具有不寻常 SGNH 基序的鞘氨醇 GDS(L)-like 水解酶的特征。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Miriam A Fingerhut, Lea Henrich, Christiane Lauber, Niklas Broel, Parviz Ghezellou, Dominik Karrer, Bernhard Spengler, Kim Langfelder, Timo Stressler, Holger Zorn, Martin Gand
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引用次数: 0

摘要

利用毛霉菌异源表达了来自基生真菌区系(Basidiomycota Pleurotus sapidus)的 GDS(L)-like 脂肪酶(PSA_Lip),其活性为 350 U L-1。等电点聚焦法测定其等电点为 5.0。新型 PSA_Lip 与之前表征的 GDSL 类酶(属于碳水化合物酯酶家族 16)中的磷脂酶、植物脂肪酶、乙酰胆碱酯酶和乙酰木糖酯酶分别只有 23.8%-25.1%、25.5%、26.6% 和 28.4%的相同性。因此,从培养上清液中纯化了该酶,并使用不同的检测方法研究了该酶的催化特性和底物特异性,以揭示其潜在功能。虽然没有检测到磷脂酶、乙酰胆碱酯酶和乙酰木聚糖酯酶活性,但对阿魏酸甲酯(约 8.3%)和阿魏酰化碳水化合物 5-O-反式阿魏酰-阿拉伯呋喃糖(约 0.8%)的水解研究表明,这些底物的转化率较低。通过研究不同链长的对硝基苯(pNP)酯的水解活性,在 65 °C、pH 值为 8 时,中等链长的对硝基苯辛酸酯的活性最高,而对硝基苯己酸酯几乎没有活性。该酶在 pH 值为 10、温度为 4 ℃ 的条件下储存至少 7 天,具有很高的稳定性。此外,通过共识序列分析和同源建模,我们可以证明 PSA_Lip 的活性位点不含通常存在于 GDS(L)-like 酶中的 SGNH 残基。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif.

Characterization of a GDS(L)-like hydrolase from Pleurotus sapidus with an unusual SGNH motif.

The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L-1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8-25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.

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来源期刊
AMB Express
AMB Express BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
7.20
自引率
2.70%
发文量
141
审稿时长
13 weeks
期刊介绍: AMB Express is a high quality journal that brings together research in the area of Applied and Industrial Microbiology with a particular interest in ''White Biotechnology'' and ''Red Biotechnology''. The emphasis is on processes employing microorganisms, eukaryotic cell cultures or enzymes for the biosynthesis, transformation and degradation of compounds. This includes fine and bulk chemicals, polymeric compounds and enzymes or other proteins. Downstream processes are also considered. Integrated processes combining biochemical and chemical processes are also published.
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