Ibrahim Abdelnasser S S, Al-Salamah Ali A, El-Tayeb Mohamed A, El-Badawi Yahya B, Antranikian Garabed
Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.
Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstrasse 12, 21073 Hamburg, Germany.
Int J Mol Sci. 2012;13(8):10505-10522. doi: 10.3390/ijms130810505. Epub 2012 Aug 22.
Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg(-1) protein, 20.0 U mg(-1) protein and 11.0 U mg(-1) protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl(2). K(m) and V(max) values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co(2+), Zn(2+), Cu(2+), Hg(2+), Ba(2+), Cd(2+), and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.
从埃及纳特鲁恩谷高盐碱苏打湖采集的样本中筛选产环糊精糖基转移酶（CGTase）的嗜碱细菌，得到了一株高效产CGTase的嗜碱细菌，命名为NPST-10。16S rDNA序列分析确定该分离株为两栖芽孢杆菌属。通过淀粉吸附和阴离子交换色谱法将CGTase纯化至同质，纯化倍数达22.1倍，产率为44.7%。使用SDS-PAGE分析，纯化后的酶为单体蛋白，估计分子量为92 kDa。该酶的环化、偶联和水解活性的催化活性分别为88.8 U mg(-1)蛋白、20.0 U mg(-1)蛋白和11.0 U mg(-1)蛋白。该酶在pH 5.0至11.0的宽pH范围内稳定，在pH 8.0时活性最高。CGTase在45℃至70℃的宽温度范围内具有活性，在50℃时活性最高，在30℃至55℃下稳定至少1小时。在CaCl(2)存在下，纯化酶的热稳定性可显著提高。以可溶性淀粉为底物，估计K(m)和V(max)值分别为1.7±0.15 mg/mL和100±2.0 μmol/min。在Co(2+)、Zn(2+)、Cu(2+)、Hg(2+)、Ba(2+)、Cd(2+)和2-巯基乙醇存在下，CGTase受到显著抑制。据我们所知，这是关于两栖芽孢杆菌属产CGTase的首次报道。两栖芽孢杆菌属NPST-10能将不溶性粗玉米淀粉高效转化为环糊精（67.2%），且主要产物为β-环糊精（86.4%），使其成为环糊精生产行业的理想选择。
