低能离子诱变选育华根霉高产L-乳酸菌株的研究

对L-乳酸产生菌华根霉(Rhizopus chinensis)适宜产酸的条件进行了初步研究和优化,并利用10 keV氮离子对出发菌L-乳酸产率进行诱变。为比较出发菌株和突变菌株在分子水平的差异,利用108条RAPD随机引物对出发菌株和突变菌株进行了比较分析。结果表明:在葡萄糖90‰、(NH4)2SO42‰、KH2PO40.6‰、Mg-SO4.7H2O 0.25‰、ZnSO4.7H2O 0.05‰、CaCO350.0‰pH值自然,接种量在5%左右,34℃,200 r/min的培养条件下,可获得较高的L-乳酸产量(28.8 g/L)。氮离子注入诱发L-乳酸产量突变的适宜剂量为1.30×1015ions/cm2。筛选获得的突变株RC-H13 L-乳酸含量为33.7 g/L,比出发菌株提高17%,且遗传稳定性较好。在RAPD分析中108条引物有3条在两池间表现多态性,差异条带的频率为0.67%。

Screening High-yield Bacteria Rhizopus chinensis Carrying L-lactic Acid by Low Energy Ions Implantation

The L-lactic acid producing condition of Rhizopus chinensis was optimized. The high-yield mutant strain was obtained by the method of implanting nitrogen ion ( N+ ) into Rhizopus chinensis in 10 keY. In order to compare differences at the molecular level between the original strain and the mutant strain, 108 pieces of random primers were designed to clarify the genetic background by randomly amplified polymorphie DNA (RAPD). The results showed that the final concentration of L-lactic aeid reached 28.8 g/L when the optimal fermentation medium consisted of glucose 90 ‰, (NH4)2SO42‰, KH2PO4 0. 6‰, MgSO4 ·7H2O 0.25 ‰, ZnSO4·7H2O 0.05 ‰, CaCO3 50.0 ‰ and 5 % volume of seeds and fostered at 34 ℃. A high-yield mutant strain was obtained using N+ implantation with the dose of 1.30×1015 ions/cm2, whose L-lactic acid yield was 17 % higher than that of the original one, which had high genetic stability. By RAPD analysis, 3 primers whose amplification products showed polymorphic eharacter were selected from 108 primers, which can be used to discern the mutant strain.