Preparation And Its Controlling Mechanism Of Arachidonic Acid Produced With Mortierlla Isabellina At High-yield

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.