南极普里兹湾深海沉积物微生物低温酶的筛选、基因克隆表达及性质分析

【作者】 张金伟；

【导师】 曾润颖；

【作者基本信息】 国家海洋局第三海洋研究所 ， 海洋生物学， 2007， 硕士

【摘要】 南极、深海等极端环境中蕴藏着丰富的资源，其中微生物资源是国内外研究的热点。它们经过长期的进化、选择，在各种极端的环境条件下形成了独特的组织结构、酶系统及代谢机制以进行生存和繁衍。对低温酶、冷适应酶独特的生理结构及机能进行研究，探讨生物适应特殊的生存环境而发生的基因变异，无论在科学上还是实际开发应用上都具有重大的意义。由于极端环境样品采集方面的限制，目前大部分的极端微生物仍未得到认识和研究。低温酶由于在低温下的高催化效率，而在工业、农业、医药卫生、洗涤、生物制剂、食品等方面都有极其广泛的应用，其中淀粉酶、脂肪酶、蛋白酶和纤维素酶等是主要酶制剂品种。本文对南极普里兹湾深海沉积物(PN5-6，74°25’E，66°55’S，900m)样品开展研究。采用传统分离微生物手段获得可培养的微生物共42株，对它们进行选择性培养，筛到17株细菌明显具有分泌水解酶的能力，其中淀粉酶产生菌6株，分别有假单胞菌、红球菌、诺卡氏菌；脂肪酶产生菌8株，分别有假单胞菌和嗜冷杆菌；蛋白酶产生菌1株，为诺卡氏菌。对这17株菌的16S rDNA进行进化树分析，12株细菌属于γ-Proteobacteria亚群，有5株细菌属于革兰氏阳性细菌。复合酶产生菌7197的产酶发酵条件得到优化；诺卡氏菌7326产的淀粉酶得到了纯化和性质分析；来自嗜冷杆菌7195的低温脂肪酶基因(lipAl)得到了克隆与表达；来自假单胞菌7323的低温脂肪酶(lipA)得到了克隆与表达；来自假单胞菌7197的淀粉酶基因(amyP)得到了克隆与表达，这些表达后的蛋白均得到纯化和性质的分析。此外，采用宏基因组技术，提取该沉积物样品的宏基因组DNA，通过设计探针从中克隆到低温脂肪酶基因(lip3)的开放阅读框完整序列，构建重组表达载体，在大肠杆菌中获得表达，表达后的重组蛋白也得到了纯化和性质分析。该研究结果及其所建立的技术为南极丰富的低温酶资源的研究开发奠定了基础。更多还原

【Abstract】 Microorganisms living in permanent cold environment, such as deep sea and Antarctic, presumably have developed particular characteristics that allow them to thrive at such environment. For example, the cold-active or cold-adaptive enzymes have attracted more and more interests as they not only are essential in some mechanism studies, but also provide potential for commercial development. Recent microbial studies of the Antarctic have led to significantly new discoveries of unusual microbial diversity, new species, and cold-adaptive enzymes. However, partially due to the great difficulties in collecting samples, it remains as one of the most unknown fields. Compared to organic synthesis, biocatalysts often have far better chemical precision, which can lead to more efficient production of single stereoisomers, fewer side reactions, and a lower environmental burden. The bulk uses of amylases, lipases, proteases and celluases in industrial sectors are including food and feed industry, peptide synthesis, leather industry, management of industrial and household waste, photographic industry.A total of 42 strains, isolated from deep sea sediment of Prydz Bay, Antarctic (74°25’E, 66°55’S, depth of 900 m), which grow only below 30℃were identified based on the morphological differences and SDS-PAGE of culture supernatant. 17 of them were found to have cold-adaptive amylase, lipase or protease activity. Six strains showing strong amylolytic activity, eight strains showing lipolytic activity, one showing proteolytic activity under 10℃were picked out for further study. They were affiliated withγ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequences. The amylaseproducing strains belonged to Pseudomonas, Rhodococcus and Nocardiopsis. The lipaseproducing strains belonged to Pseudomonas, Psychrobacter. The protease-producing strains belonged to Nocardiopsis. The optimal culture conditions for the enzyme production of strain 7197 were investigated; Cold-adaptive alpha-amylase produced by Nocardiopsis sp. 7326 was purified and characterization; Lipase encoding gene (lipA1) from Psychrobacter sp. 7195 was cloned and sequenced, and expressed in E. coll.; Lipase encoding gene (lipA) from Pseudomonas sp. 7323 was cloned and sequenced, and expressed in E. coli.; Amylase encoding gene (amyP) from Pseudomonas sp. 7197 was cloned and sequenced, and expressed in E. coli. We also obtained the lipase gene (lip3) through PCR cloning with metagenome DNA, which was extracted from the deep sea sediment of Prydz Bay. Then the gene was expressed in E. coli and characterization. The results and techniques will contribute to the further research in the abundant cold-adaptive enzymes from the Antarctic.更多还原