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 α-Amylase ApkA

作　　者：曾静[1] 郭建军[1] 袁林[1] ZENG Jing;GUO Jianjun;YUAN Lin(Institute of Microbiology, Jiangxi Academy of Sciences, Nanchang 330096, China)

机构地区：[1]江西省科学院微生物研究所,江西南昌330096

出　　处：《食品科学》2017年第6期48-54,共7页Food Science

基　　金：国家自然科学基金青年科学基金项目(31501422);江西省青年科学基金项目(20151BAB214001);江西省科学院资助项目(2014-YYB-08;2014-XTPH1-08)

摘　　要：为探索N-糖基化修饰对极端嗜热酸性α-淀粉酶Apk A酶学性质的影响,同时为构建酵母工程菌奠定基础,将Apk A缺失信号肽突变体Apk Ads及含有2个潜在N-糖基化修饰位点的突变体Apk Ads D182N/G373S在毕赤酵母(Pichia pastoris)GS115中进行表达。Apk Ads和Apk Ads D182N/G373S在Pichia pastoris GS115中大量表达并分泌到胞外,Apk Ads的表观分子质量约为45 k D,Apk Ads D182N/G373S的表观分子质量约为55 k D。酶学性质分析表明,与Apk Ads相比,Apk Ads D182N/G373S的酶学性质发生了一定的变化。其最适反应p H值由6.5降低至5.5~6.0,酸性条件下稳定性增强;最适反应温度由90℃提高至100℃;于90℃的半衰期由5 h增加至5.5 h,于100℃保温10 min后的相对酶活力由32.03%增加至49.04%。结果表明N-糖基化修饰可适当提高Apk A的酸性条件下酶活力和稳定性、最适反应温度、热稳定性。突变体Apk Ads D182N/G373S的酶学性质使其适于淀粉液化工艺的应用。This study aimed to explore the effect of N-glycosylation on enzymatic characteristics of hyperthermoacidophilicα-amylase ApkA for the purpose of establishing the basis of the development of genetically engineered yeast.Based on theamino 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.Therecombinantα-amylases ApkAds and ApkAdsD182N/G373S were expressed at high levels and secreted into the culturemedium.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis showed that the molecularweights of ApkAds and ApkAdsD182N/G373S were about45and55kD,respectively.Compared with ApkAds,the mutantApkAdsD182N/G373S showed optimal pH of5.5–6.0instead of6.5.The mutant ApkAdsD182N/G373S was more stableunder acidic conditions.Its optimal temperature was100℃compared with90℃for ApkAds.When incubated at90℃,ApkAds and ApkAdsD182N/G373S exhibited half-lives of5and5.5h,respectively.After incubated at100℃for10min,the residual activities of ApkAds and ApkAdsD182N/G373S were32.03%and49.04%,respectively.These results suggestthat N-glycosylation moderately increases enzymatic activity and stability under acidic conditions,optimal temperature,andthermostability of ApkA,and the mutant ApkAdsD182N/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 

分 类 号：Q814[生物学—生物工程]