【作者】 李雪；

【导师】 韦宇拓；

【作者基本信息】 广西大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 随着现代工业生产快速发展,许多生产过程需要在特定环境下进行,如高温、高盐或碱性环境,这就导致了极端环境工业用酶的开发和利用。嗜盐性α-淀粉酶因其能够在高盐环境中具有稳定的酶活进而能抵抗工业生产中极端环境,这一特性越来越引起研究人员的关注。从弗氏柠檬酸杆菌(Citrobacter freundii)基因组DNA中克隆到一个淀粉酶基因f2,并在大肠杆菌JM109中进行过量表达。对重组蛋白(F2)进行纯化和酶学特性测定表明其在有NaCl存在的条件下才具有酶活性,而在没有NaCl的条件下酶活基本丧失,因此鉴定F2为嗜盐性α-淀粉酶。以可溶性淀粉为底物时,F2的最适温度为50℃,最适pH为6.5,最适NaCl浓度为3mol/L,并且在NaCl浓度为6mol/L时还有大约50%的相对酶活力存在。K6为本实验室构建的来源大肠杆菌JM109的重组淀粉酶,经研究发现它也具有耐盐性。对K6进行酶学性质研究表明,它的最适NaCl浓度为2mol/L,在浓度为5.5mol/L时还有大约20%的相对活力存在。通过同源建模和定点突变技术对F2与K6的嗜盐机制进行研究。结果将F2中E130和E131以及K6中E287的酸性氨基酸E突变为中性氨基酸A后,淀粉酶的嗜盐性发生了改变,即突变酶在高盐环境中酶活力急剧下降甚至完全丧失。结果表明酸性氨基酸对嗜盐酶的嗜盐特性有影响。进一步对突变酶及原始酶进行三维结构分析表明,突变酶与原始酶相比在其突变位点附近p折叠链数由4个减少到两个,并且链的长度也变短了。初步推测酸性氨基酸和酶空间结构中p折叠链均对嗜盐酶的嗜盐特性有影响。更多还原

【Abstract】 With the fast development of modern industrial technology, many production processes need to be carried out under some certain conditions, such as high temperature, high salt, or alkaline environment, which promote the development and utilization of industrial enzymes from extreme environment. Halophilic α-amylase has attracted increasing interest among researchers because of its abilities to maintain activity in high salt condition and to withstand extreme conditions in industrial production.A amylase (f2) gene from the genomic DNA of Citrobacter freundii was cloned and overexpressed in Escherichia coli JM109and the recombinant protein (F2) was purified and characterized. F2was identified as a halophilic α-amylase because its activity depending on the presence of NaCl. The optimum temperature, pH, and NaCl concentration of F2using starch as substrate were50C,6.5, and3M, respectively. And F2could retain about50%relative activity with6M NaCl. Another recombinant amylase (K6) from E. coli JM109constructed in our laboratory also has the salt tolerance. Its optimum NaCl concentration was2M, and could retain about20%relative activity with5.5M NaCl.The halophilic mechanism of F2and K6was studied by homology modeling and directed mutagenesis. When the acidic amino acids E of E130and E131sites from F2and E287site from K6were respectively replaced by neutral amino acids A, the halophilic feature of amylase had changed (i.e. the enzyme activity of mutants sharply declined or even completely losed under a high salt condition). Furtherly, the3D structure of mutant enzymes from F2and K6showed that the numbers of β folded chain around mutant points reduced from four to two and their length became shorter than wild-type. It speculates that the acidic amino acids and β folded chain of spatial structure of halophilic enzyme are related to its halophilic characteristic.更多还原