Arachidonic acid(AA or ARA), an essential ω-6 polyunsaturated fatty acid(PUFA), can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of Δ6-desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of Δ6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of Δ6-desaturase and ARA content. Based on this, Δ6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.
We cloned and expressed bile salt hydrolase gene of Lactobacillus plantarum M1-UVS29 in Lactococcus lactis NZ9000 successfully. Gene-specific primers for amplification of L. plantarum bsh were designed by using sequence which availabled from Gen Bank. The production of PCR amplicon was confirmed by sequencing and cloned into pMD18-T vector, and then recombined into expression vector p NZ8148 and yielding vector p NZ8148-BSH. p NZ8148-BSH was transferred into Lactococcus lactis NZ9000. Sequencing indicated that the cloned bsh fragment contained 995 nucleotides, and shared 99.3% sequence homology with bsh gene from L. plantarum MBUL10. Cloned bsh fragment was successfully transduced into NICE expression system and confirmed by PCR and restriction digest. Recombinant BSH protein was analyzed by SDS-PAGE. The molecular weight of BSH protein was approximately 37 ku. Activity of the expressed protein was 0.77 μmol · min-1. The successfully expressed proteins by genetic engineering technology made the function of lactic acid bacteria be abundant and laid the foundation for further researches into cholesterol-lowering lactic acid bacterium food and probiotics.
采用模拟移动床色谱对微生物油甲酯中ARA甲酯进行分离纯化,以ARA甲酯的纯度和收率为指标优化其分离工艺。结果表明,模拟移动床色谱分离纯化ARA甲酯的最佳工艺参数为:切换时间720 s、进样流速39.3 m L/min、洗脱流速8 m L/min、解吸流速4.5 m L/min、再生流速5.5 m L/min。在该操作条件下,ARA甲酯纯度达到91%,收率达到92.9%。说明模拟移动床色谱可有效分离纯化微生物油甲酯中的ARA甲酯。
