N-糖基化改造提高Aspergillus fumigatus生淀粉糖化酶的催化效率

Improving the catalytic efficiency of Aspergillus fumigatus glucoamylase by N-glycosylation modification

生淀粉糖化酶(raw starch glucoamylase,RSGA)可以直接作用于未糊化淀粉颗粒。该研究基于Aspergillus fumigatus来源RSGA氨基酸序列分析发现,Asn121、Asn422和Asn610位点均为Asn-Xaa-Ser形式的糖基化基序,具有将其突变为Asn-Xaa-Thr形式糖基化基序的潜力。首先,利用NetNGlyc 1.0 Server评估突变序列N-糖基化的可能性,并获得突变体S123T(Asn121-Pro122-Thr123)、S424T(Asn422-Gly423-Thr424)和S612T(Asn610-Arg611-Thr612)。突变体在Komagataella phaffii中表达并完成糖基化,利用去糖基化酶EndoH处理进一步验证突变体均成功被糖基化。在此基础上,以生玉米淀粉为底物,在40℃,pH 4.6的条件下分析了突变体S123T、S424T和S612T的催化效率(k cat/K m),分别是RSGA的1.31、1.29和1.15倍,这主要是由于K m值降低导致突变体对底物的亲和力增加造成的。此外,蛋白浓度及十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)分析显示突变体S123T表达量提高了9.3%,且糖基化效果显著。酶学性质分析结果显示,重组酶的最适反应温度从70℃降低到60℃。研究结果表明,N-糖基化基序改造是一种有效的提高RSGA的催化效率的策略。

Raw starch glucoamylase(RSGA)can degrade raw starch granules directly.In this study,based on amino acid sequence analysis of Aspergillus fumigatus RSGA,it was found that the Asn121,Asn422 and Asn610 sites are all glycosylated motifs in the Asn-Xaa-Ser form.All of them have the potential to be mutated to the Asn-Xaa-Thr form of glycosylation motifs.First,the possibility of N-glycosylation for the mutant sequences were assessed using NetNGlyc 1.0 Server and mutants S123T(Asn121-Pro122-Thr123),S424T(Asn422-Gly423-Thr424),and S612T(Asn610-Arg611-Thr612)were obtained.The mutants were expressed in Komagataella phaffii and completed glycosylation,which was further verified by treatment with the deglycosylase EndoH.On this basis,the catalytic efficiency(k cat/K m)of mutants S123T,S424T,and S612T were analyzed at 40℃and pH 4.6,using raw corn starch as substrate,and increased by 1.31,1.29,and 1.15-fold compared to RSGA,respectively.This was caused by a decrease in K m value and an increase in the affinity of the mutants for the substrate.In addition,protein concentration and SDS-PAGE analysis showed a 9.3%increase in expression and a significant glycosylation effect of mutant S123T.The results of enzymatic property analysis showed that the optimum reaction temperature of the recombinant enzyme was reduced from 70℃to 60℃.These results show that N-glycosylation modification is an effective strategy to improve the catalytic efficiency of enzymes.