Lakshmi Hanumanthu Prasanna, Prasad Uppu Venkateswara, Yeswanth Sthanikam, Swarupa Vimjam, Prasad Osuru Hari, Narasu Mangamoori Lakshmi, Sarma Potukuchi Venkata Gurunadha Krishna
Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati-517 507, AP, India.
Bioinformation. 2013;9(6):281-5. doi: 10.6026/97320630009281. Epub 2013 Mar 19.
Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria.
金黄色葡萄球菌是一种重要的革兰氏阳性人类病原体，它能分泌许多表面蛋白和分泌蛋白，包括各种酶和致病因子，这些有助于其成功定殖于宿主组织并引发感染。α-淀粉酶是金黄色葡萄球菌分泌的一种酶，它催化复杂糖类分解为单糖，而单糖是该病原体在任何解剖部位定殖和存活所必需的。在本研究中，我们克隆、测序、表达并鉴定了来自金黄色葡萄球菌ATCC12600的α-淀粉酶基因。重组酶的分子量为58kDa，动力学分析显示其Vmax为0.0208±0.033（mg/ml）/mg/min，Km为10.633±0.737mg/ml。多重序列分析表明，金黄色葡萄球菌的α-淀粉酶与枯草芽孢杆菌和牛链球菌有很大差异。由于无法获得金黄色葡萄球菌α-淀粉酶的晶体结构，我们采用同源建模方法构建其结构。通过拉氏图对构建的结构进行验证，结果显示90%的残基位于允许区域，而在禁止区域未发现残基，且构建的结构与晶体结构接近，Z值为-6.85。与枯草芽孢杆菌和牛链球菌的α-淀粉酶进行结构叠加研究，结果显示存在明显差异，RMSD值分别为18.158Å和7.091Å，这与酶动力学相关，表明这些细菌中的α-淀粉酶存在差异。
