【作者】 于长青；

【导师】 邓旭明；

【作者基本信息】 吉林大学 ， 基础兽医学， 2009， 博士

【摘要】 花生四烯酸是一种人体必须的不饱和脂肪酸,具有广泛的生物活性和重要的营养作用。其传统制备方法成本较高、提取率低,微生物发酵法生产花生四烯酸可降低成本、提高产量。△5-、△6-脱饱和酶是花生四烯酸合成途径上的关键酶,评价脱饱和酶与花生四烯酸产量的关系,可进一步确立该酶在花生四烯酸生物合成中的作用。本研究的目的是选育花生四烯酸高产菌株,优化花生四烯酸提取和纯化的工艺参数,研究深黄被孢霉诱变菌株脱饱和酶的基因序列变化,探讨脱饱和酶的活性及mRNA表达量与花生四烯酸产量的关系。以深黄被孢霉为出发菌株,分别采用微波诱变、紫外诱变、原生质体再生诱变和紫外诱变原生质体等方法进行诱变,利用气相色谱分析花生四烯酸含量,最终获得四株花生四烯酸高产菌株,其中YZ-124花生四烯酸产量最高,为4.72 g/L,比出发菌株提高576 %,并且遗传性能稳定。采用CO2超临界萃取技术提取微生物油脂中的花生四烯酸,花生四烯酸提取率为45.7%,花生四烯酸含量为19.34%。采用尿素包合法纯化花生四烯酸,最终得到产品中花生四烯酸含量为36.84%。以深黄被孢霉原始菌株及高产花生四烯酸的诱变菌株为基础,利用RT-PCR及基因克隆方法获得了5株深黄被孢霉的△5、△6-脱饱和酶基因的完整cDNA,对其进行序列分析,结果表明:诱变菌株与原始菌株的核苷酸及氨基酸序列同源性较高。利用TTC-脱氢酶活性测定法及荧光定量PCR方法测定了5株深黄被孢霉的同一培养时间及高产花生四烯酸菌株YZ-124在不同培养时间的活性及mRNA表达量,结果显示:脱饱和酶的活性及表达量与培养物油脂中花生四烯酸含量呈正相关关系。更多还原

【Abstract】 Arachidonic acid is an essential dietary component for human beings,and has various physiological functions and plays an important role in infant nutrition. Arachidonic acid(ARA), widely existing in the animal kingdom,can be isolated from the lipids extracted from pig adrenal gland or pig liver and sardines as well; however, the yield of it is only 0.2%(w/w) or lower, so it is very dificult to be applied in a industrial scale and the research and application are restricted.It has been focused on the study of ARA fermented by microbe in the whole world.△5-、△6-desaturase is the key enzyme of arachidonic acid synthetic pathway. Evaluating the relation of desaturase and ARA production can further establish its function on biological Synthesis.The aim of this study is to selectively breed the high-yield ARA strain, to optimize the technology parameter of extraction and purification for ARA ,to study the gene sequence variation of Mortierlla isabellina mutant strain desaturase, to explore the activity of desaturase and the relationship between the expressed quantity and ARA production.Mortierella isabellina3.3410 was used as initial strain, microwave mutation, ultraviolet mutation, protoplast mutation and ultraviolet mutation protoplast were used to mutate, and then the content of the ARA was measured by GC. The ultimum four strains who had the most ARA yield and genetic stability were obtained. Under powe of microwave at 700W and heating time of 35s,lethality of spores of Mortierella isabellina was 78.3%.After two-time microwave mutation and repeated screen, a mutation of Mortierella isabellina W35s2-153 was obtained,whose ARA yield was 2.61g/L, 3.18 times of the control strains.The power of viltalight lamp was 20W,exposure distance was 30cm and exposure time was 80s,lethality of spores of Mortierella isabellina was 76.4%.After two-time ultraviolet mutation and repeated screen,a mutation of Mortierella isabellina Z80s2-109 was obtained, whose ARA yield was 2.74g/L, 3.77 times of the control strains.After protoplast mutation, enzyme concentration at 4%,enzymolysis temperature at 30℃and enzymolysis time at 7.5h,the rate of protoplast was 78.4%.After mutation and repeated screen,a mutation of Mortierella isabellina Y-69 was obtained whose ARA yield was 2.92g/L and the concentration in biomass were 3.56 times of the control strains. After ultraviolet mutation protoplast, the power of viltalight lamp at 20W ,exposure distance at 30cm and exposure time at 80s,lethality of spores of Mortierella isabellina was 76.4%,a mutation of YZ-124 was obtained,whose ARA yield was 4.72 g/L , 5.76 times of the control strains.The process condition optmization of arachidonic acid extraction from microbial oil Was carried out by means of supercritical CO2 fluid technology.It was used to increase the extraction rate of arachidonic acid in microbial oil. The supercritical CO2 fluid technology was used to extract the ARA from microbial oil; an orthogonal, rotatable, central composite was employed to determine the optimum conditions for extraction; GC was used to measure the content of the ARA. The results indicated that extraction temperature of 32℃, extraction pressure of 16mPa,extraction time of 101min and Taking 10% methanol as entrainer were the most Effective conditions, Which could obtain extraction rate of 44.6% and purity of 29.34%.Through the variance analysis and verification test ,it showed that mathematical model was reliable.An orthogonality experiment was employed to determine the optimum conditions for purification of ARA by urea clathration.GC was used to measure the content of the ARA. The results indicated that urea:fatty acid of 4:1, fatty acid:ethanol of 1:12, clathration temperature of -15℃and clathration time of 12h, the purity of ARA was 36.84%.The clone of△5-、△6-desaturase gene. Full cDNA of Mortierlla isabellina AS3.3410、W35s2-153、Z80s2-109、Y-69、YZ-124 5 strain△5-、△6 -desaturase gene was obtained by RT-PCR and gene clone based on the original strain of Mortierlla isabellina and the mutant strain of high-yield AA and its sequence was analyzed. The result showed that the full-length cDNA sequence of 5 strain Mortierlla isabellina△6-desaturase gene was 1374bp and 457 amino acids were coded.△5-desaturase gene was 1341bp and 446 amino acids were coded. The cDNA and amino acid sequence were analysed with biological information software. The results showed that the△5-、△6-desaturase gene sequence of Mortierlla isabellina AS3.3410 original strain had higher homology with Genebank published sequence, while nucleotide sequences of the mutant strain had low variation. Animo acid sequences of desaturase gene of the mutant strain had lower homology Compared with original strain sequence, which could show the protein of mutant strain probable had a certain variation.The relation on activity and expression levels of desaturase and ARA quantity. The activities of 5 strain Mortierlla isabellina at the same culture time and high-yield ARA strain YZ-124 at the different culture time were determined by TTC-desaturase activity determining method and the experimental condition was established by using fluorescence quantitative PCR method to determine the mRNA expression levels of Mortierlla isabellina△5-、△6 -desaturase gene. The results showed that the desaturase activities of different strain and different cultured time were different and the desaturase activity of original strain AS3.3410 was the lowest while the mutant strain YZ-124 was the highest. The activities of desaturase cultured in 3-5d increased obviously and were basicly stable after 5d.The results of fluorescence quantitative PCR testing showed the mRNA expression level of different strain△5-、△6 -desaturase gene were different.The desaturase gene mRNA expression quantity of Mortierlla isabellina YZ-124 at different culture time was different, the△5、△6 -desaturase mRNA expression quantity of 3-8d cultures gradually increased with bacterium age increasing, and the expression quantity reached the highest at 8d.The activity and expression levels of desaturase and ARA quantity in cultured oil presented a positive correlation with ARA acquisition.更多还原