植酸酶phyA^m基因结构延伸突变改善酶的热稳定性

Improving Thermostability of Aspergillus niger Phytase by Elongation Mutation

将来源于黑曲霉N25的植酸酶基因phyAm重组于大肠杆菌表达载体pET30b(+),以重组表达载体pET30bFphyAm为模板经PCR扩增获得结构延伸突变植酸酶基因phyAe(在植酸酶基因C端增加了来源于pET30bFphyAm载体上13氨基酸残基)。含突变基因的重组表达载体pPIC9kphyAe在GS115酵母中表达。纯化的突变酶PPNPe与野生型酶PPNPm8相比:PPNPAe的最适反应温度上升了3℃,75℃处理10min,热稳定性提高21%,比活力略有提高。最适反应pH为5.6,有效pH范围pH4.6到pH6.6。比未突变酶扩大了0.4单位。

The phytase gene phyAz from Aspergillus niger N25 was recombined into E. coli expression vector pET-30b（＋）. Recombined at expression vectors pET30b-FphyA^m was served as a template to amplify phytase gene, and the PCR product named elongation mutation gene phyA^e was expanded with a 13 amino acid sequence from pET-30b-FphyA^m vector at C-terminal of phytase gene phyA^m. Furthermore, phyA^e gene was recombined into expression vector pPIC9k and expressed in Pichia pastoris. The comparison experiment of mutant phytase PP-NP^e with wild-type phytase PP-NP^e-8 showed that： the optimum temperature of PP-NPe was increased by 3℃, and its thermostability was increased by 21% when it was exposed to 10min at 75℃. Its effective reaction pH range with catalysis efficiency above 70% was pH4.6-pH6.6, and wider 0.4 pH value than that of wild-type phytase.