N224糖基化对纤维酵母菌R64 α-淀粉酶活性和稳定性的影响

Q4 Pharmacology, Toxicology and Pharmaceutics
Yovin Sugijo, T. D. Rosahdi, Fernita Puspasari, W. Ismaya, Khomaini Hasan, Ihsanawati, T. Subroto, D. Natalia
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引用次数: 0

摘要

纤维酵母R64(SfamyR64)的α-淀粉酶的氨基酸序列包含两个假定的N-连接糖基化位点N153和N224。N224假设负责淀粉底物的结合,因为它在SfamyR64同源物中高度保守。测试N224是否在酶活性和稳定性中起关键作用。N224Q取代是通过定点诱变引入的。野生型和突变体在毕赤酵母KM71中独立地过量产生。比较了野生型和突变体的活性,并使用热处理评估了它们的热稳定性。研究了SfamyR64与其具有不同糖基化模式的结构同源物的进化关系。N224Q突变体的活性比野生型低约80%。突变体在50°C预孵育10分钟后没有表现出活性,而野生型SfamyR64在处理30分钟前表现出活性。Sfamy似乎比其结构同源物进化得更早。SfamyR64N224对酶活性和热稳定性至关重要。这种糖基化位点对于真菌和细菌α-淀粉酶是独特的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability
The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. To test whether N224 plays a key role in enzyme activity and stability. N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.
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来源期刊
Current Enzyme Inhibition
Current Enzyme Inhibition Pharmacology, Toxicology and Pharmaceutics-Drug Discovery
CiteScore
1.30
自引率
0.00%
发文量
30
期刊介绍: Current Enzyme Inhibition aims to publish all the latest and outstanding developments in enzyme inhibition studies with regards to the mechanisms of inhibitory processes of enzymes, recognition of active sites, and the discovery of agonists and antagonists, leading to the design and development of new drugs of significant therapeutic value. Each issue contains a series of timely, in-depth reviews written by leaders in the field, covering a range of enzymes that can be exploited for drug development. Current Enzyme Inhibition is an essential journal for every pharmaceutical and medicinal chemist who wishes to have up-to-date knowledge about each and every development in the study of enzyme inhibition.
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