【作者】 曾丽清；

【导师】 施碧红；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 本研究从新疆火焰山土样中分离到一株脂肪酶产生菌FS132，对其进行18SrRNA分子标记鉴定，进一步克隆了该菌脂肪酶基因(ATL)，并实现了该脂肪酶基因在毕赤酵母中的表达，为深入研究Aspergillus tamarii脂肪酶合成的分子机理奠定了基础，也为改造现有脂肪酶提供新的脂肪酶基因资源。主要研究内容及成果如下：(1)从新疆火焰山口土样中分离的一株脂肪酶产生菌FS132，菌落形态表明为霉菌。克隆测定了该菌18S rRNA基因序列，并将该序列进行比对和聚类分析，构建系统进化树。结果表明该菌与报道的Aspergillus tamarii具有最紧密亲缘关系。(2)ATL基因及其cDNA的克隆和序列分析：分别提取Aspergillus tamarii FS132基因组DNA和总RNA，根据资料，利用Aspergillus tamarii亲源关系较近的脂肪酶基因序列设计引物，采用PCR、RT-PCR等技术扩增了FS132脂肪酶基因(ATL)和全长cDNA序列。ATL-cDNA全长由921个碱基组成；PCR扩增获得的ATL-DNA由1742个碱基组成，包括ATL编码区，3’非翻译区和5’非翻译区基因的序列，其中ATL编码区有1024个碱基，含有2个内含子，大小分别为51 bp、52 bp。(3)为了验证所克隆到的ATL-cDNA序列的功能，构建重组表达载体，以毕赤酵母GS115为宿主进行功能表达分析。表达菌株经0.5％甲醇诱导培养，实现了Aspergillus tamarii FS132脂肪酶蛋白的分泌表达。经SDS-PAGE分析结果表明，重组酵母分泌36.7 kD左右的重组蛋白；三丁酸甘油酯检验板中出现水解圈：NaOH滴定法测定表明发酵液脂肪酶活力为20U／mL。(4)初步优化了Aspergillus tamarii FS132发酵产酶条件，产酶最适培养时间为45 h，培养基最适初始pH 7.0，最适培养温度36℃，最适摇瓶空气量25 mL／250 mL锥形瓶。更多还原

【Abstract】 A lipase-producing strain FS132 was isolated from the volcanic vent soil in SinKiang Province. The strain was identified by 18S rRNA molecular marker. The lipase gene (ATL) was further cloned and then expressed in Pichia pastoris. The results not only make a foundation for further investigating the mechanism of lipase synthesis in Aspergillus tamari, but also provide a new kind of lipase gene resource for improvement the existing lipase. The main results of this study were as follows:(1) A lipase-producing strain FS132 was isolated from the volcanic vent soil in SinKiang Province. It was a mildew based on its colony morphology. The 18S rRNA gene of FS132 was cloned and sequenced. According to the phylogenetic analysis, the 18S rRNA marker of FS 132 was showed highly homology to that ofAspergillus tamari.(2) Cloning and sequence analysis of the ATL-DNA and ATL-cDNA: the genomic DNA and total RNA of A. tamari FS 132 were extracted respectively. Specific primers were designed based on the homology lipase gene in the literature. The ATL-DNA and ATL-cDNA was amplified using the techniques of PCR and RT-PCR. The total size of ATL-cDNA was made of 921 bp, and the open read frame of ATL gene was 1024 bp, which containing 2 introns with sizes of 51 bp and 52 bp respectively.(3) To identify the function of cloned ATL-cDNA, the recombinant expression vector was constructed, and the Pichia pastoris GS115 was used as a host for expressing ATL gene. The ATL gene of A. tamarii FS132 was expressed in GS 115 and secreted into culture aider the addition of 0.5% methanol to the medium. Result of SDS-PAGE showed that a 36.7 kDa recombinant protein was secreted from the host yeast. And clear zone was developed on the tributyrin plate. The lipase activity in the supernatant of the culture was 20U/ml assayed by NaOH titrate method.(4) The optimal culture conditions for lipase producing of FS132 was obtained as following: the optimal culture temperature of 36℃, initiation pH of 7.0 for the culture medium, oxygen-consumption of 25 mL medium for each 250mL flask, and culture time of 45 hr.更多还原