BACKGROUND A strain, Gracilibacillus alcaliphilus SK51.001, which 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. RESULTS According to 16S rRNA analysis and the morphological shape the strain was identified as Gracilibacillus alcaliphilus. The β-CGTase gene was successfully cloned, sequenced and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues with 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 SDS-PAGE, while 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. G. alcaliphilus SK51.001CGTase was comparatively stable at pH between 6.0-9.0 and temperature 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 μg mL-1 and 0.13 μmol mL-1 min-1 , respectively. CONCLUSION G. alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, β-, and γ-cyclodextrin in ratio of 2: 83: 15 % respectively, this high ratio production of β-CD could allow the enzyme to be used in β-CD production. This article is protected by copyright. All rights reserved.

中文翻译：

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嗜碱芽孢杆菌 SK51 .001 嗜热 β-环糊精葡聚糖转移酶的遗传和生化特征

背景从苏丹土壤中筛选并分离出一株产β-CGTase（β-环糊精葡糖基转移酶）（EC 2.4.1.19）的菌株，嗜碱芽孢杆菌SK51.001。本研究的目的是对来自 G. alcaliphilus SK51.001 的 β-CGTase 基因进行测序和表征。结果经16S rRNA分析及形态学鉴定，该菌株为嗜碱芽孢杆菌。β-CGTase基因在大肠杆菌BL21中成功克隆、测序并表达。该基因显示706个氨基酸残基，其中33个氨基酸作为信号肽。G. alcaliphilus SK51.001CGTase 的活性位点残基使用酶建模和与产物对接进行描述。G. alcaliphilus SK51.001CGTase 的估计分子量通过 SDS-PAGE 测定约为 74 kDa，而凝胶过滤的评估显示大约 85 kDa，这意味着 G. alcaliphilus SK51.001CGTase 是单体。G. alcaliphilus SK51.001CGTase的最适温度和pH分别为60℃和7.0。G. alcaliphilus SK51.001CGTase 在 6.0-9.0 的 pH 值和 30-50 °C 的温度下相对稳定。G. alcaliphilus SK51.001CGTase 的活性被 Ni2+ 和 Co2+ 增加，但被 Al3+ 和 Fe3+ 抑制。Km 和Vmax 的动力学参数分别为2068.52 μg mL-1 和0.13 μmol mL-1 min-1。结论 G. alcaliphilus SK51.001CGTase 可以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 可以使酶用于 β -CD 制作。本文受版权保护。版权所有。