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

Abstract: This study aimed to explore the effect of N-glycosylation on enzymatic characteristics of hyperthermoacidophilic α-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 α-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 ApkAdsD182N/G373S is ideal for starch liquefication.

Key words: hyperthermoacidophilic α-amylase, Pichia pastoris GS115, N-glycosylation, secretion expression