WO2018130212A2 - 胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体 - Google Patents

胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体 Download PDF

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WO2018130212A2
WO2018130212A2 PCT/CN2018/072549 CN2018072549W WO2018130212A2 WO 2018130212 A2 WO2018130212 A2 WO 2018130212A2 CN 2018072549 W CN2018072549 W CN 2018072549W WO 2018130212 A2 WO2018130212 A2 WO 2018130212A2
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mutant
phytase
ykappa
improved
seq
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PCT/CN2018/072549
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French (fr)
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WO2018130212A3 (zh
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牛灿芳
杨培龙
姚斌
李阳阳
于大力
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
于大力
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, 于大力, 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 US16/477,907 priority Critical patent/US11028377B2/en
Priority to EP18739340.0A priority patent/EP3569704A4/en
Publication of WO2018130212A2 publication Critical patent/WO2018130212A2/zh
Publication of WO2018130212A3 publication Critical patent/WO2018130212A3/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
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia

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  • the invention relates to the field of genetic engineering, in particular to a phytase YkAPPA mutant with improved phytase mutant gastric protein resistance and improved catalytic efficiency.
  • Phytase (myo-inositol hexakisphosphate phosphohydrolase) is a class of important industrial enzymes, a general term for a class of enzymes that degrade phosphate monoester bonds in phytic acid to produce inorganic phosphorus and lower inositol phosphate. At present, most phytase enzymes are poorly resistant, resulting in a huge waste of phosphorus, increased feed costs and environmental pollution. Phytase, which is resistant to proteases and does not reduce catalytic efficiency, can reduce animal production costs and has good economic and ecological benefits.
  • the object of the present invention is to modify phytase by site-directed mutagenesis to provide a phytase YkAPPA mutant with improved phytase mutant gastric protein resistance and improved catalytic efficiency.
  • Another object of the present invention is to provide a gene encoding a phytase YkAPPA mutant having improved gastric protein resistance and improved catalytic efficiency.
  • Another object of the present invention is to provide a recombinant vector comprising the above phytase YkAPPA mutant gene with improved gastric protein resistance and improved catalytic efficiency.
  • Another object of the present invention is to provide a recombinant strain comprising the above phytase YkAPPA mutant gene with improved gastric protein resistance and improved catalytic efficiency.
  • the present invention performs site-directed mutagenesis of the Yersinia kristensenii-derived phytase YkAPPA gene, which has the amino acid sequence shown in SEQ ID NO. 1, and the mature protein is The nucleotide sequence shown in SEQ ID NO. 2 is encoded.
  • mutants which improve acid resistance and pepsin resistance are obtained by site-directed mutagenesis, and are named YkAPPA-L162G/A and YkAPPA-E230G/A/S/T/D/P, respectively.
  • /R that is, the leucine of the 162th position of YkAPPA is mutated to glycine or alanine, or the glutamic acid at position 230 is mutated to glycine, alanine, serine, threonine, aspartic acid, guanidine Acid or arginine.
  • the phytase mutant YkAPPA-L162G having improved pepsin resistance according to the present invention has an amino acid sequence as shown in SEQ ID NO.
  • amino acid sequence of the mutant YkAPPA-L162A is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230G is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230A is shown in SEQ ID NO.
  • amino acid sequence of the mutant YkAPPA-E230S is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230T is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230D is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230P is shown in SEQ ID NO.
  • the amino acid sequence of the mutant YkAPPA-E230R is shown in SEQ ID NO.
  • the present invention also provides a gene sequence encoding the above-described phytase mutant YkAPPA-L162G having improved acid resistance and pepsin resistance, the nucleotide sequence of which is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-L162A is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230G is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230A is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230S is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230T is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230D is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230P is shown in SEQ ID NO.
  • the gene sequence of the mutant YkAPPA-E230R is shown in SEQ ID NO.
  • a preferred vector of the invention is pET-22b(+), resulting in a recombinant prokaryotic expression plasmid of the mutant.
  • a preferred host strain of the invention is BL21 (DE3). The nucleotide sequence of the phytase mutant is located downstream of and regulated by the T7-lac promoter.
  • the pepsin resistance of the phytase mutants YkAPPA-L162G/A and YkAPPA-E230G/A/S/T/D/P/R according to a specific embodiment of the present invention is significantly improved compared to the wild type, and YkAPPA
  • the catalytic efficiency of -L162G and YkAPPA-E230G/A is increased by up to 2.1 times, which is conducive to the development of a conservation feed enzyme industry.
  • Figure 1 is a comparison of protease resistance of phytase before and after transformation
  • Figure 2 shows the hydrolysis ability of phytase before and after the modification.
  • Enzymes and other biochemical reagents DNA purification kits, and LADNA polymerase were purchased from TaKaRa, endonuclease and ligase, sodium phytate and pepsin (p0685).
  • E. coli medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.
  • the gene sequence (SEQ ID NO. 2) derived from Y. kristensenii phytase YkAPPA was engineered, and the mutation was introduced by Overlap PCR to obtain mutants YkAPPA-L162G/A and YkAPPA- Mutant gene of E230G/A/S/T/D/P/R.
  • the PCR reaction parameters were: 95 ° C for 5 min; 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30-90 sec, 30 cycles; 72 ° C for 10 min.
  • the mutation uses 20 PCR primers, including the upstream and downstream primers Yk-F and Yk-R, which amplify the complete coding sequence of the mutant gene, and the upstream and downstream primers L162G-F, L162G-R, L162A-F, L162A which introduce mutation at a specific position.
  • the primer sequences are as follows:
  • L162G-F 5'-cgggggtatgtaaa ggc gacccagagaaaac-3'
  • L162G-R 5'-gttttctctgggtc gcc tttacatacccccg-3'
  • L162A-F 5'-cgggggtatgtaaa gcg gacccagagaaaac-3'
  • L162A-R 5'-gttttctctgggtc cgc tttacatacccccg-3'
  • E230G-F 5'-tcgaggtaaataaa ggc gggacaaaagtctc-3'
  • E230G-R 5'-gagacttttgtccc gcc tttatttacctcga-3'
  • E230A-F 5'-tcgaggtaaataaa gcg gggacaaaagtctc-3'
  • E230A-R 5'-gagacttttgtccc cgc tttatttacctcga-3'
  • E230S-F 5'-tcgaggtaaataaa tct gggacaaaagtctc-3'
  • E230S-R 5'-gagacttttgtccc aga ttatttacctcga-3'
  • E230T-F 5'-tcgaggtaaataaa acc gggacaaaagtctc-3'
  • E230T-R 5'-gagacttttgtccc ggt tttatttacctcga-3'
  • E230D-F 5'-tcgaggtaaataaa gat gggacaaaagtctc-3'
  • E230D-R 5'-gagacttttgtccc atc tttatttacctcga-3'
  • E230P-F 5'-tcgaggtaaataaa ccg gggacaaaagtctc-3'
  • E230P-R 5'-gagacttttgtccc cgg tttatttacctcga-3'
  • E230R-F 5'-tcgaggtaaataaa cgt gggacaaaagtctc-3'
  • E230R-R 5'-gagacttttgtccc acg tttatttacctcga-3 '
  • the italics in the primers represent the restriction enzyme sites EcoR I and Not I, and the underlined nucleotide sequence is the mutation site.
  • the mutated gene product was recovered by over-PCR amplification and ligated to the pEASY-T3 vector and confirmed by sequencing.
  • 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 the phytase activity of the mutant of the present invention
  • the activity unit (U) of a phytase defines the amount of enzyme required to release 1 ⁇ mol of inorganic phosphorus per minute under certain conditions.
  • 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 ratio of protease to phytase is between 1/1000 and 1/1.
  • the phytase activity was measured using the ferrous sulfate molybdenum blue method.
  • the 9 mutant enzymes of the present invention can maintain the residual enzyme activity higher than that of the wild enzyme under different pepsin concentrations, and the experimental results show that the pepsin resistance of the mutant enzyme is obviously improved.
  • 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 8 mutant phytase have an optimum pH (pH 4.5) similar to that of the wild enzyme except that the optimum pH of the mutant YkAPPA-E230R is shifted downward by 0.5 pH units.
  • the stability of the mutant enzymes YkAPPA-E230G/A/R and YkAPPA-L162G/A under acid treatment was significantly higher than that of wild-type enzyme.
  • the mutant enzymes YkAPPA-E230G/A/R and YkAPPA- L162G/A retains more than 85% of enzyme activity, while wild enzymes only retain 64% of enzyme activity.
  • the tolerance of YkAPPA-E230P/S/T/D to acid is basically the same as that of wild enzyme.
  • the reaction rate (V max ) and conversion rate (K cat ) of the mutant enzyme YkAPPA-E230G increased by 1.9 times that of the wild enzyme, and the catalytic efficiency (K cat /k m ) was 2.1 times higher than that of the wild enzyme.
  • the reaction rate, conversion rate and catalytic efficiency of the mutant enzyme YkAPPA-L162G increased by 1.6-1.8 times compared with the wild enzyme.
  • the reaction rate, conversion rate and catalytic efficiency of the mutant enzyme YkAPPA-E230A were lower than that of the wild enzyme (about 1.3 times).
  • the catalytic properties of other mutant enzymes are basically consistent with wild enzymes.
  • Example 3 Determination of the phytase activity of the mutant of the present invention

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Abstract

本发明涉及基因工程领域，具体涉及植酸酶突变体胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA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、丙氨酸、脯氨酸、精氨酸、丝氨酸、苏氨酸或天冬氨酸。植酸酶突变体胃蛋白酶抗性明显提高，且催化效率增加高达2.1倍，有利于发展节约型饲料酶工业。

Description

胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体 技术领域
本发明涉及基因工程领域，具体涉及植酸酶突变体胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体。
背景技术
植酸酶(myo-inositol hexakisphosphate phosphohydrolase)是一类重要的工业酶，降解植酸中磷酸单酯键生成无机磷和低级磷酸肌醇的一类酶的总称。目前大多数植酸酶的蛋白酶抗性较差，造成磷的巨大浪费，饲料成本增加和环境污染。抗蛋白酶且不降低催化效率的植酸酶能够降低动物生产成本，具有良好的经济效益和生态效益。
随着蛋白结构和分子生物学的发展，运用定向进化和理性设计的手段对酶分子进行人工改造已是当前酶工程领域研究的热点。
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，提供植酸酶突变体胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体。
本发明另一目的是提供编码上述胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA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改良和催化效率提高的植酸酶YkAPPA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改良和催化效率提高的植酸酶YkAPPA突变体基因的重组菌株。
本发明对克氏耶尔森氏菌(Yersinia kristensenii)来源植酸酶YkAPPA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，采用定点突变的方法，获得了提高耐酸性和胃蛋白酶抗性的突变体，分别命名为YkAPPA-L162G/A和YkAPPA-E230G/A/S/T/D/P/R，即YkAPPA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、丙氨酸、丝氨酸、苏氨酸、天冬氨酸、脯 氨酸或精氨酸。
因此根据本发明的胃蛋白酶抗性提高的植酸酶突变体YkAPPA-L162G，其氨基酸序列如SEQ ID NO.3所示。
突变体YkAPPA-L162A的氨基酸序列如SEQ ID NO.4所示。
突变体YkAPPA-E230G的氨基酸序列如SEQ ID NO.5所示。
突变体YkAPPA-E230A的氨基酸序列如SEQ ID NO.6所示。
突变体YkAPPA-E230S的氨基酸序列如SEQ ID NO.7所示。
突变体YkAPPA-E230T的氨基酸序列如SEQ ID NO.8所示。
突变体YkAPPA-E230D的氨基酸序列如SEQ ID NO.9所示。
突变体YkAPPA-E230P的氨基酸序列如SEQ ID NO.10所示。
突变体YkAPPA-E230R的氨基酸序列如SEQ ID NO.11所示。
本发明还提供了编码上述耐酸性和胃蛋白酶抗性提高的植酸酶突变体YkAPPA-L162G的基因序列，其核苷酸序列如SEQ ID NO.12所示。
突变体YkAPPA-L162A的基因序列如SEQ ID NO.13所示。
突变体YkAPPA-E230G的基因序列如SEQ ID NO.14所示。
突变体YkAPPA-E230A的基因序列如SEQ ID NO.15所示。
突变体YkAPPA-E230S的基因序列如SEQ ID NO.16所示。
突变体YkAPPA-E230T的基因序列如SEQ ID NO.17所示。
突变体YkAPPA-E230D的基因序列如SEQ ID NO.18所示。
突变体YkAPPA-E230P的基因序列如SEQ ID NO.19所示。
突变体YkAPPA-E230R的基因序列如SEQ ID NO.20所示。
将上述编码耐酸性和胃蛋白酶抗性提高的植酸酶突变体基因的阅读框插入到所述载体的EcoRI和NotI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(+)，得到突变体的重组原核表达质粒。本发明优选的宿主菌为BL21(DE3)。所述植酸酶突变体的核苷酸序列位于T7-lac启动子的下游并受其调控。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YkAPPA-L162G/A和YkAPPA-E230G/A/S/T/D/P/R的胃蛋白酶抗性明显提高，且YkAPPA-L162G和YkAPPA-E230G/A的催化效率增加高达2.1倍，有利于发展节约型饲料酶工业。
附图说明
图1为改造前、后的植酸酶的蛋白酶抗性比较；
图2为改造前、后的植酸酶的水解能力比较。
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)。
2、酶类及其它生化试剂：DNA纯化试剂盒，和LADNA聚合酶购自TaKaRa，内切酶和连接酶，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
以来源于克氏耶尔森氏菌(Y.kristensenii)植酸酶YkAPPA的基因序列(SEQ ID NO.2)进行改造，采用Overlap PCR方法引入突变，获得突变体YkAPPA-L162G/A和YkAPPA-E230G/A/S/T/D/P/R的突变基因。PCR反应参数为：95℃ 5min；94℃ 30 sec,55℃ 30 sec,72℃ 30-90 sec,30个循环；72℃ 10min。突变使用20条PCR引物，包括扩增突变基因完整编码序列的上下游引物Yk-F和Yk-R，及在特定位置引入突变的上下游引物L162G-F、L162G-R、L162A-F、L162A-R、E230G-F、E230G-R、E230A-F、E230A-R、E230S-F、E230S-R、E230T-F、E230T-R、E230D-F、E230D-R、E230P-F、E230P-R、E230R-F和E230R-R。引物序列如下：
Yk-F:5’-cgcgaattcgcaccgcttgcagcacaatctac-3’
Yk-R:5’-gatgcggccgcttaaatatggcaggctggctcg-3’
L162G-F:5’-cgggggtatgtaaa ggcgacccagagaaaac-3’
L162G-R:5’-gttttctctgggtc gcctttacatacccccg-3’
L162A-F:5’-cgggggtatgtaaa gcggacccagagaaaac-3’
L162A-R:5’-gttttctctgggtc cgctttacatacccccg-3’
E230G-F:5’-tcgaggtaaataaa ggcgggacaaaagtctc-3’
E230G-R:5’-gagacttttgtccc gcctttatttacctcga-3’
E230A-F:5’-tcgaggtaaataaa gcggggacaaaagtctc-3’
E230A-R:5’-gagacttttgtccc cgctttatttacctcga-3’
E230S-F:5’-tcgaggtaaataaa tctgggacaaaagtctc-3’
E230S-R:5’-gagacttttgtccc agattatttacctcga-3’
E230T-F:5’-tcgaggtaaataaa accgggacaaaagtctc-3’
E230T-R:5’-gagacttttgtccc ggttttatttacctcga-3’
E230D-F:5’-tcgaggtaaataaa gatgggacaaaagtctc-3’
E230D-R:5’-gagacttttgtccc atctttatttacctcga-3’
E230P-F：5’-tcgaggtaaataaa ccggggacaaaagtctc-3’
E230P-R：5’-gagacttttgtccc cggtttatttacctcga-3’
E230R-F：5’-tcgaggtaaataaa cgtgggacaaaagtctc-3’
E230R-R：5’-gagacttttgtccc acgtttatttacctcga-3’
引物中斜体代表限制性酶切位点EcoR I和Not I，下划线标记的核苷酸序列为突变位点。通过over-PCR扩增得到突变基因产物回收后连接到pEASY-T3载体上并经测序证实。
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活力的测定
1个植酸酶的活性单位(U)定义：在一定条件下，每分钟释放出1μmol无机磷所需的酶量。
突变植酸酶的蛋白酶抗性的测定
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。蛋白酶和植酸酶比例在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950ul1.5mmol/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。对照是在加酶液之前先加入TCA混匀使酶变性，其它相同。根据显色后700nm下的光吸收值计算植酸酶活性。结果(图1中的A和B)表明，突变酶较原酶的胃蛋白酶抗性均得到了明显提高。在1/1000至1/20的胃蛋白浓度下具有较小侧链的突变酶YkAPPA-E230G、YkAPPA-E230A、YkAPPA-L162G、YkAPPA-L162A、YkAPPA-E230S、YkAPPA-E230D和YkAPPA-E230T分别剩 余83％、76％、50％、42％、34％、12％和12％以上的酶活，具有较强刚性侧链的突变酶YkAPPA-E230P和YkAPPA-E230R在1/1000至1/20的胃蛋白浓度下分别剩余64％和49％以上的酶活。而当胃蛋白酶浓度从1/1000升至1/20时，野生酶YkAPPA几乎完全丧失酶活力。本发明的9个突变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，该实验结果显示突变酶的胃蛋白酶抗性确有明显提高。
突变植酸酶的pH和温度活性模式的测定
纯化的突变体植酸酶在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YkAPPA-E230R的最适pH向下移动了0.5个pH值单位，其它8个突变植酸酶具有与野生酶类似的最适pH值(pH 4.5)。突变酶YkAPPA-E230G/A/R和YkAPPA-L162G/A在酸处理下的稳定性明显高于野生酶，在pH 1.0-1.5下处理1h时，突变酶YkAPPA-E230G/A/R和YkAPPA-L162G/A可保持85％以上酶活，而野生酶仅保持64％酶活。YkAPPA-E230P/S/T/D对酸的耐受性与野生酶基本一致。
表1 pH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 PCTCN2018072549-appb-000001
突变植酸酶k m值、V max、K cat及催化效率(K cat/k m)的测定
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Km值及Vmax，再根据酶的理论分子量求出kcat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YkAPPA-E230G的反应速度(V max)和转换率(K cat)提高达野生酶的1.9倍，其催化效率(K cat/k m)较野生酶提高了2.1倍。突变酶YkAPPA-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YkAPPA-E230A的反应速度、转换率和催化效率较野生酶增加了较低水平(约1.3倍)。而其它突变酶的催化性质(包括反应速度、周转率和催化效率)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 PCTCN2018072549-appb-000002
实施例3：本发明所述的突变植酸酶活力的测定
用不同pH(1.0-5.5)和不同的胃蛋白浓度(胃蛋白酶与植酸酶的比例为1/1和1/100)的条件来模拟动物的肠胃环境，以玉米粉为底物，检测突变植酸酶YkAPPA-E230G的水解能力。结果(图2)表明，突变植酸酶YkAPPA-E230G与野生酶YkAPPA在pH 4.5下水解玉米粉能释放最多的无机磷；且突变植酸酶YkAPPA-E230G在pH 4.5下释放的无机磷高于野生酶，不加胃蛋白酶时，突变植酸酶YkAPPA-E230G在pH 4.5下释放的磷酸是野生酶的2倍，添加1/0和1/1的胃蛋白酶后，突变植酸酶YkAPPA-E230G在pH 4.5下释放的无机磷较野生酶分别提高达11倍和24倍。

Claims (8)

  1. 胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体，其特征在于，所述突变体的氨基酸序列为氨基酸序列如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. 胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体，其特征在于，所述突变体的氨基酸序列为氨基酸序列如SEQ ID NO.1所示的植酸酶的第230位谷氨酸突变为甘氨酸、丙氨酸、脯氨酸、精氨酸、丝氨酸、苏氨酸或天冬氨酸。
  3. 胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体基因，其特征在于，编码权利要求1或2所述的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体。
  4. 根据权利要求3所述的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19或SEQ ID NO.20所示。
  5. 包含权利要求3所述的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体基因的重组载体。
  6. 包含权利要求3所述的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体基因的重组菌株。
  7. 一种制备胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA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)培养重组菌株，诱导表达胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体；以及
    3)回收并纯化所表达的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体。
  8. 权利要求1或2所述的胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体基因的应用。
PCT/CN2018/072549 2017-01-15 2018-01-15 胃蛋白抗性改良和催化效率提高的植酸酶YkAPPA突变体 WO2018130212A2 (zh)

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