Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu, 180001, India.
Appl Microbiol Biotechnol. 2010 May;86(6):1821-8. doi: 10.1007/s00253-009-2404-y. Epub 2010 Jan 7.
The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 degrees C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.
对新型生物催化剂的需求不断增加，刺激了对土壤资源的探索。宏基因组学是一种不依赖培养的方法，为从未培养的微生物中发现新型生物催化剂提供了无尽的资源。在这项研究中，构建了一个包含 90700 个重组体的土壤衍生宏基因组文库，并对其进行了脂酶、纤维素酶、蛋白酶和淀粉酶活性的筛选。在筛选了 35000 个大肠杆菌克隆后，发现了一个编码淀粉酶的 909bp 基因（pAMY）。氨基酸序列比较和系统发育分析表明，pAMY 与未培养细菌密切相关。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计 pAMY 的分子量约为 38 kDa。使用可溶性淀粉、直链淀粉、糖原和麦芽糖作为底物测定淀粉酶活性。在接近中性 pH 值的条件下，pAMY 在 40°C 时的最大活性（2.46 U/mg）为直链淀粉；而在所有研究中使用的底物中，pAMY 在低温下保留了 90%的活性。pAMY 在低温下工作的能力是迄今为止从培养和未培养微生物中报道的淀粉酶所特有的。
