鉴定隶属于糖苷水解酶13家族(glycoside hydrolase family 13, GH13)的潮滩发光杆菌的β-半乳糖苷酶

Identification of a novel GH13 β-galactosidase from Photobacterium gaetbulicola

摘要:

【背景】糖苷水解酶13家族(glycoside hydrolase family 13, GH13)是已知最大的α-淀粉酶家族，不含有半乳糖苷酶。【目的】对海洋细菌潮滩发光杆菌(Photobacterium gaetbulicola)的一个蛋白BgalPg进行鉴定。【方法】通过保守位点分析和系统发育树确定BgalPg蛋白的家族分类；通过克隆、表达和纯化测定重组BgalPg蛋白的酶学性质并鉴定功能。【结果】BgalPg的蛋白序列新颖，与已知的碳水化物酶无同源性。序列分析结果表明该蛋白具有GH13家族的典型特性，并且隶属于GH13_38亚家族。BgalPg对α-淀粉酶家族酶的相关底物均无催化活性，却能水解含有β-半乳糖苷键的底物pNP-β-Gal [(2.8±0.4) U/mg]和oNP-β-Gal[(1.4±0.3) U/mg]，并且能水解乳糖[(0.40±0.01) U/mg]，表现出典型的β-半乳糖苷酶活性。同时，该酶在pH 7.0-8.5稳定性好，60 ℃的半衰期为1.5 h。【结论】发现隶属于GH13家族的β-半乳糖苷酶。

Abstract:

[Background] Glycoside hydrolase family 13 (GH13), known as the largest α-amylase family, does not contain galactosidases. [Objective] To identify a protein BgalPg from the marine bacterium Photobacterium gaetbulicola. [Methods] The family of BgalPg was identified by conserved motif analysis and a phylogenetic analysis. The BgalPg gene was cloned and expressed in Escherichia coli. The enzymatic properties of purified BgalPg were determined. [Results] BgalPg possessing the conserved motifs of GH13 family is a new member of the GH13_38 subfamily, although it showed low sequence identity with characterized α-amylase family proteins. BgalPg demonstrated no catalytic activity against any substrates of the α-amylase family, while it had the activities towards pNP-β-d-galactopyranoside [pNP-β-Gal, (2.8±0.4) U/mg] and oNP-β-d-galactopyranoside [oNP-β-Gal, (1.4±0.3) U/mg]. Moreover, the enzyme hydrolyzed lactose [(0.40±0.01) U/mg], which represented a typical β-galactosidase activity. BgalPg exhibited high stability in pH 7.0–8.5 and the half-life time of 1.5 h at 60 ℃. [Conclusion] This is the first time that an enzyme of GH13 family was biochemically defined as a novel β-galactosidase.