食品科学2017，Vol.38Issue(6)：48-54,7.DOI:10.7506/spkx1002-6630-201706008
N-糖基化修饰对极端嗜热酸性α-淀粉酶ApkA酶学性质的影响
Effect of N-Glycosylation on Enzymatic Characteristics of Hyperthermoacidophilic a-Amylase ApkA
摘要
Abstract
This study aimed to explore the effect of N-glycosylation on enzymatic characteristics of hyperthermoacidophilic a-amylase ApkA for the purpose of establishing the basis of the development of genetically engineered yeast.Based on the amino acid sequence analysis of ApkA,a signal peptide deleted mutant ApkAds and a double site mutant ApkAdsD182N/ G373S containing two potential N-glycosylation sites were constructed and expressed in Pichia pastoris GS115.The recombinant a-amylases ApkAds and ApkAdsD182N/G373S were expressed at high levels and secreted into the culture medium.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weights of ApkAds and ApkAdsD182N/G373S were about 45 and 55 kD,respectively.Compared with ApkAds,the mutant ApkAdsD182N/G373S showed optimal pH of 5.5-6.0 instead of 6.5.The mutant ApkAdsD182N/G373S was more stable under acidic conditions.Its optimal temperature was 100 ℃ compared with 90 ℃ for ApkAds.When incubated at 90 ℃,ApkAds and ApkAdsD182N/G373S exhibited half-lives of 5 and 5.5 h,respectively.After incubated at 100 ℃ for 10 min,the residual activities of ApkAds and ApkAdsD182N/G373S were 32.03％ and 49.04％,respectively.These results suggest that N-glycosylation moderately increases enzymatic activity and stability under acidic conditions,optimal temperature,and thermostability of ApkA,and the mutant ApkAdsD 182N/G373S is ideal for starch liquefication.关键词
极端嗜热酸性α-淀粉酶/毕赤酵母GS115/N-糖基化修饰/分泌表达Key words
hyperthermoacidophilic a-amylase/Pichia pastoris GS115/N-glycosylation/secretion expression分类
生物科学引用本文复制引用
曾静,郭建军,袁林..N-糖基化修饰对极端嗜热酸性α-淀粉酶ApkA酶学性质的影响[J].食品科学,2017,38(6):48-54,7.基金项目
国家自然科学基金青年科学基金项目(31501422) （31501422）
江西省青年科学基金项目(20151BAB214001) （20151BAB214001）
江西省科学院资助项目(2014-YYB-08 （）
2014-XTPH1-08) （）
