【作者】 杨秀霞；

【导师】 陈大刚； 黄晓航；

【作者基本信息】 中国海洋大学 ， 渔业资源， 2004， 博士

【摘要】 在极端环境中生存的微生物，在与环境长期抗争的适应过程中形成了许多普通微生物所没有的独特特性。自20世纪70年代以来，极端环境微生物引起了人们的广泛关注，成为当今科学研究的热点之一。南极地区是一个潜在的、重要的微生物资源库，它不仅是微生物新种属的生存繁衍地，也是适应独特生态环境的微生物的生存繁衍地，更是产生新型生物活性物质如酶、抗生素、多糖及不饱和脂肪酸等菌株的潜在种源地。在南极异常寒冷的自然环境中，以嗜冷菌、耐冷菌为主的低温微生物在生态学方面具有明显的优势，它们能耐受经常性的、大幅度、剧烈的温度变化，很重要的一个原因就是这些微生物体内参与代谢的低温酶的存在。与中温酶相比，低温酶在0℃～30℃能保持活性，K_cat值和生理效率（K（cat）／K_m）高，有着更高的催化活性；具有有限的热稳定性，在高温下很快失活，并且对热表现出更强的敏感性；低温酶的活化能较低等。正是因为具备如上的特征，使得低温酶具备了极高的应用价值。 本研究从南极第18次科学考察所采水样中分离出500株低温细菌，对其进行低温脂肪酶筛选，得到120株产酶菌株，从中进一步筛选到5株酶活较高的细菌，进行产酶分析，发现皆为诱导产酶类型。随后对5株酶活较高的细菌进行了分子鉴定，通过16S rDNA序列的比较分析，菌株1-2-8-1、2-5-10-1、2-2-2-1应属于Moritella属，1-1-10-2属于假交替单胞菌属（Pseudoalteromonas sp．），9-2属于嗜冷杆菌属（Psychrobacter sp．）。由系统发育分析可见，本研究5株产低温脂肪酶的菌株均属于细菌域（Bacteria）中的紫细菌（Proteobacteria）类群中的γ-亚群。 我们最终选择了目前国际上研究较少的Moritella属的2-5-10-1作为实验材料，进行了其生长、产酶和酶的性质研究。研究发现菌株2-5-10-1最适生长温度为5℃，最适pH为7～8，此时分泌的胞外脂肪南极细菌启b厂jtella sP.低温脂肪酶基因的克隆与表达酶最多。添加IWeen80、橄榄油可显著诱导和促进脂肪酶的产生。该脂肪酶的最适作用温度为35℃，在0一20℃范围内具有酶活性，0℃下可保持37%的相对酶活;酶的最适作用pH为7.5，在pH6一9的范围内均存在较高酶活;该脂肪酶的米氏常数计算为2.7 x 10一M，活化自由能较低，仅20kJ/mof，对热较敏感，在60℃保温巧min可丧失50%以上的酶活。添加EDTA对脂肪酶活力无影响，说明该脂肪酶的催化作用不需要金属离子的参与，但Cu2+和Zn2+对酶活有着强烈的抑制作用。 对菌株2一5一10一1酶性质研究发现，该酶具有典型的低温脂肪酶特性，显示出很好的应用前景。考虑到低温培养的局限性，我们对低温脂肪酶基因进行了中温宿主的克隆与表达研究。采用限制性内切酶Sau3AI酶切染色体DNA，洗脱2一gkb的DNA片段，然后与事先经BamHI酶切且去磷酸化的质粒载体pBluescriPt连接，导入大肠杆菌DHSQ中，以此建立基因组DNA文库，产生1 8700个重组克隆，然后采用常规的透明圈法筛选目的克隆。培养条件为25℃培养至长出可见菌落，然后4℃培养4d，最终筛选到1个有显著透明圈的克隆。提取克隆质粒DNA并测序发现，该核昔酸序列包含完整的脂肪酶的可读框长837bp，编码279个氨基酸。该酶含有一个绝大多数脂肪酶都存在的以Ser为中心的共有特征序列和一个保守的His一Gly二肤。上游有关启动子的保守序列一10序列TAAATA和一35序列r刀汀CCT以及SD序列AAGTG，说明此基因是靠自己的启动子在大肠杆菌中得到了表达。酶活研究发现，重组的脂肪酶基因表现出明显的低温活性。 借助不同的网络服务器和生物学软件，对低温脂肪酶基因进行了蛋白质一级结构解析，二级结构预测等项分析，探讨了低温脂肪酶的结构与功能。更多还原

【Abstract】 The microorganiams living in the extreme environment have their own special characters, compared with those of common microorganisms, to survive and adapt in harsh environment. Since 1970s, they become the focus of scientific interests and being the one of the hot research fields. Their habitat, the Antarctic region, has prolific biodiversity, provides unidentified new microbe species which might be the source of producing many bioactive substances such as low-temperature enzyme, antibiotic, polysaccharide, polyunsaturated fatty acid (PUFA), etc. Cold-adapted microorganisms inhabiting in Antarctica can tolerant the drastic temperature fluctuation, this property is possibly maintained by the activity of cold adapted enzymes which keeps the normal metabolism of these microorganism in extreme environment.Cold-adapted enzymes are characteried by higher Kcat and physiological efficiency (Kcat/Km) and by a lower and rather constant Km at temperatures from 0 to 30C. In addition, cold-adapted enzymes display an apparent optimal activity at lower temperatures and manifest pronounced heat lability. These unique properties potentially make them a great economical value as being utilized in a temperature range from 0 to 30C at which homologous mesophilic enzymes are essentially inactive.Our study started from the screening of bacteria demonstrating lipase activity. More than 500 strains were selected from samples collected by CTD during Chinese 18th Antarctic Scientific Expedition in the Southern Ocean, of which 120 strains showed lipolytic activity. Through comparison, 5 strains were selected for their higher lipase activity. Theycan produce lipase only if there existed lipase inducer. Through 16S rDNA sequences analysis of 5 strains, the strain 1-1-10-2 and 9-2 were classified as genus Pseudoalteromonas sp. and genus Psychrobacter sp. respectively. The strain 2-5-10-1, 2-2-2-1 and 1-2-8-1 were determined as genus Moritella sp. Phylogenetic analysis indicated that all of these five strains belong to Bacteria, r-Proteobacteria.To our knowledge, there are little published researches on Antarctica Moritella sp., thus Moritella sp.2-5-10-1 was chosen as the model for the following studies. The highest lipase secretion of this strain is observed at temperature 5C and pH 7-8, which is also for their optimal growth. Tween 80 and olive oil can induce lipase secretion. The optimal temperature and pH for lipase activity are 35C and 7.5C, respectively. The lipase keeps active in the temperature range of 0-20C and still has 37% relative lipase activity at 0C. The enzymatic kinetic parameter Km of the lipase is 3.13X10-5M and activation energy Ea is 20kJ/mol. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 C for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2+ and Zn2+All these characteristics proved that the lipase secreted by strain 2-5-10-1 is cold-adapted lipase and this enzyme will have great industrial prospect if manipulated by genetic engineering. The chromosomal DNA was extracted from strain 2-5-10-1, partially digested with Sau3AI. The DNA fragments of 2-9kb were isolated and then ligated with pBluescript vector that had been previously digested with BamHI and dephosphorylated with bacterial alkaline phosphatase. The resultant plasmids were introduced into E.coli DH5 a producing a gene library containing 18700 recombinant clones. The clones producing a lipase were detected due to the formation of halos around the colonies on LB agarplates supplemented with ampicillin, IPTG and tributyrin. After incubation at 25 C until clones became visible, the plates were incubated further at 4 C for 4 days. 1 clone showed a clearest halos was selected for DNA sequence analysis. We determined its nucleotide sequence and found an open reading frame comprising 837bp, which encodes a putative protein of 279 amino acids. The nucleotide sequence have a unique c更多还原