大肠杆菌K12植酸酶热稳定性突变体在毕赤酵母中的高效表达和性质研究

摘要

植酸酶广泛用作工业饲料添加剂.通过基因优化和两步法基因合成了E.coli K12植酸酶的耐热突变体AppA NR,将其克隆到毕赤酵母表达载体pHBM905A上,然后将构建好的质粒转入毕赤酵母GSll5中表达.经过筛选得到了一株高表达毕赤酵母工程茵GS115/AppA NR.产量达到10 mg,mL-l,活力为980 U.mL-1.该重组酶在95℃加热10 min,仍保留90％的活力,相比野生型植酸酶.更能抵抗高温.同时.该酶具有较宽的pH值范围和抗胃酶水解的能力.所有这些性质的改善表明该重组酶比较适合用作工业饲料添加剂.%Phytase is widely used as a feed additive in industry.Here by introducing a mutant AppA NR derived from Escherichia coli K12 and optimizing its codon usage based on the preferred codons of Pichia pastoris,the gene was synthesized by two-step PCR,and then cloned into the expression vector pHBM905A.A high-level expression recombinant P.pastoris GSll5/AppA NR was screened,with the yield of 10 mg · mL-1 and the activity of 980 U · mL-1.The recombinant phytase was heated at 95 ℃ for 10 min,and still remained 90％ of its activity,which was more thermostable than the wild type,AppA.Meanwhile,the enzyme exhibited broad pH value stability from 1.5 to 8.0 and was resistant to pepsin.All these improvements made it possible to be used as a feed additive in the feed industry.