Based on the codon bias of Pichia pastoris,the gene encoding a thermoacidophilic α-amylase BD5088 was modified for the optimal expression in P.pastoris.The gene was cloned into the vector of pPIC9K to produce the recombinant vector pPIC9K-BD5088 and then transformed into chromosome of P.pastoris GS115 strain.Regulated by the α-factor,promoter of AOX1 gene and termination signal of yeast genomic,the recombinant α-amylase PrBD5088 was expressed and secreted into the culture with a max yield of 200 mg/L after 5 days induction.Comparing to the recombinant amylase ErBD5088 expressed by E.coli,the optimum temperature of PrBD5088 increased from 80℃ to 90℃.Although the optimum pH of PrBD5088 was same to ErBD5088,but PrBD5088 have more broad stable range of pH than ErBD5088.More than 50% of its maximal activity was retained in the pH 4.0~8.0 of PrBD5088 and pH 5.0~7.0 of ErBD5088.The PrBD5088 is so thermostable that over 80 % of its activities was retained after disposed at 100℃ for 1 h.But the half-life of ErBD5088 at 100℃,pH5.6 was only 20 min.High concentration of Zn2＋ and Ca2＋ slightly increased its activity,while Cu2＋ partially inhibited its activity at high concentration.SDS-PAGE result showed the molecular weight of PrBD5088 was about 56 ku,which was bigger than the calculated molecular mass 49 ku of ErBD5088.PrBD5088 is not modified by N-linked glycosylation but by O-linked glycosylation.The O-linked glycosylation had influence on thermostability,the optimal temperature and optimal pH range of PrBD5088.%把密码子优化后的超耐热酸性α-淀粉酶的基因BD5088,引入载体pPIC9K中,将正确构建的重组质粒pPIC9K-BD5088转化毕赤酵母GS115细胞,得到酵母工程菌株。在酵母α-Factor及AOX1基因启动子和终止信号的调控下,重组超耐热酸性α-淀粉酶PrBD5088在甲醇酵母中大量表达并分泌到胞外。该酶受甲醇的严格调控和诱导,在诱导培养5 d后酶活力达到最大值,表达量可达到约200 mg/L。与原核表达产物ErBD5088相比,PrBD5088的酶学性质发生了一定的改变。其最适反应温度由80℃增加到90℃。最适反应pH值仍为pH5.6,但范围更宽,在pH值5.0～6.5之间,其相对酶活在90%以上,在pH4.0～8.0范围内酶活性保留50%以上。而ErBD5088则只在pH5.0～7.0范围内能维持活性的50%以上。且PrBD5088的温度稳定性远高于ErBD5088。PrBD5088在100℃条件下热处理1 h仍具有80%以上的酶活力,而ErBD5088相同条件下的半衰期只有20 min。此外,Ca2＋和Zn2＋对维持rBD5088活性和稳定性会产生轻微促进作用,该酶的这些优点使其非常适于在工业生产上应用。SDS-PAGE测得该酶的分子量为56 ku,高于原核表达的酶蛋白。重组α-淀粉酶PrBD5088不存在N-连接的糖基化,但是存在O-连接的糖基化现象。本实验结果表明O-连接糖基化可适当提高PrBD5088的热稳定性、最适温度和酸性pH条件下酶活性。
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