嗜热酸性生淀粉α-淀粉酶Gt-amy的C末端结构域功能及生淀粉结合位点分析

doi:10.7506/spkx1002-6630-20190115-159

食品科学 ›› 2020, Vol. 41 ›› Issue (6): 108-115.doi: 10.7506/spkx1002-6630-20190115-159

嗜热酸性生淀粉α-淀粉酶Gt-amy的C末端结构域功能及生淀粉结合位点分析

曾静，郭建军，涂熠坤，袁林

（1.江西省科学院微生物研究所，江西南昌 330096；2.南京工业大学化学与分子工程学院，江苏南京 211800）

出版日期:2020-03-25 发布日期:2020-03-23
基金资助:
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）；江西省科学院杰出青年项目（2018-JCQN-02）；江西省科学院产学研合作项目（2018-YCXY-02）

Function Evaluation of C-Terminal Domain of Thermoacidiphilic Raw Starch Degrading α-Amylase Gt-amy and Identification of Raw Starch Binding Sites

ZENG Jing, GUO Jianjun, TU Yikun, YUAN Lin

(1. Institute of Microbiology, Jiangxi Academy of Sciences, Nanchang 330096, China; 2. School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211800, China)

Online:2020-03-25 Published:2020-03-23

摘要/Abstract

摘要： 为确定嗜热酸性生淀粉α-淀粉酶Gt-amy中参与生淀粉结合的结构区域，对Gt-amy的C末端结构域（C-terminal domain，CTD）进行缺失突变，并比较Gt-amy及CTD缺失突变体Gt-amy-T的酶学性质。在与Gt-amy相同的条件下，Gt-amy-T不能结合和降解玉米淀粉，CTD可有效结合玉米淀粉。以可溶性淀粉为底物，Gt-amy-T的kcat值约为Gt-amy的77.9%。CTD的系统进化关系分析显示CTD不是典型的淀粉结合结构域，但是本研究证实CTD属于生淀粉结合结构域，在Gt-amy结合并降解生淀粉中发挥重要作用，并且CTD有利于Gt-amy发挥可溶性淀粉酶活力。此外，本研究将CTD中Tyr残基定点突变为Ala残基，通过比较CTD与突变体的玉米淀粉结合能力以及Gt-amy与Gt-amy W501A/W514A的玉米淀粉结合率和降解率，确定CTD中W501和W514可能是生淀粉结合位点。

关键词: 嗜热酸性生淀粉α-淀粉酶, 生淀粉结合域, 生淀粉结合位点, 定点突变

Abstract: To identify the raw starch binding region of the thermoacidiphilic raw starch degrading α-amylase Gt-amy, C-terminal domain (CTD) truncated mutant Gt-amy-T was constructed and its enzymatic properties were characterized and compared with those of Gt-amy. The abilities of Gt-amy and Gt-amy-T to bind to and hydrolyze raw corn starch to evaluate the role of CTD as a raw starch binding domain. Both Gt-amy and CTD displayed comparable affinities for raw corn starch whereas Gt-amy-T was unable to bind to raw corn starch. Gt-amy hydrolyzed raw corn starch efficiently, while Gt-amy-T did not. The kcat of Gt-amy-T was 77.9% of that of Gt-amy with soluble starch as the substrate. Phylogenetic analysis of CTD revealed that CTD was not a typical starch binding domain, but Gt-amy’s CTD was experimentally verified to be raw starch binding domain, which played an important role in raw starch binding and hydrolysis by Gt-amy. CTD contributed to the hydrolysis of soluble starch by Gt-amy. In addition, mutational analysis of Tyr residues in CTD was performed to identify the raw starch binding sites in CTD. The binding capacities of CTD and the mutants to raw corn starch were assayed. The raw corn starch binding and hydrolysis capacities of Gt-amy and Gt-amy W501A/W514A were investigated. The results revealed that the residues W501 and W514 were probably raw starch binding sites in CTD.

Key words: thermoacidiphilic raw starch degrading α-amylase, raw starch binding domain, raw starch binding site, mutational analysis

中图分类号:

Q814

曾静，郭建军，涂熠坤，袁林. 嗜热酸性生淀粉α-淀粉酶Gt-amy的C末端结构域功能及生淀粉结合位点分析[J]. 食品科学, 2020, 41(6): 108-115.

ZENG Jing, GUO Jianjun, TU Yikun, YUAN Lin. Function Evaluation of C-Terminal Domain of Thermoacidiphilic Raw Starch Degrading α-Amylase Gt-amy and Identification of Raw Starch Binding Sites[J]. FOOD SCIENCE, 2020, 41(6): 108-115.

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嗜热酸性生淀粉α-淀粉酶Gt-amy的C末端结构域功能及生淀粉结合位点分析

Function Evaluation of C-Terminal Domain of Thermoacidiphilic Raw Starch Degrading α-Amylase Gt-amy and Identification of Raw Starch Binding Sites

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