N末端结构模块缺失对嗜热酸性III型普鲁兰多糖水解酶TK-PUL酶学性质的影响  

Effect of Truncation of N-Terminal Structural Modules on Enzymatic Properties of Thermoacidiphilic Type Ⅲ Pullulan Hydrolase TK-PUL

作　　者：曾静[1] 何础阔 郭建军[1] 袁林[1] ZENG Jing;HE Chukuo;GUO Jianjun;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》2021年第10期201-208,共8页Food Science

基　　金：江西省杰出青年基金项目(20202ACBL215001);国家自然科学基金青年科学基金项目(31501422);江西省青年科学基金项目(20171BAB214003)。

摘　　要：通过对嗜热酸性Ⅲ型普鲁兰多糖水解酶TK-PUL进行N末端截短突变,并比较TK-PUL与突变酶的酶学性质,确定N末端结构模块的缺失对酶学性质的影响。结构模块N1的缺失改良了TK-PUL的催化特性,其α-淀粉酶比活力提高至TK-PUL的1.11倍,普鲁兰酶比活力提高至TK-PUL的1.12倍,于100℃的半衰期延长至TK-PUL的1.15倍。结构模块N2的缺失提高了酶的热稳定性,于100℃的半衰期延长至TK-PUL的1.25倍。但是结构域N2的缺失降低了酶的pH值稳定性、酶的底物结合能力以及比活力。并且结构域N2影响了酶的底物选择性,导致其α-淀粉酶活性与普鲁兰酶活性的比值由0.49提高至0.60。结果表明,TK-PUL的N末端结构模块N1和N2均不是其发挥催化活性所必需的结构区域,但是对酶的底物结合能力、底物降解能力以及稳定性具有重要影响。In this study, the enzymatic properties of TK-PUL, a thermoacidiphilic type Ⅲ pullulan hydrolase produced by the extremely thermophilic archaea Thermococcus kodakarensis KOD1 were compared with those of its N-terminal truncated mutants, in order to determine the effect of the truncation of N-terminal structural modules on the enzymatic properties of TK-PUL. The truncation of the structural module N1 improved the catalytic characteristics of TK-PUL, resulting in a 1.11-fold increase in α-amylase specific activity, a 1.12-fold increase in pululanase specific activity and a 1.15-fold increase in half-life at 100 ℃. The truncation of the structural module N2 enhanced the thermal stability of the enzyme, and extended the halflife at 100 ℃ by 1.25 times, whilst it decreased the pH stability, substrate binding capacity and specific activity of TK-PUL. Moreover, the truncation of the structural module N2 changed the substrate specificity of TK-PUL, resulting in an increase in the ratio of α-amylase to pullulanase activity from 0.49 to 0.60. The results showed that neither the structural module N1 nor N2 of TK-PUL was the essential structural regions for enzymatic catalysis, but both of them had important effects on the substrate binding capacity, catalytic activity and stability of the enzyme.

关 键 词：Ⅲ型普鲁兰多糖水解酶 N末端结构模块 缺失突变 催化特性 

分 类 号：Q814[生物学—生物工程]