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 GH13β-galactosidase from Photobacterium gaetbulicola

【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。

[Background]Glycoside hydrolase family 13(GH13),known as the largestα-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 characterizedα-amylase family proteins.BgalPg demonstrated no catalytic activity against any substrates of theα-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 typicalβ-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 novelβ-galactosidase.