高产γ-氨基丁酸酵母菌株的亚硝基胍诱变选育

Breeding of High-yield GABA Saccharomyces Cerevisiae Strain by NTG Mutagenesis

目的:探讨亚硝基胍诱变选育高产γ-氨基丁酸酵母菌株的方法。方法:使用亚硝基胍对酵母菌株进行诱变;采用含溴甲酚绿的YEPD培养基筛选突变菌,采用薄层层析法和比色法鉴定变异菌株发酵液中的γ-氨基丁酸及其含量;对突变菌株连续继代培养4代,测定各代发酵液中γ-氨基丁酸的含量,鉴定诱变菌株的遗传稳定性。结果:亚硝基胍诱变酵母的最佳浓度为1.0g.L-1,最佳诱变时间为15min;获得了5株突变菌株,菌落呈绿色;薄层层析法鉴定突变菌株都能产γ-氨基丁酸;诱变菌发酵液中的γ-氨基丁酸含量各异,但高于对照,且增长幅度很大;对突变菌株后代遗传稳定性进行了鉴定,结果表明突变菌株4遗传性较稳定。结论:采用1.0g.L-1的亚硝基胍溶液处理酵母菌15min,经筛选鉴定,获得了一株遗传稳定的高产γ-氨基丁酸的酵母菌株。

Objective： The research aimed to study on the breeding method of the High-yieldGABA（Y- aminobutyric acid） Saccharomyces cerevisiae strain by NTG Mutagenesis. Methods： The strains of Saccharomyces cerevisiae were mutated by NTG,and then were cultured on YEPD medium plate with bromocresol green to screen the mutants. The GABA production in fermentation broth of mutant strains were determined by thin layer chromatography and spectrophotometry. In order to determine the hereditary character of the GABA-producing mutants, after four generations of successive culture, the GABA production in fermentation broth of mutant strains were measured. Results： The optimum concentration of NTG for mutation was 1.0 g.L^-1 and the optimum time was 15 min. Five mutants of the characteristics overproducing GABA were screened out. The typical colonies of mutants were green. In contrast with parent strains, the GABA production by the mutants were increased largely, and were different with each other. The highly GABA-producing mutant 4 was stable through the experiment of genetic stability. Conclusion：; A mutant Yeast strain overproducing GABA was screened from its parent strain by 1.0 g.L^-1 NTG mutation for 15 min.