嗜热酸性Ⅲ型普鲁兰多糖水解酶TK-PUL保守基序中关键氨基酸残基对其催化性质的影响  

Effect of Key Amino Acid Residues in the Conserved Regions of Thermoacidiphilic Type Ⅲ Pullulan Hydrolase TK-PUL on Its Catalytic Properties

作　　者：曾静[1] 何础阔 郭建军[1] 侯安伟 聂俊辉 袁林[1] ZENG Jing;HE Chukuo;GUO Jianjun;HOU Anwei;NIE Junhui;YUAN Lin(Institute of Microbiology,Jiangxi Academy of Sciences,Nanchang 330096,China;College of Food Science and Engineering,Hainan University,Haikou 570228,China)

机构地区：[1]江西省科学院微生物研究所,江西南昌330096 [2]海南大学食品科学与工程学院,海南海口570228

出　　处：《食品科学》2022年第22期113-120,共8页Food Science

基　　金：国家自然科学基金地区科学基金项目(32160579);江西省杰出青年科学基金项目(20202ACBL215001);江西省主要学科学术和技术带头人培养计划项目(20212BCJ23033)。

摘　　要：对嗜热酸性Ⅲ型普鲁兰水解酶TK-PUL保守基序中非保守氨基酸残基进行定点突变,通过比较TK-PUL与突变体的酶学性质,确定保守基序中关键氨基酸残基对其催化性质的影响。TK-PUL保守基序Ⅱ中I500W位点变化不影响其最适反应pH值、pH值稳定性、最适反应温度、热稳定性,但使其α-淀粉酶活性和普鲁兰酶活性均明显降低。动力学常数测定结果显示,突变体I500W以麦芽三糖为底物的k_(cat)/K_(m)值基本不变,以异潘糖为底物的k_(cat)/K_(m)值约为TK-PUL的64.78%。结果表明,TK-PUL保守基序II中第500位氨基酸残基Ile对其水解糖苷键的偏好性起到重要作用。TK-PUL中I500W位点变化不影响其α-1,4-糖苷键水解活性,但使其α-1,6-糖苷键水解活性明显降低。本研究有助于深入理解TK-PUL的双功能催化机制,也可为TK-PUL的分子改造提供理论依据和设计思路。In this study,site-directed mutagenesis of non-conserved amino acid residues in the conserved regions of thermoacidophilic type Ⅲ pullulan hydrolase TK-PUL was performed,and the effect of key amino acid residues in the conserved regions on its catalytic properties was determined by comparing the enzymatic properties of TK-PUL with those of its mutants.The I500W mutation in the conserved sequence region II of TK-PUL did not affect the optimum pH,pH stability,optimum temperature or thermal stability of TK-PUL,but significantly decreased theα-amylase and pullulanase activity.The kinetic parameters of TK-PUL and the mutant I500W TK-PUL were determined.The k_(cat)/K_(m) value of the mutant I500W for maltotriose was basically unchanged,and the k_(cat)/K_(m) value of the mutant I500W for isopanose was about 64.78%of TK-PUL.The results showed that Ile at residue 500 was important for the preference forα-1,4-andα-1,6-glycosidic linkages of TK-PUL.The I500W mutation did not affect theα-1,4-activity of TK-PUL,but significantly reduced theα-1,6-activity.This study is helpful for an in-depth understanding of the bifunctional catalytic mechanism of TK-PUL,and can also provide a theoretical basis and design ideas for the molecular modification of TK-PUL.

关 键 词：Ⅲ型普鲁兰多糖水解酶 保守基序 定点突变 催化活性 同源模建 

分 类 号：Q814[生物学—生物工程]