【作者】 刘海霞；

【导师】 赵国芬；

【作者基本信息】 内蒙古农业大学 ， 微生物学， 2011， 硕士

【摘要】 共轭亚油酸(conjugated linoleic acids简称CLA)是一种具有多种生理活性的天然脂肪酸,CLA具有抗癌、降低胆固醇、增强机体免疫力等多种生理功能。亚油酸异构酶可将亚油酸转化为生物活性的CLA。目前,亚油酸异构酶的结构和性质的研究以及利用生物工程和基因工程对亚油酸异构酶进行有效地改造已成为热点。但是在微生物发酵生产CLA的代谢途径和机理方面研究甚少,有待进一步深入研究。本实验以植物乳杆菌(Lactobacillus plantarum subsp. plantarum P8)基因组为模板,设计引物,通过PCR扩增得到亚油酸异构酶基因,将其克隆到pMD19-T载体测序鉴定后,再亚克隆到表达载体pQE30中,双酶切鉴定表达载体pQE30-LAI成功构建。用http: //www. ncbinlm.nih.gov/blast和vecterNTI等软件工具对亚油酸异构酶进行生物信息学分析并预测酶关键氨基酸,预测结果确定亚油酸异构酶的必要氨基酸为193位苯丙氨酸,将其定为突变位点,改变578位碱基T为C,将苯丙氨酸极性氨基酸突变为非极性氨基酸丝氨酸。设计突变引物,以重组质粒pQE30-LAI为模板,用Transgen公司开发的QuickChange定点突变试剂盒进行定点突变,成功构建突变载体pQE30-F193LAI。突变前后的亚油酸异构酶在大肠杆菌中经IPTG诱导表达,分别用提取的粗酶和菌体直接催化测定酶活力,酶催化的结果分别为野生型1999.79 U/ml,突变型1564.53 U/ml,菌体催化的结果分别为野生型117.11 U/ml,突变型87.96 U/ml。两种结果均表明,重组菌pQE30-LAI的酶活力大于突变后重组菌pQE30-F193LAI,表明突变体表现出了酶活被破坏的特性,193 Phe位点在酶的催化过程中都发挥着不可替代的作用。将表达的野生型和突变型的酶提取后检测,结果表明,野生型和突变型均有68KD的条带,说明亚油酸异构酶均得到表达。更多还原

【Abstract】 CLA (conjugated linoleic acids) is a natural fatty acids with a variety of physiological activities, such as anti-cancer, lowering cholesterol, enhancing immunity. Linoleate isomerase can convert linoleic acid into biologically active CLA. At present, the researches of linoleic acid isomerase structure and properties and transforming linoleic acid isomerase effectively with biological engineering and genetic engineering techniques have become a hot spot. However, it is still to make further study for fewer researches in the metabolic pathway and mechanism of microbial fermentation production of CLA.In this study, the Lactobacillus (Lactobacillus plantarum subsp. plantarum P8) genome was selected as a template, then primers were designed and linoleic acid isomerase gene was amplified with PCR. The PCR production was cloned into the vector pMD19-T to identify the sequence, then it was subcloned into the expression vector pQE30. It showed the expression vector pQE30-LAI was successfully constructed by double enzyme digestion analysis.Using the software tool such as http://www.ncbinlm.nih.gov/blast and vecterNTI,bioinformatic analysis of linoleic acid isomerase was made to predict Key amino acids, It was confirmed that phenylalanine 193, as the Key amino acids was mutated and the polar phenylalanine could be converted into non-polar serine by mutation of T to C at the position of 578.The mutation primers were designed and the recombinant plasmid pQE30-LAI was selected as a template , then site-directed mutagenesis was made with the site-directed mutagenesis kit QuickChange from Transgen. Finally, mutant vector pQE30-F193LAI was successfully constructed.Before and after mutation, enzyme activity of linoleic acid isomerase about the crude enzyme and the bacteria was determined after its expression induced by IPTG in E. coli. The results about the crude enzyme were 1999.79 U/ml and 1564.53 U/ml. The results about the bacteria were 117.11 U/ml and 87.96U/ml. The enzyme activity of recombinant plasmid pQE-LAI was all greater than the mutation plasmid pQE30-F193LAI, it showed that enzyme activity of the mutant was destroyed and phenylalanine 193 have played an irreplaceable role in the catalytic process of linoleic acid isomerase. The wild type and mutant expressed enzymes was extracted and detected. The results showed that both wild type and mutant had band at 68Kd and the linoleate isomerase had been expressed.更多还原