WO2016019834A1 - 植酸酶变体 - Google Patents

植酸酶变体 Download PDF

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WO2016019834A1
WO2016019834A1 PCT/CN2015/085748 CN2015085748W WO2016019834A1 WO 2016019834 A1 WO2016019834 A1 WO 2016019834A1 CN 2015085748 W CN2015085748 W CN 2015085748W WO 2016019834 A1 WO2016019834 A1 WO 2016019834A1
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phytase
phya
km71h
amino acid
variant
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李峰
李凤梅
王忆平
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北京大学
众望百奥生物科技（北京）有限公司
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    • C12R2001/84—Pichia

Definitions

  • the present invention relates to phytase variants, an amino acid sequence encoding a phytase variant and a nucleotide sequence encoding a phytase and uses thereof.
  • Phytic acid also known as phytic acid, contains six phosphate groups with abundant phosphorus.
  • Phytic acid is an important storage form of phosphorus in feed.
  • Phytase (EC 3.1.3.8), a phytate hydrolase, catalyzes the hydrolysis of phytic acid and phytate to inositol and phosphoric acid.
  • the phytase derived from Aspergillus niger has good heat resistance and is resistant to the above short-term high temperature process. But due to the source of Aspergillus The specific activity of phytase is lower than that of bacterial-derived phytase. Therefore, the specific activity of phytase derived from Aspergillus niger is further increased to enhance fermentation activity, which is a research hotspot for feed phytase. one. At the same time, further improvement of heat resistance and reduction of enzyme activity loss during granulation is also an important research direction of feed phytase.
  • the invention modifies the amino acid sequence of the phytase derived from Aspergillus niger by a method of gene mutation, so that the modified phytase has an improved specific activity or heat resistance.
  • the expression vector and the strain were constructed to efficiently express the phytase after transformation, and finally reached the requirements of industrial production.
  • the invention adopts the mutation screening of phytase by Aspergillus niger phytase PHYA.
  • the phytase-derived PHYA gene derived from Aspergillus niger has a full length of 1401 bp (GeneBank number: P34752) and encodes 467 amino acids (see, for example, SEQ ID NO: 1).
  • the 19 amino acids at the N-terminus are signal peptides, and the Aspergillus niger phytase from which the signal peptide is removed can be secreted and expressed by Pichia pastoris to obtain a mature protein having phytase activity.
  • the phytase variant of the invention has an amino acid modification selected from at least one of the following positions relative to the amino acid sequence SEQ ID No: 1 of the wild type phytase: position 51, 84, 86, 91, 126, 165, 202, 211, and 300.
  • the amino acid modification is selected from the group consisting of: Y51S, T (Y51S, T indicates that amino acid residue Y at position 51 is preferably replaced by S or T, the same applies hereinafter); A84R, S, G; Y86P, G, W; K91N, T, V, A, G, S, M; D126E, N; R165K, W; E202D, Q; D211K; and K300E, G, L, I, V, M.
  • the invention also relates to conservative substitution variants of the above phytase variants.
  • the conservative substitution variant retains increased specific activity and/or heat resistance of the phytase variants of the invention. It will be apparent to those skilled in the art that such substitutions can occur in regions other than the above sites while still retaining the corresponding activity.
  • the conservative substitution variant has an amino acid conservative substitution of at least one position.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine).
  • basic amino acids such as arginine, lysine, and histidine
  • acidic amino acids such as glutamic acid and aspartic acid
  • polar amino acids Such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (
  • the invention also relates to a phytase variant having a certain amino acid homology to the amino acid sequence of a phytase variant described above, eg, at least about 80% homology, more preferably at least about 81% homology.
  • it is at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, more preferably at least about 86%, more preferably at least about 87%, more preferably at least about 88%, More preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, and most preferably at least about 95%, most Preferably at least about 96%, most preferably at least about 97%, most preferably at least about 98%, and most preferably at least about 99%, as long as the variant having the homology retains the increased specific activity of the phytase variant of the invention And / or heat resistance.
  • retaining increased specific activity and/or heat resistance of a phytase variant of the invention means retaining at least about 20% of the specific activity and/or heat resistance of a phytase variant of the invention. Preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, most Preferably 100%.
  • the invention also provides a method for increasing specific activity or heat resistance, which comprises: introducing a wild type phytase or an amino acid sequence similar to the wild type phytase with more than 90% amino acid sequence of phytase Or multiple amino acid mutations to increase specific activity or temperature tolerance. For example, at positions 51, 84, 86, 91, 126, 165, 202, 211, and 300 of phytase.
  • Y51S, T indicates that the amino acid residue Y at the 51st position is preferably replaced by S or T, the same applies hereinafter).
  • the wild-type phytase encoding gene has the nucleotide sequence set forth in SEQ ID No: 2, which does not include the signal peptide sequence of the wild-type phytase.
  • the nucleotide sequence is designed according to the expression host yeast preferred codon, which effectively increases the expression efficiency of the gene. This sequence encodes a mature phytase after the initiation codon or in the correct reading frame of the protein expression system. All site mutations are preferably made on the basis of the codon-optimized gene sequences described above.
  • the invention provides a method of making an improved phytase, the method comprising: Synthesis or cloning of the coding gene, construction of the recombinant vector, transformation of the recipient cell, screening of the mutant strain, and large-scale fermentation expression of the optimal expression strain.
  • the recombinant strain is Pichia pastoris strain KM71H.
  • the high-copy transformants were screened with a high concentration of zeocin plates, and the selected transformants were subjected to shake flask and fermentation experiments, and finally the high expression strains were identified by screening.
  • the optimized and improved phytase of the invention has great application potential in the feed and food industries.
  • Figure 1 shows the recombinant vector pPHYA (i.e., pPICZ ⁇ -phyA) containing a phytase variant of the invention.
  • Figure 2 shows the amino acid sequence of SEQ ID NO: 1 derived from the wild type phytase of Aspergillus niger.
  • the parental signal peptide sequence of phytase in parentheses is not included in the scope of the present invention, and the letter box is a mutation site.
  • Figure 3 shows a codon-optimized nucleotide sequence encoding the phytase amino acid sequence of SEQ ID No: 1, starting from amino acid 20 and containing no signal peptide sequence.
  • E. coli strain Top10 Pichia pastoris KM71H, vector pPICZ ⁇ were purchased from Invitrogen.
  • PCR enzyme, plasmid extraction, gel purification kit was purchased from Tiangen, restriction endonuclease and ligase were purchased from Thermo, and the antibiotics were purchased from Invitrogen.
  • the point mutation kit was purchased from Quanjin Company.
  • the E. coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LB-zeromycin
  • Yeast medium is YPD (1% yeast extract, 2% peptone, 2% glucose), yeast transformation medium is YPDS zeromycin (1% yeast extract, 2% peptone, 2% glucose, 100ug/ml zero mold) Prime).
  • the yeast screening medium was YPD-nomycin (YPD + 100-5000 ug/ml zirconia).
  • Yeast medium BMGY 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (V/V)
  • modified induction medium BMMY divided by 2% methanol instead of glycerol, the rest of the ingredients The same as BMGY.
  • the whole gene sequence of SEQ ID NO: 2 was synthesized according to the optimal codon of Pichia pastoris, and the whole gene synthesis service was provided by Beijing Yingmao Shengye.
  • the primers at both ends of the gene contained EcoRI and NotI restriction sites, respectively. And cloned on the TA vector.
  • the obtained bands were purified by gel electrophoresis using EcoRI and NotI.
  • the amplified fragment was cloned into the EcoRI and NotI sites of pPICZ ⁇ , and the resulting recombinant vector was named pPHYA (i.e., pPICZ ⁇ -phyA). Sequencing determines the correctness of its amino acid sequence.
  • Example 1 introduction of site-directed mutagenesis and transformation of yeast KM71H on the basis of the synthesized phyA gene
  • amino acid residues 51, 84, 86, 91, 126, 165, 202, 211, and 300 of the amino acid sequence have important functions, and site-directed mutagenesis is performed on the above sites.
  • the mutation was based on the pPHYA plasmid and was run using a full-scale gold site site-directed mutagenesis kit.
  • the primers used are listed below:
  • TOP10 was transformed using a full-point gold-based site-directed mutagenesis kit, and an LB-zero-mycin plate was coated. After the transformants were correctly sequenced, LB culture and extraction of 5 ug or more plasmids with plasmid extraction kit were linearized with SacI, and the fragments were purified and electroporated to transform yeast KM71H, and YPDS-zero-mycin plates were coated, and 10 transformants were collected for each transformation. Test tube screening. At the same time, KM71H was transformed with pPHYA as a standard control and named KM71H-PHYA.
  • yeast transformants of all 51, 84, 86, 91, 126, 165, 202, 211, and 300 site mutants are established, preferably containing Y51S, T; A84R, S, G; Y86P, G, W. ; K91N, T, V, A, G, S, M; D126E, N; R165K, W; E202D, Q; D211K; K300E, G, L, I, V mutant yeast transformants.
  • Two or more mutation site combinations can be obtained after multiple rounds of mutation.
  • Yeast transformants containing different phyA mutations were inserted into 250 containing 15 ml BMGY medium In a ml shake flask, incubate at 250 ° C for 30 h at 30 ° C, then add 15 ml of modified induction BMMY medium and continue to culture for 24 h. The bacterial cells and the supernatant were collected separately, and the supernatant was tested for phytase activity according to the national standard GB/T 18634-2002 of the People's Republic of China. The activity of KM71H-PHYA was 100%, and the preferred phytase variants were relative. The enzyme activity is shown in the table below.
  • mutant mutation Relative enzyme activity (%) KM71H-PHYA —— 100 KM71H-PHYA-Y51S Y51S 122 KM71H-PHYA-Y51T Y51T 167 KM71H-PHYA-A84R A84R 118 KM71H-PHYA-A84S A84S 127 KM71H-PHYA-A84G A84G 115 KM71H-PHYA-Y86P Y86P 143 KM71H-PHYA-Y86G Y86G 108 KM71H-PHYA-Y86W Y86W 149 KM71H-PHYA-K91N K91N 133 KM71H-PHYA-K91T K91T 142 KM71H-PHYA-K91V K91V 142 KM71H-PHYA-K91A K91A 134 KM71H-PHYA-K91G K91G 127 KM71H-PHYA-K91S K91S 144 KM71H-
  • the mutant yeast transformant having an increased enzyme activity obtained in Example 3 was selected for representative heat resistance identification.
  • the shake flask culture supernatant was diluted to 2 U/ml with 0.2 M pH 5.5 acetate buffer, one portion was incubated in a 85 ° C water bath for 5 min, immediately placed on ice, and the other directly placed on ice as a control.
  • the phytase activity was measured using the national standard GB/T 18634-2002, and the enzyme-diluted solution without heat treatment was normalized to 100%, and the residual enzyme activity ratio was measured. This method can reflect the heat resistance of different mutants.

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Abstract

提供了黑曲霉植酸酶PHYA的变体，其与野生型氨基酸序列SEQ ID NO: 1相比，具有在选自以下的至少一个位点的氨基酸修饰：位点51、84、86、91、126、165、202、211和300。还提供了编码该植酸酶变体的核苷酸序列以及该植酸酶变体的应用。

Description

植酸酶变体
本申请要求申请号为201410381678.9，发明名称为“植酸酶变体”的中国专利申请的优先权。
技术领域
本发明涉及植酸酶变体，编码植酸酶变体的氨基酸序列和编码植酸酶的核苷酸序列及其应用。
背景技术
在谷物和豆类中，磷主要以植酸或植酸盐(Phytate)的形式储存。植酸又称肌醇六磷酸，含有6个磷酸基团，带有丰富的磷，植酸是饲料中磷的重要贮存形式。植酸酶(EC3.1.3.8)即肌醇六磷酸水解酶，催化植酸及植酸盐水解成肌醇与磷酸。
磷是动物生长的必需元素。单胃动物如猪、家禽和鱼不能直接吸收植酸盐，导致大量植物来源的磷元素排放于环境中，造成环境富营养化。同时，动物吸收磷酸盐不足会影响生长，为了补偿这些动物的磷酸盐的缺乏，需在饲料中补充磷酸盐，增加了养殖成本并加剧了对环境的不利影响。通过在动物饲料中使用植酸酶，水解了植酸盐，可改善动物对磷的吸收，并降低磷排放。因此，植酸酶具备一举两得的功效，已在动物饲料中普遍应用，具有十分重大的经济价值。
自然界中植酸酶的来源多种多样。通过基因工程手段，特别是DNA重组技术的应用，使各种微生物来源的植酸酶大规模廉价生产和实际应用成为可能。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细菌的植酸酶两种。其中来源于黑曲霉的植酸酶PHYA具有高耐热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。
在颗粒饲料生产过程中有一个短暂的80-90℃的造粒过程。黑曲霉来源的植酸酶耐热性良好，能够抵抗住上述短时高温过程。但由于黑曲霉来源 的植酸酶比活性较低，相比细菌来源的植酸酶比活性相差较大，因此进一步提高黑曲霉来源的植酸酶的比活性从而提升发酵活力，是饲用植酸酶的研究热点之一。同时，进一步提升耐热性，降低造粒时的酶活损失，也是饲用植酸酶的重要研究方向。
发明内容
本发明通过基因突变的方法对黑曲霉来源植酸酶的氨基酸序列进行改造，使改造后的植酸酶具有提高的比活性或者耐热性能。构建表达载体和菌株，使改造后的植酸酶高效表达，最终达到工业化生产的要求。
本发明是以黑曲霉植酸酶PHYA为起始植酸酶进行突变筛选。来源于黑曲霉的植酸酶PHYA基因全长1401bp(GeneBank号码:P34752)，编码467个氨基酸(参见例如SEQ ID NO:1中所示)。N端的19个氨基酸为信号肽，去除信号肽的黑曲霉植酸酶可以利用毕赤酵母进行分泌表达获得具有植酸酶活性的成熟蛋白。
在一个实施方案中，本发明的植酸酶变体，相对于野生型植酸酶的氨基酸序列SEQ ID No:1而言，具有选自以下的至少一个位点的氨基酸修饰：位点51、84、86、91、126、165、202、211、和300。优选地，所述的氨基酸修饰选自：Y51S,T(Y51S,T表示第51位的氨基酸残基Y优选S或T进行替换，以下同)；A84R,S,G；Y86P,G,W；K91N,T,V,A,G,S,M；D126E,N；R165K,W；E202D,Q；D211K；和K300E,G,L,I,V,M。
在一个实施方案中，本发明还涉及上述植酸酶变体的保守取代变体。优选地，所述的保守取代变体保留本发明植酸酶变体的提高的比活性和/或耐热性。对于本领域的技术人员而言很明显，这种取代可以在上述位点以外的区域发生，而仍保留相应活性。优选地，所述保守取代变体具有至少一个位置的氨基酸保守取代。保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。最常见的氨基酸互换有氨基酸G至A；A至G,S；V至I,L,A,T,S；I至V,L,M；L至l,M,V；M至L,I,V；P至A,S,N；F 至Y,W,H；Y至F,W,H；W至Y,F,H；R至K,E,D；K至R,E,D；H至Q,N,S；D至N,E,K,R,Q；E至Q,D,K,R,N；S至T,A；T至S,V,A；C至S,T,A；N至D,Q,H,S；Q至E,N,H,K,R的互换，以及它们的相反的互换。
在一个实施方案中，本发明还涉及与上述植酸酶变体的氨基酸序列具有一定的氨基酸同源性的植酸酶变体，例如，同源性至少约80％，更优选至少约81％，更优选至少约82％，更优选至少约83％，更优选至少约84％，更优选至少约85％，更优选至少约86％，更优选至少约87％，更优选至少约88％，更优选至少约89％，更优选至少约90％，更优选至少约91％，更优选至少约92％，更优选至少约93％，更优选至少约94％，最优选至少约95％，最优选至少约96％，最优选至少约97％，最优选至少约98％，最优选至少约99％，只要具有所述同源性的变体保留本发明植酸酶变体的提高的比活性和/或耐热性。
在本文中，“保留本发明植酸酶变体的提高的比活性和/或耐热性”是指，保留本发明植酸酶变体的比活性和/或耐热性的至少约20％，优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％，更优选至少约90％，最优选100％。
本发明同时提供了一种提高比活性或者耐热性的方法，该方法包括：在野生型植酸酶或与野生型植酸酶氨基酸序列相似性超过90％以上植酸酶氨基酸序列中引入一个或多个氨基酸突变来提高比活性或者耐温性。例如，在植酸酶的第51、84、86、91、126、165、202、211、300位。优选Y51S,T；A84R,S,G；Y86P,G,W；K91N,T,V,A,G,S,M；D126E,N；R165K,W；E202D,Q；D211K；K300E,G,L,I,V(Y51S,T表示第51位的氨基酸残基Y优选S或T进行替换，以下同)。
另一方面，本发明提供了本发明所述植酸酶的编码基因。
在一个优选实施方案中，所述野生型植酸酶的编码基因具有SEQ ID No:2所示的核苷酸序列，其不包括野生型植酸酶的信号肽序列。该核苷酸序列根据表达宿主酵母偏爱密码子设计，有效提高该基因的表达效率。该序列在起始密码子后或蛋白表达系统正确阅读框内编码成熟的植酸酶。所有的位点突变优选在上述密码子优化后的基因序列基础上进行。
另一方面，本发明提供了一种改良的植酸酶的制备方法，该方法包括： 编码基因的合成或克隆，重组载体的构建，受体细胞的转化，突变菌株的筛选，及最佳表达菌株大规模发酵表达。
作为本发明的一个最优选的实施方案，为了使植酸酶基因在毕赤酵母中高效表达，我们将黑曲霉PHYA原有的信号肽序列去除，使改造后植酸酶基因插入到带有alpha因子信号肽序列的酵母表达载体pPICZαA上的Eco R I和Not I限制性酶切位点之间。经转化酵母细胞，稳定整合到酵母染色体上。优选重组菌株是毕赤酵母菌株KM71H。用高浓度的零霉素(zeocin)平板筛选高拷贝的转化子，将筛选到的转化子进行摇瓶及发酵实验，最终筛选确定高表达菌株。
本发明的优化改良的植酸酶在饲料、食品行业具有巨大的应用潜力。
除另有说明外，本申请中的所有科技术语的都具有与本发明所属领域普通技术人员通常理解相同的含义。尽管与本申请中描述类似或等同的方法及材料都可用于实施或检验本发明，但是下文仍还是对合适的方法和材料进行了描述。本申请中引用的全部出版物、专利申请、专利及其他参考文献其全部内容在此引入作为参考。如有抵触，包括定义，以本申请为准。
附图说明
图1显示含本发明所述植酸酶变体的重组载体pPHYA(即pPICZα-phyA)。
图2显示来源于黑曲霉的野生型植酸酶的氨基酸序列SEQ ID NO:1。括号内为植酸酶的原始信号肽序列，不含在本发明范围之内，字母带框的为突变位点。
图3显示编码植酸酶氨基酸序列SEQ ID No:1的经密码子优化的核苷酸序列，所述植酸酶从第20位氨基酸开始，不含信号肽序列。
具体实施方式
下列实施例旨在进一步举例说明实现本发明的具体方式，而决不构成对本发明的限制。本领域技术人员应该理解的是，在不违背本发明的精神和原则的前提下，对本发明进行改动得到的技术方案都将落入本发明的待批权利 要求范围内。
实施例
实验材料和试剂：
1、菌株与载体
大肠杆菌菌株Top10、毕赤酵母KM71H、载体pPICZα，均购自Invitrogen公司。
2、酶与试剂盒
PCR酶，质粒提取，胶纯化试剂盒购自天根，限制性内切酶、连接酶购自Thermo，零霉素抗生素均购自Invitrogen公司，点突变试剂盒购自全式金公司。
3、培养基
大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LB-零霉素
为LB培养基加50ug/mL零霉素。
酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)，酵母转化培养基为YPDS零霉素(1％酵母提取物，2％蛋白胨，2％葡萄糖，100ug/ml零霉素)。酵母筛选培养基为YPD零霉素(YPD+100-5000ug/ml零霉素)。
酵母培养基BMGY(1％酵母提取物、2％蛋白胨、1.34％YNB、0.00004％生物素、1％甘油(V/V))和改良诱导培养基BMMY(除以2％甲醇代替甘油，其余成份相与BMGY相同)。
实施例1
黑曲霉来源的野生型phyA基因(不含信号肽)的密码子优化及全基因合成
根据毕赤酵母最适密码子合成SEQ ID NO:2的全基因序列，由北京英茂盛业提供全基因合成服务，基因两端引物分别含有EcoRI和NotI酶切位点， 并克隆在TA载体上。用EcoRI和NotI双酶切，凝胶电泳纯化获得的条带。将扩增得到的片段克隆到pPICZα的EcoRI和NotI位点，得到的重组载体命名为pPHYA(即pPICZα-phyA)。测序确定其氨基酸序列的正确性。
实施例2
在实施例1中在合成的phyA基因基础上引入定点突变并转化酵母KM71H
根据结构信息，本发明认为氨基酸序列的第51、84、86、91、126、165、202、211、300位氨基酸残基具有重要功能，对上述位点实行定点突变。优选Y51S,T；A84R,S,G；Y86P,G,W；K91N,T,V,A,G,S,M；D126E,N；R165K,W；E202D,Q；D211K；K300E,G,L,I,V进行突变。该突变基于pPHYA质粒，使用全式金公司的定点突变试剂盒进行操作，所使用的引物列表如下：
引物名称 5’-3’序列
Y51S-for TCCCACTTGTGGGGTCAATCTGCCCCATTCTTCTCTTTG
Y51T-for TCCCACTTGTGGGGTCAAACTGCCCCATTCTTCTCTTTG
Y51-rev TTGACCCCACAAGTGGGAGGTC
A84R-for GTTTTGTCTAGACACGGAAGAAGATACCCTACTGACTCC
A84S-for GTTTTGTCTAGACACGGATCCAGATACCCTACTGACTCC
A84G-for GTTTTGTCTAGACACGGAGGTAGATACCCTACTGACTCC
A84-rev TCCGTGTCTAGACAAAACTTG
Y86P-for TCTAGACACGGAGCTAGACCACCTACTGACTCCAAGGGT
Y86G-for TCTAGACACGGAGCTAGAGGTCCTACTGACTCCAAGGGT
Y86W-for TCTAGACACGGAGCTAGATGGCCTACTGACTCCAAGGGT
Y86-rev TCTAGCTCCGTGTCTAGACAAAAC
R165K-for AGAAGTTCCGGTTCCTCTAAGGTGATTGCTTCCGGTAAG
R165W-for AGAAGTTCCGGTTCCTCTTGGGTGATTGCTTCCGGTAAG
R165-rev AGAGGAACCGGAACTTCTGATG
K91A-for AGATACCCTACTGACTCCGCTGGTAAGAAATACTCCGCTC
K91G-for AGATACCCTACTGACTCCGGTGGTAAGAAATACTCCGCTC
K91M-for AGATACCCTACTGACTCCATGGGTAAGAAATACTCCGCTC
K91N-for AGATACCCTACTGACTCCAACGGTAAGAAATACTCCGCTC
K91S-for AGATACCCTACTGACTCCTCTGGTAAGAAATACTCCGCTC
K91T-for AGATACCCTACTGACTCCACCGGTAAGAAATACTCCGCTC
K91V-for AGATACCCTACTGACTCCGTCGGTAAGAAATACTCCGCTC
K91-rev GGAGTCAGTAGGGTATCTAGC
D126E-for AACTACTCTTTGGGAGCCGAAGACTTGACTCCATTCGGTG
D126N-for AACTACTCTTTGGGAGCCAACGACTTGACTCCATTCGGTG
D126-rev GGCTCCCAAAGAGTAGTTGTAG
E202D-for ATCGACGTAGTCATCTCTGACGCTTCCTCCTCTAACAAC
E202Q-for ATCGACGTAGTCATCTCTCAAGCTTCCTCCTCTAACAAC
E202-rev AGAGATGACTACGTCGATCTTTG
D211K-for TCCTCTAACAACACCTTGAAGCCTGGTACTTGTACCGTC
D211-rev CAAGGTGTTGTTAGAGGAGGAAG
K300E-for GACTACTTGCAGTCCTTGGAGAAGTACTACGGTCACGGTGCC
K300G-for GACTACTTGCAGTCCTTGGGTAAGTACTACGGTCACGGTGCC
K300I-for GACTACTTGCAGTCCTTGATCAAGTACTACGGTCACGGTGCC
K300L-for GACTACTTGCAGTCCTTGTTGAAGTACTACGGTCACGGTGCC
K300M-for GACTACTTGCAGTCCTTGATGAAGTACTACGGTCACGGTGCC
K300V-for GACTACTTGCAGTCCTTGGTCAAGTACTACGGTCACGGTGCC
k300-rev CAAGGACTGCAAGTAGTCGTAG
以pPHYA为模板，使用全式金公司的定点突变试剂盒，转化TOP10，涂布LB-零霉素平板。转化子测序正确后，用LB培养并用质粒提取试剂盒提取5ug以上质粒用SacI线性化，片段纯化后电击转化酵母KM71H，涂布YPDS-零霉素平板，每个转化收集10个转化子用于试管筛选。同时用pPHYA转化KM71H作为标准对照，命名为KM71H-PHYA。按照此方法，建立全部51、84、86、91、126、165、202、211、300位点突变体的酵母转化子，优选建立含有Y51S,T；A84R,S,G；Y86P,G,W；K91N,T,V,A,G,S,M；D126E,N；R165K,W；E202D,Q；D211K；K300E,G,L,I,V突变的酵母转化子。
可以经过多轮突变，获得两个及两个以上的突变位点组合。
实施例3
包含不同phyA突变的酵母转化子的摇瓶表达以及酶活性提高的转化子的测定
包含不同phyA突变的酵母转化子接入含有15ml BMGY培养基的250 ml摇瓶中，30℃摇床250rpm培养24h，再加入15ml改良诱导BMMY培养基，继续培养24h。分别收集菌体和上清，上清根据中华人民共和国国家标准《GB/T 18634-2002》检测植酸酶活性，以KM71H-PHYA的活性归一为100％，优选的植酸酶变体相对酶活如下表所示。
突变体 突变 相对酶活(％)
KM71H-PHYA —— 100
KM71H-PHYA-Y51S Y51S 122
KM71H-PHYA-Y51T Y51T 167
KM71H-PHYA-A84R A84R 118
KM71H-PHYA-A84S A84S 127
KM71H-PHYA-A84G A84G 115
KM71H-PHYA-Y86P Y86P 143
KM71H-PHYA-Y86G Y86G 108
KM71H-PHYA-Y86W Y86W 149
KM71H-PHYA-K91N K91N 133
KM71H-PHYA-K91T K91T 142
KM71H-PHYA-K91V K91V 142
KM71H-PHYA-K91A K91A 134
KM71H-PHYA-K91G K91G 127
KM71H-PHYA-K91S K91S 144
KM71H-PHYA-K91M K91M 135
KM71H-PHYA-D126E D126E 116
KM71H-PHYA-D126N D126N 161
KM71H-PHYA-R165K R165K 121
KM71H-PHYA-R165W R165W 147
KM71H-PHYA-E202D E202D 107
KM71H-PHYA-E202Q E202Q 123
KM71H-PHYA-D211K D211K 157
KM71H-PHYA-K300E K300E 138
KM71H-PHYA-K300G K300G 129
KM71H-PHYA-K300L K300L 133
KM71H-PHYA-K300I K300I 130
KM71H-PHYA-K300V K300V 133
KM71H-PHYA-K300M K300M 139
KM71H-PHYA-K91R.K300V K91R K300V 142
实施例4
耐热性提高的酵母转化子的筛选
由于耐热性也是植酸酶的一个重要指标，如果酶活提高但是耐热性降低了，那么其应用价值将大打折扣，因此找到酶活性和耐热性均提高的突变子。在实施例3中获得的酶活性提高的突变体酵母转化子，选取其中有代表性的进行耐热性鉴定。将摇瓶培养的上清用0.2M pH5.5的乙酸缓冲液稀释至2U/ml，一份置于85℃水浴中温育5min，立即置冰，另一份直接置冰作为对照。用国家标准《GB/T 18634-2002》检测植酸酶活性，以未经热处理的酶稀释液归一为100％，测得残留酶活比例。该法即可反映不同突变体的耐热性。
突变体 突变 残留酶活(％)
KM71H-PHYA —— 61
KM71H-PHYA-Y51S Y51S 77
KM71H-PHYA-Y51T Y51T 81
KM71H-PHYA-A84R A84R 61
KM71H-PHYA-A84S A84S 59
KM71H-PHYA-A84G A84G 60
KM71H-PHYA-Y86P Y86P 43
KM71H-PHYA-Y86G Y86G 70
KM71H-PHYA-Y86W Y86W 68
KM71H-PHYA-K91N K91N 66
KM71H-PHYA-K91T K91T 80
KM71H-PHYA-K91V K91V 79
KM71H-PHYA-K91A K91A 70
KM71H-PHYA-K91G K91G 65
KM71H-PHYA-K91S K91S 84
KM71H-PHYA-K91M K91M 81
KM71H-PHYA-D126E D126E 72
KM71H-PHYA-D126N D126N 63
KM71H-PHYA-R165K R165K 65
KM71H-PHYA-R165W R165W 57
KM71H-PHYA-E202D E202D 63
KM71H-PHYA-E202Q E202Q 85
KM71H-PHYA-D211K D211K 77
KM71H-PHYA-K300E K300E 71
KM71H-PHYA-K300G K300G 59
KM71H-PHYA-K300L K300L 76
KM71H-PHYA-K300I K300I 73
KM71H-PHYA-K300V K300V 68
KM71H-PHYA-K300M K300M 75
KM71H-PHYA-K91R.K300V K91R K300V 72

Claims (9)

  1. 植酸酶变体，其与野生型氨基酸序列SEQ ID No:1相比，具有在选自以下的至少一个位点的氨基酸修饰：位点51、84、86、91、126、165、202、211、和300。
  2. 根据权利要求1的植酸酶变体，相对于SEQ ID No:1的氨基酸序列，所述植酸酶变体具有选自以下的至少一个修饰：Y51S,T；A84R,S,G；Y86P,G,W；K91N,T,V,A,G,S,M；D126E,N；R165K,W；E202D,Q；D211K；和K300E,G,L,I,V,M。
  3. 根据权利要求1或2的植酸酶变体，其进一步在其它位点具有保守取代。
  4. 根据前面权利要求1-3任一项的植酸酶变体，比SEQ ID No:1所示的植酸酶有增加的酶活性或者增加的热稳定性。
  5. 核苷酸序列，编码根据权利要求1-3任一项的植酸酶。
  6. 重组表达载体，其包含根据权利要求5的核苷酸序列。
  7. 重组宿主细胞，其包含根据权利要求5的核苷酸序列或者根据权利要求6的载体。
  8. 动物饲料添加剂，其包含根据权利要求1-3任一项的植酸酶中的至少一种。
  9. 动物饲料，其包含根据权利要求1-3任一项的植酸酶中的至少一种。
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