基于晶体结构的超嗜热古菌高温α-淀粉酶热稳定性不依赖钙离子的分子机制研究

项目名称： 基于晶体结构的超嗜热古菌高温α-淀粉酶热稳定性不依赖钙离子的分子机制研究

项目编号： No.31271929

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 王淑军

作者单位： 淮海工学院

项目金额： 78万元

中文摘要： 大部分微生物的α-淀粉酶的热稳定性依赖Ca2+，仅有少数菌分泌的α-淀粉酶热稳定性不依赖Ca2+。目前工业应用的高温α-淀粉酶热稳定性依赖Ca2+是淀粉深加工的瓶颈，获得热稳定性不依赖Ca2+的高温α-淀粉酶是解决该问题的最有效途径之一。本研究组发现一株超嗜热古菌所分泌的高温α-淀粉酶（TSA）热稳定性不依赖Ca2+。关于酶的热稳定性不依赖Ca2+的机制以及这两种酶的蛋白晶体的结构异同，尚无研究报道。本研究拟通过制备TSA晶体，测定和分析TSA晶体结构，并与热稳定依赖Ca2+的其他α-淀粉酶的晶体结构进行比较分析，来研究TSA热稳定性不依赖Ca2+的分子机制。并通过对TSA中热稳定性不依赖Ca2+的关键氨基酸进行定点突变，验证所获得的分子机制。以期对一些热稳定性好、但热稳定性依赖Ca2+的α-淀粉酶进行分子改造，为获得适用于淀粉制糖工业的高温α-淀粉酶提供依据。

中文关键词： 超嗜热α-淀粉酶；晶体结构；热稳定性不依赖钙离子；分子机制；

英文摘要： Most of the microbial α-amylase used in industry require addition of calcium ion (Ca2+) for their thermostability. Few microbial α-amylase thermostability does not depend on Ca2 +. It is a conspicuous drawback in the application of enzymes for the deep processing of starch. The acquirement of α-amylases which have good thermostability and don't require addition of Ca2+ for its thermostability is one of the most effective way to resolve the problem. A extremely thermophilic anaerobic archaeon strain, HJ21, from a deep-sea hydrothermal vent, could produce hyperthermophilic α-amylase(TSA) that do not require Ca2+ for thermostability. There are no research reports for the principle ofα-amylase thermostability not depinging on Ca2 + and the difference of crystal structures between the two kind of α-amylase yet. The purpose of this project is to investigate the molecular mechanism of the hyperthermophilic α-amylase of the marine hyperthermophilic archaea that does not require Ca2+ for thermostability by analyzing their crystal structure determined by X-ray single crystal diffraction of the TSA. Then point mutation will be produced on the key amino acid for the TSA do not require Ca2+ for thermostability. The mutant enzymes will be purified and characteried to confirm the validity of the results. The results would be e

英文关键词： hyperthermophilic α-amylase；Crystal structure；Don’t require additional Ca2+ for thermostability；Molecular mechanism；