State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
International Joint Laboratory of Food Science and Safety, Jiangnan University, Wuxi, China.
J Sci Food Agric. 2021 Jun;101(8):3308-3318. doi: 10.1002/jsfa.10960. Epub 2020 Dec 16.
Gracilibacillus alcaliphilus SK51.001, a strain that produces β-CGTase (β-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the β-CGTase gene from G. alcaliphilus SK51.001.
According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The β-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni , and Co but inhibited by Al and Fe . The kinetic parameters of K and V were 2068.52 μg mL and 0.13 μmol mL min , respectively.
Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, β-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of β-CD could allow the enzyme to be used in β-CD production. © 2020 Society of Chemical Industry.
从苏丹土壤中筛选并分离到一株产β-CGTase（β-环糊精葡萄糖基转移酶）（EC 2.4.1.19）的菌株，名为嗜碱芽胞杆菌 SK51.001。本研究的目的是对来自嗜碱芽胞杆菌 SK51.001 的β-CGTase 基因进行测序和表征。
根据该菌株的 16S rRNA 分析及其形态，将其鉴定为嗜碱芽胞杆菌。成功克隆、测序并在大肠杆菌 BL21 中表达了 β-CGTase 基因。该基因含有 706 个氨基酸残基，包括 33 个氨基酸的信号肽。利用酶建模和与产物对接，描述了嗜碱芽胞杆菌 SK51.001CGTase 的活性位点残基。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，嗜碱芽胞杆菌 SK51.001CGTase 的估计分子质量约为 74 kDa，凝胶过滤的评估表明约为 85 kDa，这意味着嗜碱芽胞杆菌 SK51.001CGTase 是单体。嗜碱芽胞杆菌 SK51.001CGTase 的最适温度和 pH 分别为 60°C 和 7.0。嗜碱芽胞杆菌 SK51.001CGTase 在 pH 6.0-9.0 和 30-50°C 的温度范围内相对稳定。Ni 和 Co 能提高嗜碱芽胞杆菌 SK51.001CGTase 的活性，而 Al 和 Fe 则抑制其活性。嗜碱芽胞杆菌 SK51.001CGTase 的 K 和 V 动力学参数分别为 2068.52 μg mL 和 0.13 μmol mL min 。
嗜碱芽胞杆菌 SK51.001CGTase 可将可溶性淀粉水解成 α-、β-和 γ-环糊精，比例分别为 2:83:15%。这种高比例的 β-CD 生成使该酶可用于 β-CD 的生产。 © 2020 化学工业协会。
