多形拟杆菌α-L-鼠李糖苷酶序列分析与酶学性质  

Sequence Analysis and Characterization of Two Novelα-L-Rhamnosidases from Bacteroides thetaiotaomicron VPI-5482

作　　者：李彬春[1] 吉亚茹 李艳琴[1] 丁国斌[1] 

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006

出　　处：《中国生物化学与分子生物学报》2017年第4期391-399,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31400684);山西省基础研究计划项目(No.2014021030-3);山西大学引进人才建设项目(No.206533801001)资助~~

摘　　要：α-L-鼠李糖苷酶在生物技术领域具有很好的生物催化应用前景。目前,仅16个细菌源GH78家族α-L-鼠李糖苷酶基因被报道,对α-L-鼠李糖苷酶的序列特征与分子催化机制了解尚不完善。人体肠道细菌多形拟杆菌可利用多种多糖与糖苷,其基因组包括6个预测的α-L-鼠李糖苷酶基因,本文集中于连续定位于一个基因座的2个α-L-鼠李糖苷酶基因BtRha78D和BtRha78E,通过研究二者的序列特征和酶学特性旨在确定此基因座的生物学意义。序列分析显示,BtRha78D和BtRha78E的序列组成与长度差异性大,与已发现的α-L-鼠李糖苷酶序列一致性较低,分子进化关系也较远;酶学性质研究表明,BtRha78D和BtRha78E可高效水解底物p NPR,最适p H分别为p H6.0与p H 7.0。BtRha78E在p H 7.5~8.0范围内,仍能保持52%以上的活性。BtRha78D和BtRha78E最适温度为50℃,对pNPR的kcat分别为14.78 s-1和5.48 s-1,Km分别为0.14 mmol/L和0.44 mmol/L,kcat/Km分别为105 600 s-1L/mol和12 500 s-1L/mol。BtRha78D和BtRha78E耐受低浓度(1%)有机溶剂,10%浓度显著降低酶活性,对二甲亚砜具有很好的耐受性,10%浓度下仍能分别保持42%和53%的活性。本研究揭示,α-L-鼠李糖苷酶BtRha78D和BtRha78E间的序列组成与长度差异性很大,具有相同的催化活性而酶学性质却不同,一个包含2个α-L-鼠李糖苷酶基因的基因座可能参与多形拟杆菌降解利用含α-L-鼠李糖基的多糖与糖苷化合物。α-L-Rhamnosidase has good potential for biocatalytic application in biotechnology. At present only 16 bacterial α-L-rhamnosidase genes belonging to glycoside hydrolase family 78 （GH78） have been reported. Sequence information and catalytic mechanism for GH78 α-L-rhamnosidase have been incompletely investigated. Genome mining showed that there were six predicted α-L-rhamnosidase genes in the genome of human gut bacteria Bacteroides thetaiotaomicron VPI-5482. Here we focused on the genetic locus containing two sequential α-L-rhamnosidase genes BtRha78D and BtRha78E to prospect biological function of the genetic locus by exploring the sequence features and enzymatic properties of two α-L-rhamnosidases. Sequence analysis suggested that amino acid composition and residuenumber of BtRha78D and BtRha78E were different. Sequence identities of BtRha78D and BtRha78E with cloned bacterial α-L-rhamnosidases were low, and BtRha78D and BtRha78E were distinct from cloned bacterial α-L-rhamnosidases. BtRha78D and BtRha78E could efficiently hydrolyze the substrate pNPR. Optimum pH of BtRha78D and BtRha78E was pH 6.0 and pH 7.0, respectively. BtRha78E maintained the catalytic activity of more than 52% in pH 7.5 - 8.0. Optimum temperature of BtRha78D and BtRha78E was 50 ℃.koat of BtRha78D and BtRha78E on pNPR were 14.78 s^-1 and 5.48 s^-1, Km were 0.14 mmol/L and 0.44 mmol/L, and koat/Km were 105 600 s^-1 L/mol and 12 500 s^-1 L/tool, respectively. BtRha78D and BtRha78E could tolerate various organic solvents at low concentration （1%）. Most organic solvents significantly lowered catalytic activity at the concentration of 10%. However, BtRha78D and BtRha78E were well tolerant of dimethyl sulfoxide （DMSO） , and remained catalytic activity of 42% and 53% at the concentration of 10% , respectively. This study revealed that amino acid compositions and residuenumber of BtRha78D and BtRha78E were different, and they displayed same catalytic activity and different enzymatic properties. Thus, the genetic locus which co

关 键 词：多形拟杆菌 α-L-鼠李糖苷酶 糖苷水解酶家族78 基因座 

分 类 号：Q556[生物学—生物化学]