WO2018130211A1 - 胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体 - Google Patents

胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体 Download PDF

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WO2018130211A1
WO2018130211A1 PCT/CN2018/072540 CN2018072540W WO2018130211A1 WO 2018130211 A1 WO2018130211 A1 WO 2018130211A1 CN 2018072540 W CN2018072540 W CN 2018072540W WO 2018130211 A1 WO2018130211 A1 WO 2018130211A1
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phytase
yeappa
resistance
mutant
catalytic efficiency
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French (fr)
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牛灿芳
杨培龙
姚斌
李阳阳
杜永凯
罗会颖
黄火清
王亚茹
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中国农业科学院饲料研究所
牛灿芳
杨培龙
姚斌
李阳阳
杜永凯
罗会颖
黄火清
王亚茹
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Application filed by 中国农业科学院饲料研究所, 牛灿芳, 杨培龙, 姚斌, 李阳阳, 杜永凯, 罗会颖, 黄火清, 王亚茹 filed Critical 中国农业科学院饲料研究所
Priority to EP18738844.2A priority Critical patent/EP3569703A4/en
Priority to US16/477,903 priority patent/US11155793B2/en
Publication of WO2018130211A1 publication Critical patent/WO2018130211A1/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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • 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)

Definitions

  • the invention relates to the field of genetic engineering, in particular to a phytase YeAPPA mutant with improved gastric protein resistance and acid resistance and improved catalytic efficiency.
  • Phytase can hydrolyze phosphate residues from phytic acid, destroying the combination of phytic acid and mineral elements, thereby improving nutrient utilization. Therefore, phytase with high catalytic efficiency against protease can produce good economic and ecological benefits, and it is bound to have a broad market in the feed industry.
  • the catalytic function of the enzyme is directly related to its molecular structure.
  • the study of the crystal structure of different phytic acid degrading enzymes will deepen our understanding of the structure and function of phytase.
  • the crystal structure of several phytic acid degrading enzymes with significantly different structures has been reported. Some structural components of the enzyme molecule are necessary for catalysis, and some non-essential parts can also be adapted to catalyze different substrates.
  • the object of the present invention is to modify phytase by site-directed mutagenesis to obtain a phytase YeAPPA mutant having improved gastric protein resistance, acid resistance improvement and catalytic efficiency.
  • Another object of the present invention is to provide a gene encoding a phytase YeAPPA mutant having the above-described gastric protein resistance and acid resistance improvement and catalytic efficiency.
  • Another object of the present invention is to provide a recombinant vector comprising the phytase YeAPPA mutant gene having the above-described gastric protein resistance and acid resistance improvement and catalytic efficiency improvement.
  • Another object of the present invention is to provide a recombinant strain comprising the phytase YeAPPA mutant gene having the above-described gastric protein resistance and acid resistance improvement and catalytic efficiency improvement.
  • the present invention performs site-directed mutagenesis of the phytase YeAPPA gene derived from Yersinia enterocolitica, the mature protein of the phytase having the amino acid sequence shown in SEQ ID NO. 1, which is composed of Encoded as the nucleotide sequence set forth in SEQ ID NO.
  • five mutants which improve acid resistance and pepsin resistance are obtained by site-directed mutagenesis, and are named YeAPPA-L162G/A and YeAPPA-E230G/P/R, respectively, ie, YeAPPA.
  • the leucine at position 162 is mutated to glycine or alanine, or the glutamic acid at position 230 is mutated to glycine, valine or arginine.
  • the phytase mutant YeAPPA-L162G has improved phytase resistance and acid resistance and catalytic efficiency, wherein the leucine at position 162 is mutated to glycine, and the amino acid sequence thereof is shown in SEQ ID NO.
  • a phytase mutant YeAPPA-L162A having improved pepsin resistance and acid resistance according to the present invention wherein the leucine at position 162 is mutated to alanine, and the amino acid sequence thereof is as shown in SEQ ID NO.
  • the phytase mutant YeAPPA-E230G according to the present invention, wherein the leucine at position 230 is mutated to alanine, and the amino acid sequence thereof is as shown in SEQ ID NO. .
  • a phytase mutant YeAPPA-E230P having an improved pepsin resistance and acid resistance according to the present invention wherein the leucine at position 230 is mutated to alanine, and the amino acid sequence thereof is shown in SEQ ID NO.
  • the leucine at position 230 is mutated to alanine, and the amino acid sequence thereof is shown in SEQ ID NO.
  • the present invention also provides a gene sequence encoding the above phytase mutant having improved acid resistance and pepsin resistance or improved catalytic efficiency, the nucleotide sequences of which are SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID, respectively. Shown as NO.10, SEQ ID NO.11 and SEQ ID NO.12.
  • the above reading frame of the phytase mutant YeAPPA-L162G/A or YeAPPA-E230G/P/R encoding improved acid and pepsin resistance and improved catalytic efficiency is inserted into the vector in an appropriate and correct reading frame. Between the restriction sites, the nucleotide sequence is operably linked to the expression control sequence.
  • a preferred vector of the present invention is pET-22b(+), and the engineered phytase gene is inserted between the EcoRI and NotI restriction sites of the plasmid pET-22b(+) such that the nucleotide sequence is located at T7. Downstream and regulated by the -lac promoter, recombinant E. coli expression plasmids of each mutant were obtained.
  • a preferred host strain of the invention is Escherichia coli BL21 (DE3).
  • the pepsin resistance and acid resistance of the phytase mutants YeAPPA-L162G/A and YeAPPA-E230G/P/R according to a specific embodiment of the present invention are significantly improved compared to the wild type, and YeAPPA-L162G and YeAPPA-
  • the catalytic efficiency of E230G increased by 1.6 times and 2.4 times, respectively, which is conducive to the development of a conservation feed enzyme industry.
  • Figure 1 shows the comparison of phytase activity before and after transformation by pepsin.
  • Figure 2 shows the comparison of phytase stability before and after modification by pepsin.
  • the prokaryotic expression vector pET-22b (+) and the host cell BL21 (DE3) were purchased from Novagen and from Tiangen.
  • Enzymes and other biochemical reagents endonuclease, ligase, DNA purification kit, LA DNA polymerase, sodium phytate and pepsin (p0685).
  • E. coli medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.
  • the gene sequence of Y. enterocolitica phytase YeAPPA (SEQ ID NO. 2) was modified, and the mutation was introduced by Overlap PCR to obtain mutants YeAPPA-L162G/A and YeAPPA-E230G. /P/R gene.
  • the Overlap PCR method in the implementation was completed by two rounds of PCR reaction using the reaction parameters of 95 ° C for 5 min, 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30-90 sec, 30 cycles, and 72 ° C for 10 min.
  • the mutation uses 12 PCR primers, of which Ye-F and Ye-R are upstream and downstream primers for amplifying the complete coding sequence of the mutant gene, L162G-F, L162G-R, L162A-F, L162A-R, E230G-F, E230G- R, E230P-F, E230P-R, E230R-F and E230R-R are upstream and downstream primers that introduce mutations at specific positions.
  • the primer sequences are as follows:
  • L162G-F 5'-cgggggtctgtaaa ggc gactcagcgaaaac-3'
  • L162G-R 5'-gttttcgctgagtc gcc tttacagacccccg-3'
  • L162A-F 5'-cgggggtctgtaaa gcg gactcagcgaaaac-3'
  • L162A-R 5'-gttttcgctgagtc cgc tttacagacccccg-3'
  • E230G-F 5'-ttaaggtaaacgaa ggc ggtactaaagtttc-3'
  • E230G-R 5'-gaaactttagtacc gcc ttcgtttaccttaa-3'
  • E230P-F 5'-ttaaggtaaacgaa ccg ggtactaaagtttc-3'
  • E230P-R 5'-gaaactttagtacc cgg ttcgtttaccttaa-3'
  • E230R-F 5'-ttaaggtaaacgaa cgt ggtactaaagtttc-3'
  • E230R-R 5'-gaaactttagtacc acg ttcgtttaccttaa-3 '
  • the primers contain restriction enzyme sites EcoR I and Not I (in italics), or a mutated nucleotide sequence (underlined).
  • the mutated gene product was recovered by Overlap PCR amplification and ligated to the pEASY-T3 vector, and sent to the company for sequencing verification.
  • the coding region of the mutant enzyme was inserted into the expression vector pET-22b(+), and transformed into E. coli BL21 (DE3) cells, and cultured with 1 mM IPTG for 5 h at 24 ° C to induce expression of phytase.
  • the crude enzyme solution was purified by a Ni-NTA (nickel-nitrilotriacetic acid) column and a DEAE (diethylaminoethyl) column to obtain a protein molecular weight consistent with the wild enzyme.
  • Example 3 Determination of gastric protein resistance of mutant phytase according to the present invention
  • the gastric protein resistance of the mutant phytase was evaluated according to the enzyme activity and protein amount of the remaining mutant phytase after different concentrations of pepsin treatment.
  • the purified mutant phytase was treated with different concentrations of pepsin at pH 2 for 2 h, and the effect of protease on phytase activity was studied by residual enzyme activity assay.
  • the mass ratio of pepsin to phytase used is between 1/1000 and 1/1.
  • the phytase activity was measured using the ferrous sulfate molybdenum blue method.
  • mutant phytase retained more enzyme activity than the wild phytase after treatment with different concentrations of pepsin for 2 h, when the concentration of pepsin increased from 1/1000 to 1/20.
  • the mutant phytase YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A retained 30%, 22%, 17%, 17% and 10% of the enzyme activity, respectively, while the wild enzyme YeAPPA was at 1/ 1000 pepsin treatment had completely lost activity at 2 h.
  • the five mutant phytase of the present invention can maintain the residual enzyme activity higher than that of the wild enzyme under different pepsin concentrations. The experimental results show that the pepsin resistance of the mutant phytase enzyme is obviously improved.
  • the purified mutant phytase was treated with different concentrations of pepsin at pH 2 for 2 h.
  • the remaining mutant phytase protein was detected by PAGE electrophoresis, and the gray value of the phytase protein band was evaluated by Image J software.
  • the ratio of the gray value of the remaining phytase protein band to the gray value of the untreated phytase band indicates the amount of residual protein of the mutant phytase after gastric protein treatment.
  • the mutant phytase was treated with different concentrations of pepsin for 2h, and more phytase protein remained than the wild enzyme (Fig. 2A and B; data not shown).
  • mutant phytase YeAPPA-E230G, YeAPPA-E230P, YeAPPA-E230R, YeAPPA-L162G and YeAPPA-L162A have more protein than wild enzyme, remaining at 1/100 pepsin concentration
  • the mutant phytase was 0.54, 0.43, 0.33, 0.35, 0.25, respectively, of the amount of untreated enzyme.
  • the five mutant phytase described in the present invention maintained more protein than the wild enzyme under different pepsin concentrations, indicating that the five mutant phytase have better pepsin resistance than the wild enzyme.
  • Example 4 Determination of acid resistance of mutant phytase according to the present invention
  • the purified mutant phytase was subjected to an enzymatic reaction at 37 ° C in a substrate of different pH to determine its optimum pH.
  • the buffer used was: 0.1 mol/L glycine-hydrochloric acid buffer, pH 1-3; 0.1 mol/L sodium acetate-acetate buffer, pH 3-6; 0.1 mol/L Tris-HCl buffer, pH 6- 8; 0.1 mol/L glycine-sodium hydroxide buffer, pH 8-12.
  • Table 1 indicate that the other five mutant phytase have an optimum pH (pH 5.0) similar to that of the wild enzyme except that the optimum pH of the mutant YeAPPA-E230R is shifted downward by one pH unit.
  • the stability of the mutant enzymes YeAPPA-L162G/A and YeAPPA-E230G/P/R was significantly higher than that of the wild enzyme.
  • the mutant phytase enzymes YeAPPA-L162G/A and YeAPPA were treated at pH 1.0-2.0 for 1 h.
  • -E230G/P/R maintains 18-32% of enzyme activity, while wild enzymes only maintain 12% of enzyme activity.
  • the purified mutant phytase was reacted at the optimum pH and temperature (30-80 ° C) for 30 min, and its temperature activity pattern was examined.
  • the results show that the optimum temperature of YeAPPA-E230P is 50 ° C, which is 5 ° C higher than the optimum pH of wild enzyme, YeAPPA-L162G/A and YeAPPA-E230G/R.
  • YeAPPA-E230P, YeAPPA-E230G and YeAPPA-E230R maintained 12%, 21% and 9% of enzyme activity, respectively, while YeAPPA-L162G/A and wild-type enzymes were essentially inactive. Therefore, YeAPPA-E230P, YeAPPA-E230G and YeAPPA-E230R exhibited better thermal stability than wild enzymes and YeAPPA-L162G/A.
  • Example 5 Determination of kinetic parameters of mutant phytase according to the present invention
  • the reaction rate (V max ) and conversion rate (K cat ) of the mutant enzyme YeAPPA-E230G were most significantly increased, about 2.5 times that of the wild enzyme, and the catalytic efficiency (K cat /k m ) was 2.4 times higher than that of the wild enzyme.
  • the reaction rate, conversion rate and catalytic efficiency of the mutant enzyme YeAPPA-L162G increased by 1.6-1.8 times compared with the wild enzyme.
  • the reaction rates, conversion rates and catalytic efficiencies of the mutant enzymes YeAPPA-L162A, YeAPPA-E230P and YeAPPA-E230R were basically consistent with wild-type enzymes.

Abstract

一种胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体。氨基酸序列如SEQ ID NO.1所示的植酸酶的第162位亮氨酸突变为甘氨酸或丙氨酸，或第230位谷氨酸突变为甘氨酸、脯氨酸或精氨酸。相比于野生型，突变体的胃蛋白酶抗性和耐酸性明显提高，催化效率分别增加达1.6倍和2.4倍，有利于发展节约型饲料酶工业。

Description

胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体 技术领域
本发明涉及基因工程领域，具体涉及胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体。
背景技术
植酸酶可从植酸上水解出磷酸残基，破坏植酸对矿质元素的结合，从而提高营养利用率。因此，抗蛋白酶高催化效率的植酸酶能产生良好经济和生态效益，势必在饲料工业有广阔市场。
酶的催化功能与其分子结构有着直接联系，不同植酸降解酶晶体结构的研究将加深我们对植酸酶结构与功能的了解。目前已经有几种结构明显不同的植酸降解酶的晶体结构被报道。酶分子中有些结构成分是催化所必须的，而有些非必要部分也可以通过改变来适应对不同底物的催化。
发明内容
本发明的目的是通过定点突变的方法对植酸酶进行改造，获得胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体。
本发明另一目的是提供编码上述胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体的基因。
本发明的另一目的是提供包含上述胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因的重组载体。
本发明的另一目的是提供包含上述胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因的重组菌株。
本发明对小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)来源植酸酶YeAPPA基因进行定点突变，该植酸酶的成熟蛋白具有如SEQ ID NO.1所示的氨基酸序列，该成熟蛋白是由如SEQ ID NO.2所示的核苷酸序列编码的。
根据本发明的具体实施方式，蛋白酶抗性和耐酸性或催化性提高的植酸酶YeAPPA的突变体，氨基酸序列如SEQ ID NO.1所示的植酸酶的第162位的亮氨酸突变为甘氨酸或丙氨酸，或第230位的谷氨酸突变为甘氨酸、脯氨酸或精氨酸。
根据本发明的具体实施方式，采用定点突变的方法，获得了5个提高耐酸 性和胃蛋白酶抗性的突变体，分别命名为YeAPPA-L162G/A和YeAPPA-E230G/P/R，即YeAPPA的第162位的亮氨酸突变为甘氨酸或丙氨酸，或第230位的谷氨酸突变为甘氨酸、脯氨酸或精氨酸。
因此根据本发明的胃蛋白酶抗性和耐酸性及催化效率提高的植酸酶突变体YeAPPA-L162G，其中第162位的亮氨酸突变为甘氨酸，其氨基酸序列如SEQ ID NO.3所示。
根据本发明的胃蛋白酶抗性和耐酸性提高的植酸酶突变体YeAPPA-L162A，其中第162位的亮氨酸突变为丙氨酸，其氨基酸序列如SEQ ID NO.4所示
根据本发明的胃蛋白酶抗性和耐酸性及催化效率提高的植酸酶突变体YeAPPA-E230G，其中第230位的亮氨酸突变为丙氨酸，其氨基酸序列如SEQ ID NO.5所示。
根据本发明的胃蛋白酶抗性和耐酸性提高的植酸酶突变体YeAPPA-E230P，其中第230位的亮氨酸突变为丙氨酸，其氨基酸序列如SEQ ID NO.6所示。
因此根据本发明的胃蛋白酶抗性和耐酸性提高的植酸酶突变体YeAPPA-E230R，其中第230位的亮氨酸突变为丙氨酸，其氨基酸序列如SEQ ID NO.7所示。
本发明还提供了编码上述耐酸性和胃蛋白酶抗性改良或催化效率提高的植酸酶突变体的基因序列，其核苷酸序列分别如SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12所示。
将上述编码耐酸性和胃蛋白酶抗性改良及催化效率提高的植酸酶突变体YeAPPA-L162G/A或YeAPPA-E230G/P/R的阅读框以合适和正确的读码框插入到所述载体的限制性酶切位点之间，使其核苷酸序列可操作的与表达调控序列相连接。本发明优选的载体为pET-22b(+)，使改造的植酸酶基因插入到质粒pET-22b(+)的EcoRI和NotI限制性酶切位点之间，使该核苷酸序列位于T7-lac启动子的下游并受其调控，得到各突变体的重组大肠杆菌表达质粒。本发明优选的宿主菌为大肠杆菌BL21(DE3)。相比于野生型，根据本发明的具体实施方式的植酸酶突变体YeAPPA-L162G/A和YeAPPA-E230G/P/R的胃蛋白酶抗性和耐酸性明显提高，且YeAPPA-L162G和YeAPPA-E230G的催化效率分别增加达1.6倍和2.4倍，有利于发展节约型饲料酶工业。
附图说明
图1为改造前、后的植酸酶活性受胃蛋白酶影响比较。
图2为改造前、后的植酸酶稳定性受胃蛋白酶影响比较。
具体实施方式
1、菌株及载体：原核表达载体pET-22b(+)和宿主细胞BL21(DE3)分别购自Novagen公司和自天根公司。
2、酶类及其它生化试剂：内切酶、连接酶、DNA纯化试剂盒，LA DNA聚合酶，植酸钠和胃蛋白酶(p0685)。
3、培养基：大肠杆菌培养基LB(1％蛋白胨、0.5％酵母提取物、1％NaCl，pH7.0)。
实施例1：突变基因的获得
对小肠结肠炎耶尔森氏菌(Y.enterocolitica)植酸酶YeAPPA的基因序列(SEQ ID NO.2)进行改造，通过Overlap PCR方法引入突变，获得突变体YeAPPA-L162G/A和YeAPPA-E230G/P/R的基因。实施中的Overlap PCR方法通过两轮PCR反应完成，所用PCR反应参数为：95℃5min；94℃30sec,55℃30sec,72℃30-90sec,30个循环；72℃10min。突变使用12条PCR引物，其中Ye-F和Ye-R是扩增突变基因完整编码序列的上下游引物，L162G-F、L162G-R、L162A-F、L162A-R、E230G-F、E230G-R、E230P-F、E230P-R、E230R-F和E230R-R是在特定位置引入突变的上下游引物。引物序列如下：
Ye-F:5’-cgcgaattcgccccgattgctacaccgcc-3’
Ye-R:5’-gatgcggccgcttaaatatggcaggctggctcga-3’
L162G-F:5’-cgggggtctgtaaa ggcgactcagcgaaaac-3’
L162G-R:5’-gttttcgctgagtc gcctttacagacccccg-3’
L162A-F:5’-cgggggtctgtaaa gcggactcagcgaaaac-3’
L162A-R:5’-gttttcgctgagtc cgctttacagacccccg-3’
E230G-F:5’-ttaaggtaaacgaa ggcggtactaaagtttc-3’
E230G-R:5’-gaaactttagtacc gccttcgtttaccttaa-3’
E230P-F:5’-ttaaggtaaacgaa ccgggtactaaagtttc-3’
E230P-R:5’-gaaactttagtacc cggttcgtttaccttaa-3’
E230R-F:5’-ttaaggtaaacgaa cgtggtactaaagtttc-3’
E230R-R:5’-gaaactttagtacc acgttcgtttaccttaa-3’
引物中含有限制性酶切位点EcoR I和Not I(斜体表示)，或突变的核苷酸序列(下划线标记)。通过Overlap PCR扩增得到突变基因产物回收后连接到pEASY-T3载体上，送公司测序验证。
实施例2：突变植酸酶在大肠杆菌中的表达
将突变酶的编码区插入到表达载体pET-22b(+)上，并转化到大肠杆菌BL21(DE3)细胞中，用1mM IPTG在24℃下培养5h诱导表达植酸酶。粗酶液经Ni-NTA(镍-次氮基三乙酸)柱和DEAE(二乙基氨基乙基)柱纯化，获得与野生酶一致的蛋白分子量。
实施例3：本发明所述的突变植酸酶的胃蛋白抗性的测定
突变植酸酶的胃蛋白抗性，根据不同浓度胃蛋白酶处理后剩余突变植酸酶的酶活力和蛋白量进行评价。
突变植酸酶的活性受胃蛋白酶影响的测定
纯化好的突变植酸酶经不同浓度的胃蛋白酶在pH 2下处理2h后，通过剩余酶活性检测研究蛋白酶对植酸酶活性的影响。所用胃蛋白酶与植酸酶的质量比在1/1000到1/1之间。植酸酶活性测定使用硫酸亚铁钼蓝法。50ul酶液加入到950ul 1.5mmol/L植酸钠底物中，在37℃下反应30min，用1mL 10％TCA终止反应，再用2mL显色液(1％四水合钼酸铵，3.2％浓硫酸，7.32％硫酸亚铁)进行显色。根据显色后700nm下的光吸收值计算植酸酶活性。1个植酸酶的活性单位(U)定义为在一定条件下每分钟释放出1μmol无机磷所需的酶量。结果(图1A和B)表明，用不同浓度胃蛋白酶处理2h后突变植酸酶较野生植酸酶保留了较多的酶活，当胃蛋白酶的浓度从1/1000升至1/20时，突变植酸酶YeAPPA-E230G、YeAPPA-E230P、YeAPPA-E230R、YeAPPA-L162G和YeAPPA-L162A分别剩余30％、22％、17％、17％和10％的酶活，而野生酶YeAPPA在1/1000胃蛋白酶处理2h时已经完全失去活性。本发明所述的5个突变植酸酶在不同胃蛋白酶浓度处理下均可保持高于野生酶的剩余酶活力，该实验结果显示该突变植酸酶酶的胃蛋白酶抗性确有明显提高。
突变植酸酶的稳定性受胃蛋白酶影响的测定
纯化好的突变植酸酶经不同浓度的胃蛋白酶在pH 2下处理2h后，经PAGE电泳检测剩余的突变植酸酶蛋白，并用Image J软件评价植酸酶蛋白条带 的灰度值。剩余植酸酶蛋白条带的灰度值与未处理植酸酶条带灰度值的比值，表示胃蛋白处理后突变植酸酶的剩余蛋白量。突变植酸酶经不同浓度胃蛋白酶处理2h后可剩余较野生酶更多的植酸酶蛋白(图2A和B；数据未显示)，当用1/1000的胃蛋白酶时，野生酶剩余未处理酶量的0.1的蛋白，而突变植酸酶YeAPPA-E230G、YeAPPA-E230P、YeAPPA-E230R、YeAPPA-L162G和YeAPPA-L162A剩余较野生酶更多的蛋白，在1/100的胃蛋白酶浓度下剩余突变植酸酶分别是未处理酶量的0.54、0.43、0.33、0.35、0.25。本发明所述的5个突变植酸酶在不同胃蛋白酶浓度处理下保持了较野生酶更多的蛋白，显示了5个突变植酸酶具有较野生酶更优良的胃蛋白酶抗性。
实施例4：本发明所述的突变植酸酶的耐酸性测定
纯化的突变植酸酶在37℃下，不同pH的底物中进行酶学反应，以测定其最适pH。所用的缓冲液为：0.1mol/L甘氨酸-盐酸缓冲液，pH 1-3；0.1mol/L醋酸钠-醋酸缓冲液，pH 3-6；0.1mol/L Tris-盐酸缓冲液，pH 6-8；0.1mol/L甘氨酸-氢氧化钠缓冲液，pH 8-12。结果(表1)表明，除了突变体YeAPPA-E230R的最适pH向下移动了1个pH值单位，其它5个突变植酸酶具有与野生酶类似的最适pH值(pH 5.0)。突变酶YeAPPA-L162G/A和YeAPPA-E230G/P/R在酸处理下的稳定性明显高于野生酶，在pH 1.0-2.0下处理1h时，突变植酸酶酶YeAPPA-L162G/A和YeAPPA-E230G/P/R可保持18-32％以上的酶活，而野生酶仅保持12％的酶活。
纯化的突变植酸酶在最适pH和温度(30-80℃)下反应30min，检测其温度活性模式。结果(表1)显示，YeAPPA-E230P的最适温度为50℃，较野生酶、YeAPPA-L162G/A和YeAPPA-E230G/R的最适pH高出5℃。在60℃下30min，YeAPPA-E230P、YeAPPA-E230G和YeAPPA-E230R分别可保持12％、21％和9％的酶活，而YeAPPA-L162G/A与野生酶基本失去活性。因此，YeAPPA-E230P、YeAPPA-E230G和YeAPPA-E230R表现出较野生酶和YeAPPA-L162G/A更优良的热稳定性。
表1pH和温度对改造前后的植酸酶的酶活和稳定性的影响的比较
Figure PCTCN2018072540-appb-000001
Figure PCTCN2018072540-appb-000002
实施例5：本发明所述的突变植酸酶的动力学参数测定
用不同浓度的植酸钠(0.0625、0.1、0.125、0.2、0.25、0.5、1.0和1.5mmol/L)为底物，在最适条件下测定酶活，用米氏方程双倒数法求k m值及V max，再根据酶的理论分子量求出K cat值。结果(表2)表明，不同突变酶对底物的亲和力(k m)与野生酶基本一致。突变酶YeAPPA-E230G的反应速度(V max)和转换率(K cat)提高最明显，为野生酶的约2.5倍，其催化效率(K cat/k m)较野生酶提高了2.4倍。突变酶YeAPPA-L162G的反应速度、转换率和催化效率较野生酶增加了1.6-1.8倍。突变酶YeAPPA-L162A、YeAPPA-E230P和YeAPPA-E230R的反应速度、转换率和催化效率与野生酶基本一致。
表2 改造前后的植酸酶的酶学性质比较
Figure PCTCN2018072540-appb-000003

Claims (8)

  1. 胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体，其特征在于，氨基酸序列如SEQ ID NO.1所示的植酸酶的第162位亮氨酸突变为甘氨酸或丙氨酸。
  2. 胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体，其特征在于，氨基酸序列如SEQ ID NO.1所示的植酸酶的第230位谷氨酸突变为甘氨酸、脯氨酸或精氨酸。
  3. 胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因，其特征在于，编码权利要求1或2所述的胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体。
  4. 根据权利要求3所述的胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或SEQ ID NO.12所示。
  5. 包含权利要求3所述的胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因的重组载体。
  6. 包含权利要求3所述的胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体基因的重组菌株。
  7. 一种制备胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体的方法，其特征在于，包括以下步骤：
    1)用权利要求5的重组载体转化宿主细胞，得重组菌株；
    2)诱导并表达胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体；以及
    3)纯化胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体。
  8. 权利要求1或2所述的胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体的应用。
PCT/CN2018/072540 2017-01-15 2018-01-15 胃蛋白抗性和耐酸性改良及催化效率提高的植酸酶YeAPPA突变体 WO2018130211A1 (zh)

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