摘要
植酸酶是一种能将植物性饲料中植酸(盐)降解为肌醇和无机磷酸盐的酶类,其作为单胃动物的饲料添加剂,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作用。但目前酶活力和稳定性较低是植酸酶在生产运用中的一个瓶颈,因此通过基因工程和蛋白质工程技术对植酸酶进行改造具有重要的科学和实用价值。定向进化技术是在体外模拟达尔文自然进化过程,该技术已成为改造酶分子的一种有效策略,在农业、工业和医药等领域都展现了其巨大的潜力。
定向进化技术改造植酸酶基因的过程中的一个关键是构建高通量的筛选方法。本研究在常规钼钒法测定植酸酶活性的基础上,进行微板法酶活微量测定研究,并在摇瓶培养基因工程菌的基础上进行微板上的微量培养研究,构建起一套针对植酸酶酶活力改造的高通量筛选方法。结果表明,酶活微量测定法和常规钼钒测定法的稳定性基本一致;无机磷的最低检出值前者为0.67μg/mL,后者为0.30μg/mL,两种方法灵敏度均较高;精确度前者为8.8%,后者为8.2%,两种方法的精确度差异不显著。无机磷浓度测定的线性范围前者为0.67μg/mL-48.14μg/mL,后者为0.30μg/mL-25.50μg/mL,微板法测定的线性范围略大于常规钼钒法;测定值的变异系数前者为12.1%,后者为10.5%,两种方法的变异系数差异不显著。并且微板法还可以大大提高工作效率、降低实验成本。微量培养所得样品酶液的酶活力低于用三角瓶进行的摇瓶培养发酵所得样品酶液的酶活力,但是在高通量的初步筛选中,本研究关注的是突变菌株与原始出发菌株的比较,因此这种微量培养方法是可行的。
本研究采用定向进化技术中较为成熟的一种方法——易错PCR对植酸酶基因进行改造。通过优化,易错PCR体系中Mn~(2+)浓度为0.1mmol/L,Mg~(2+)浓度为7mmol/L,dATP,dGTP浓度与dCTP,dTTP浓度比例为1:5。将易错PCR产物构建入pPIC9K载体质粒中,经线性化后转化毕赤酵母工程菌GS115,形成突变体库。利用高通量筛选方法进行筛选,获得了一株较高活性的产植酸酶突变基因工程菌PP-NP~(ep)-6A,其酶活力达到82034U/mL,较出发菌株PP-NP~m-8(63667U/mL)提高了29%。该酶的最适反应温度为55℃,最适反应pH为5.6。经DNA测序分析表明PP-NP~(ep)-6A中突变植酸酶基因发生了9个碱基突变,并引起了4个氨基酸的变化,分别是Glu156Gly,Thr236Ala,Gln396Arg,Leu406Thr。经蛋白质二级结构预测发现,突变基因工程菌PP-NP~(ep)-6A表达的植酸酶二级结构与未突变一致,理化性质和等电点无明显变化。通过同源建模预测三维结构发现,突变菌株PP-NP~(ep)-6A表达的植酸酶与未突变具有相同的多肽骨架,三维结构一致,活性中心未见改变。而四个氨基酸突变可能使酶蛋白结构发生了微变化,增加了酶蛋白结构柔性,从而引起酶活力提高。
本研究利用易错PCR的方法,在毕赤酵母表达载体pPIC9K上构建了突变体库,并筛选出酶活提高的突变株,为进一步通过定向进化改造植酸酶奠定了基础。
Phytases belong to the family of histidine acid phosphatases which catalyze the release of phosphate from phytic acid. As a kind of feed additive phytases have been added to the feed to meet the phosphorus requirements of some monogastric animals like poultry, pigs and it play an important part in reducing phosphorous contamination as well. But at present, the activity of phytase still need to improve to meet the needs. Directed evolution mimics the process of Darwinian evolution in a test tube, and it's has become a effective strategies to engineer enzymes for the requirements of industrial, medical and research application.
The building of effective high-throughput screening method is the key factor of success for directed evolution of phytase. This research stablished a high-throughput screening method for phytase engineering. On the basis of general Molybdenum-Vanadium method for detecting phytase activity, the microplate method was developed for the determination of trace activity of phytase, and the microculture method was developed for culture of phytase on the basic of flask culture. The results shows that both of the microplate method and Molybdenum-Vanadium methods had similar accuracy、sensitivity and stability, and the microplate method can greatly improve the efficiency and lower the cost of experiments. The samples gained from microculture had enzyme activity and their activity were lower than the flask culture samples,but what we focused is the enzyme activity comparion between mutant strain and original strain in preliminary high-throughput screening, so the microplate method is feasible.
This research engineed the activity of phytase with ep-PCR which is a mature technology of directed evolution. After optimization,the concentration of Mn~(2+) is 01.mmol/L and the concentration of Mg~(2+) is 7mmol/L in ep-PCR reaction system. Structured ep-PCR products into plasmid vector pPIC9K and transformed the recombinant palsmid into Pichia pastris GS115 through electroporation after linearization. After high-throughput screening we acquired a mutant strain PP-NP~(ep)-6A (82034U/mL) which has 29% higher phytase enzyme activity than original strain PP-NP~m-8 (63667U/mL) . And the optimum temperature for catalytic reaction of mutant phytase is 55℃,the optimum pH is 5.6. After DNA sequencing we found that 9 bases have changed in mutant sequence,and the change result in 4 amino acid change which were Glu156Gly. Thr236Ala, Gln396Arg, Leu406Thr. After protein secondary structure prediction we found that mutant phytase has the similar physicochemical property and isoelectric point compared with original phytase. After protein three dimensional structure prediction we found that mutant phytase has the same backbone and 3D structure with orignal phytase,and the active centre has no change. The mutation of 4 amino acid may lead to micro-variation on protein structure and increase the flexibility of enzyme constitution, that may result in the increasing of enzyme activity.
This research acquired a mutant strain which has higher enzyme activity through ep-PCR,that will laid the foundation for further studiy on directed evolution of phytase.
引文
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