米曲霉腺苷酸脱氨酶的过量生产、纯化及特性研究

Overproduction, Purification and Characterization of Adenylate Deaminase from Aspergillus oryzae.

作者信息

Li Shubo, Qian Yi, Liang Yunlong, Chen Xinkuan, Zhao Mouming, Guo Yuan, Pang Zongwen

机构信息

College of Light Industry and Food Engineering, Guangxi University, Nanning, 530004, China.

College of Life Science and Technology, Guangxi University, Nanning, 530004, China.

出版信息

Appl Biochem Biotechnol. 2016 Dec;180(8):1635-1643. doi: 10.1007/s12010-016-2192-7. Epub 2016 Aug 18.

DOI:10.1007/s12010-016-2192-7

PMID:27539573

Abstract

Adenylate deaminase (AMPD, EC 3.5.4.6) is an aminohydrolase that widely used in the food and medicine industries. In this study, the gene encoding Aspergillus oryzae AMPD was cloned and expressed in Escherichia coli. Induction with 0.75 mM isopropyl β-D-l-thiogalactopyranoside resulted in an enzyme activity of 1773.9 U/mL. Recombinant AMPD was purified to electrophoretic homogeneity using nickel affinity chromatography, and its molecular weight was calculated as 78.6 kDa. Purified AMPD exhibited maximal activity at 35 °C, pH 6.0 and 30 mM K, with apparent K and V values of 2.7 × 10 M and 77.5 μmol/mg/min under these conditions. HPLC revealed that recombinant AMPD could effectively catalyse the synthesis of inosine-5'-monophosphate (IMP) with minimal by-products, indicating high specificity and suggesting that it could prove useful for IMP production.

摘要

腺苷酸脱氨酶（AMPD，EC 3.5.4.6）是一种氨基水解酶，广泛应用于食品和医药行业。在本研究中，编码米曲霉AMPD的基因被克隆并在大肠杆菌中表达。用0.75 mM异丙基-β-D-硫代半乳糖苷诱导后，酶活性达到1773.9 U/mL。重组AMPD通过镍亲和层析纯化至电泳纯，其分子量计算为78.6 kDa。纯化后的AMPD在35℃、pH 6.0和30 mM K条件下表现出最大活性，在此条件下其表观K 和V 值分别为2.7×10 M和77.5 μmol/mg/min。高效液相色谱显示重组AMPD能有效催化5'-肌苷酸（IMP）的合成，副产物极少，表明其具有高特异性，提示其可用于IMP生产。