含氨基酸的取代：36，69，89，91，111，202，213，225，238，243，253，258，266。
  2. 如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少91％，92％，93％，94％，95％，96％，97％，98％，或至少99％的同一性。
  3. 如权利要求1所述的突变体，其特征在于，所述突变体的氨基酸序列与SEQ ID NO:1相比具有至少99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，或至少99.9％的同一性。
  4. 如权利要求1所述的突变体，其特征在于，所述突变体包含下组中至少一个氨基酸的取代：A36P，D69F，D69Q，V89T，E91Q，T111P，A202P，L213F，L213W，Q225Y，T238R，W243V，W243L，Q253Y，Q258E，S266P。
  5. 如权利要求4所述的突变体，其特征在于，所述突变体包含的取代或取代的组合选自下述取代和取代的组合：
    Q258E；
    Q258E/S266P；
    V89T/Q258E；
    E91Q/Q258E；
    Q225Y/Q258E；
    Q253Y/Q258E；
    V89T/E91Q/Q258E；
    V89T/Q225Y/Q258E；
    V89T/Q253Y/Q258E；
    V89T/Q258E/S266P；
    E91Q/Q225Y/Q258E；
    E91Q/Q253Y/Q258E；
    E91Q/Q258E/S266P；
    Q225Y/Q253Y/Q258E；
    Q225Y/Q258E/S266P；
    V89T/E91Q/Q225Y/Q258E；
    V89T/E91Q/Q253Y/Q258E；
    V89T/E91Q/Q258E/S266P；
    E91Q/Q225Y/Q253Y/Q258E；
    E91Q/Q225Y/Q258E/S266P；
    V89T/Q225Y/Q253Y/Q258E；
    V89T/Q225Y/Q258E/S266P；
    E91Q/Q225Y/Q253Y/Q258E；
    E91Q/Q225Y/Q258E/S266P；
    E91Q/Q253Y/Q258E/S266P；
    V89T/Q253Y/Q258E/S266P；
    V89T/E91Q/Q225Y/Q253Y/Q258E；
    V89T/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/Q253Y/Q258E/S266P；
    V89T/E91Q/Q225Y/Q258E/S266P；
    E91Q/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
    S266P；
    V89T/S266P；
    E91Q/S266P；
    Q225Y/S266P；
    Q253Y/S266P；
    V89T/E91Q/S266P；
    V89T/Q225Y/S266P；
    V89T/Q253Y/S266P；
    E91Q/Q225Y/S266P；
    E91Q/Q253Y/S266P；
    Q225Y/Q253Y/S266P；
    V89T/E91Q/Q225Y/S266P；
    V89T/E91Q/Q253Y/S266P；
    V89T/Q225Y/Q253Y/S266P；
    V89T/E91Q/Q225Y/Q253Y/S266P；
    V89T/E91Q；
    V89T/Q253Y；
    E91Q/Q253Y；
    V89T/E91Q/Q253Y；
    V89T/E91Q/Q225Y；
    V89T/Q225Y/Q253Y；
    E91Q/Q225Y/Q253Y；
    V89T/E91Q/Q225Y/Q253Y；
    V89T/E91Q/A202P/Q253Y；
    V89T/E91Q/L213F/Q253Y；
    V89T/E91Q/L213W/Q253Y；
    V89T/E91Q/W243V/Q253Y；
    V89T/E91Q/W243L/Q253Y；
    D69F/V89T/E91Q/Q253Y；
    D69Q/V89T/E91Q/Q253Y；
    V89T/E91Q/T111P/Q253Y；
    V89T/E91Q/T238R/Q253Y；
    A36P/V89T/E91Q/Q253Y；
    V89T/E91Q/A202P/L213F/Q253Y；
    V89T/E91Q/A202P/L213W/Q253Y；
    V89T/E91Q/A202P/W243V/Q253Y；
    V89T/E91Q/A202P/W243L/Q253Y；
    V89T/E91Q/L213F/W243V/Q253Y；
    V89T/E91Q/L213W/W243L/Q253Y；
    V89T/E91Q/L213F/W243L/Q253Y；
    V89T/E91Q/L213W/W243V/Q253Y；
    V89T/E91Q/A202P/L213F/W243V/Q253Y；
    V89T/E91Q/A202P/L213W/W243L/Q253Y；
    V89T/E91Q/A202P/L213F/W243L/Q253Y；
    V89T/E91Q/A202P/L213W/W243V/Q253Y；
    V89T/E91Q/A202P/Q225Y/Q253Y；
    V89T/E91Q/L213F/Q225Y/Q253Y；
    V89T/E91Q/L213W/Q225Y/Q253Y；
    V89T/E91Q/W243V/Q225Y/Q253Y；
    V89T/E91Q/W243L/Q225Y/Q253Y；
    D69F/V89T/E91Q/Q225Y/Q253Y；
    D69Q/V89T/E91Q/Q225Y/Q253Y；
    V89T/E91Q/T111P/Q225Y/Q253Y；
    V89T/E91Q/T238R/Q225Y/Q253Y；
    A36P/V89T/E91Q/Q225Y/Q253Y；
    V89T/E91Q/A202P/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/L213F/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/L213W/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/W243V/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/W243L/Q225Y/Q253Y/Q258E/S266P；
    D69F/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
    D69Q/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/T111P/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/T238R/Q225Y/Q253Y/Q258E/S266P；
    A36P/V89T/E91Q/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213F/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213W/Q225Y/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/Q225Y/W243V/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/Q225Y/W243L/Q253Y/Q258E/S266P；
    V89T/E91Q/L213F/Q225Y/W243V/Q253Y/Q258E/S266P；
    V89T/E91Q/L213W/Q225Y/W243L/Q253Y/Q258E/S266P；
    V89T/E91Q/L213F/Q225Y/W243L/Q253Y/Q258E/S266P；
    V89T/E91Q/L213W/Q225Y/W243V/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213F/Q225Y/W243V/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213W/Q225Y/W243L/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213F/Q225Y/W243L/Q253Y/Q258E/S266P；
    V89T/E91Q/A202P/L213W/Q225Y/W243V/Q253Y/Q258E/S266P；
    A202P；
    L213F；
    L213W；
    W243V；
    W243L；
    D69Q；
    D69F；
    T111P；
    T238R；
    A36P；
    A202P/L213F；
    A202P/L213W；
    A202P/W243L；
    A202P/W243V；
    A202P/L213F/W243L；
    A202P/L213F/W243V；
    A202P/L213W/W243V；
    A202P/L213W/W243L。
  6. 如权利要求4或5所述的突变体，其特征在于，所述的突变体还进一步包含下组中至少一个氨基酸的取代：A25F，D35Y，W46E，Q62W，G70E，A73P，K75C，S80P，T114H，N126D，N137V，D142R，S146E，R159Y，T161P，N176P，K180N，S187P，V211W，Q253V，Y255D，T327Y，A380P。
  7. 如权利要求6所述的突变体，其特征在于，所述突变体进一步包含的取代选自下组中所述氨基酸的取代组合：
    W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
    A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D；
    W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
    A25F/W46E/Q62W/G70E/A73P/K75C/T114H/N137V/D142R/S146E/R159Y/Y255D/A380P；
    W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
    A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/N176P/S187P/Y255D/A380P；
    W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P；
    A25F/W46E/Q62W/G70E/A73P/K75C/S80P/T114H/N137V/D142R/S146E/R159Y/T161P/N176P/S187P/Y255D/A380P。
  8. 编码权利要求1-7所述任一植酸酶突变体的DNA分子。
  9. 具有如权利要求8所述DNA分子的载体。
  10. 一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求9所述的载体。
PCT/CN2020/074735 2019-02-18 2020-02-11 植酸酶突变体 WO2020168943A1 (zh)

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