Screening Of Rhizopus Oryzae Overproducing L-lactic Acid And Optimization Of Fermentation Conditions

Rhizopus oryzae As3.3461 was mutagenized with ultraviolet radiation(UV) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) together, and two kinds of selecting methods were used according to largest throughput screen and metabolic controlling screen theory that were agar-indicator plate and Yeast-Peptone-Dextrose (YPD) plate containing allyl alcohol. As a result, four and eight mutant strains overproducing L-lactic acid were obtained respectively by using above two methods. Compared with other mutants, the 12th mutant strain (named as HBF-12) obtained from the latter method shows the highest L-lactic acid yield 88.4g/L which increases 30%than that of the parent strain, but ethanol yield is only 4.4g/L which decereases 74%. The efficient screen model of YPD plate containing allyl alcohol was founded.By unifactor and orthogonal design of experiment, the fermentation conditions of the mutant HBF-12 were optimized with flask. The results showed that the optimum fermentation medium are glucose 120g/L, (NH₄)SO₄4g/L, MgSO₄-7H₂O0.4g/L, ZnSO₄-7H₂O0.1g/L, KH₂PO-40.3g/L, CaCO₃60g/L,and the optimum culture conditions are liquid volume 20%, rotating rate 200r/min, culture temperature 32℃, under the optimum conditions, the L-lactic acid yield of mutant HBF-12 reachs 96.2lg/L. Hereditary stability test demonstrated that the productivities of mutant strain from 1st to 6 ^st serial passage are stable.Fermentation curves were investigated by using 7L bioreactor under the optimum conditions.By contrast with the parent strain, the ethanol yield and the Alcohol Dehydrogenase (ADH) activity of HBF-12 decrease 73.6% and 74%, respectively. Whereas, the L-lactic acid yield and the Lactate dehydrogenase (LDH )activity of HBF-12 increase 39.6% and 16. 2% than those of the parent strain, respectively.Because the activity of ADH in mutant HBF-12 decreases markedly,the enzymology characteristic of ADH from Rhizopus oryzae were studied in order to know more about mutagenesis mechanism. The optimum activity of ADH is observed at the temperature25℃ and at pH7.5. The range of temperature stability is below 35℃, the range of pH stability is from 6.5 to 7.5 .The activity of enzyme is activated by Zn²⁺ dramatically and inhibited by Mg²⁺ slightly. The K_m constants based on the substrats of aldehyde and NADH are 1.750×10^-3 mol/L and 2.049× 10^-3 mol/L respectively.