Metabolic Control For The Production Of L-lactic Acid By Rhizopus Oryzae

L-lactic acid is a versatile biological chemical which has been found wide applications in food processing, pharmaceutical industries, and chemical engineering, et al. Recently, L-(+)-lactic acid production has received much attention because of the use of L(+)-lactic acid as a starting material of polylactic acid (PLA), a new biodegradable plastic, which will solve one of the worldwide environmental problems in the near future. It is speculated that the yields of lactate would exceed the production scale of citrate in recent years and received the first place in organic acid market.Rhizopus oryzae is an important filamentous fungus which synthesize high optical-purity L-lactic acid under aerobic conditions, but it suffers from some limitations such as longer fermentation period, lower conversion rate from glucose to lactate, strain degeneration, and unstable fermentation parameter, et al. In this dissertation, the preparations of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) from Rhizopus oryzae mycelium were obtained respectively and their properties were investigated. The regulation characterization of ions of Mg²⁺ and Zn²⁺, and aeration conditions on fermentation behavior were also researched based on the theories of metabolic control fermentation. The Rhizopus oryzae mutant with high-producing lactate was screened out under the guidance of metabolic control breeding. The main conclusions were as follows:(1) The method of EDTA titration for determining the lactic acid level in Rhizopus oryzae fermentation broth was modulated. The results indicate that lactic acid in fermentation broth had to be neutralized with excessive CaCO₃ before titration, then the pH value of solution was adjusted to over 12.0 with NaOH, and calcein as the indicator, a good reproducible result and recovery rate could be achieved. A reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed for the separation of 5 organic acids and quantitative determination of lactic acid in fermentation broth. The results showed that this method is accurate, simple, rapid and the results are reproducible.(2) The crude LDH preparation from mycelium of Rhizopus oryzae had optimum reaction pH of 7.5 and optimum catalysis temperature of 30～50℃. Mg²⁺ and Ca²⁺ could increase the activity of LDH, but K⁺ and Zn²⁺ reduce its activity. The K_m constants based on the substrates of NADH and pyruvate were 7.22×10^-4mol/L and 1.24×10^-3mol/L respectively. The optimum reaction temperature of The crude ADH preparation of Rhizopus oryzae was 25℃, its activity decreased rapidly when temperature was above 30℃. The pH value of reaction solution also had a marked influence on ADH activity, the optimum pH was 7.5. Addition of 1μmol EDTA, Mg²⁺, Ca²⁺ of Zn²⁺ to reaction system could reduce the activity of ADH. The K_m constant of ADH based on the substrata of acetaldehyde was 6.90×10^-4 mol/L.(3) An increase in activity of LDH was observed in broth of Rhizopus oryzae AS3.3461 by the addition of Mg²⁺.The maximum activity was found and production of lactic acid was 63.17g/L in flasks and 65.33g/L in bioreactor, respectively, when Mg²⁺ concentration was at 0.04%. The activity of LDH was affected by the addition of a little Zn²⁺. The activity of LDH was improved when Zn²⁺concentration was at 0.03%. The maximum production of lactic acid was observed in broth. A decrease in activity of LDH was observed and the production of lactic acid decreased significantly with the increase in Zn²⁺ concentration. The maximum production of lactic acid was 64.24g/L in flasks and was 66.37g/L in 5 L bioreactor when Mg²⁺concentration and Zn²⁺concentration were 0.04% and 0.03%, respectively.(4) The maximum production of lactic acid was 69.33g/L and the activity of LDH was 6.14U/mL when 10% volume of medium was used in cultures of Rhizopus oryzae AS3.3461 in flasks. The production of lactic acid and the activity of LDH were both low when the volume of medium reached 30%. The ethanol concentration was enhanced with an increase in volume of medium. It was 17.48g/L at 30% volume of medium. The maximum production of lactic acid was 68.14g/L in cultures shaken at 220r/min. The lactic acid concentration and the activity of LDH were 67.5g/L and 5.83U/mL, respectively, which increased by 64.4% and 49.9%, respectively, by single film fermentation. The ethanol concentration and the activity of ADH increased by 30.0% and 25.5%, respectively, by dual film sealed fermentation compared with single film fermentation. The maximum production of lactic acid reached 69.95g/L at 30% volume of medium and the least production of lactic acid was 64.80g/L at 50% volume of medium in 5 L bioreactor. The maximum production of lactic acid and the highest activity of LDH were 71.23g/L and 6.79U/mL, respectively, in cultures shaken at 600r/min in bioreactor. The maximum production of lactic acid was 71.78g/L in cultures aerated at 0.7L/(L.min). The maximum production of lactic acid was 68.14g/L and the activity of LDH, ethanol concentration and the activity of ADH were 5.79g/L, 16.28U/mL and 15.44U/mL, respectively, in cultures of flasks at 20% volume of medium and shaken at 220 r/min. The maximum production of lactic acid was 71.78g/L and the activity of LDH, ethanol concentration and the activity of ADH were 6.22U/mL, 14.83g/L and 13.69U/mL in cultures of 5 L bioreactor at 40% volume of medium, shaken at 600 r/min and aerated at 0.7 L/(L.min).(5) During L-lactic acid fermentation with Rhizopus oryzae, there existed a branch pathway by which pyruvate was transformed to ethanol catalyzed by pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), so the flux of pyruvate to lactic acid decreased, and the by-product ethanol accumulated. The mutant with lower ADH activity were screened out by mutagenizing the spores of Rhizopus oryzae with nitrosoguanidine (NTG) and ultraviolet radiation. It was found that the appropriate dosage of NTG to mutagenize Rhizopus oryzae was 0.1mg/mL and the optimum radiation time of UV wan 180s. The screened mutant swsp-1-2 with decreased ADH activity had a higher lactate concentration of 81.0g/L, and a decreased alcohol content of 5.6g/L in fermentation broth compared with the parent strain.(6) Using PCR method, the DNA fragment containing ldhL gene was obtained from Rhizopus oryzae genomic DNA. After sequenced, the fragment contains 998 bp, which is reported in GenBank under accession numbers EF152288. Based on T/A cloning strategy, the PCR product was cloned into PMD18-T vector firstly, and then the positive recombinant plasmid DNA was cleaved with two restrict enzymes. After purification, the cleaved DNA fragment was inserted into the pET17b, which is an expression plasmid and had been cleaved with the same enzymes. The recombinant plasmid was named as pET17b-ldhL. Then, the pET17b-ldhL was transformed into E. coli, BL21 strain, and induced with IPTG to obtain the recombinant ldhL protein. After SDS-PAGE, the result showed expected recombinant ldhL protein band. The above results demonstrated that the ldhL of Rhizopus oryzae could be expressed in E. coli using recombinant strategy.The above results can provide some theoretical basis for the establishment of metabolic control system of Rhizopus oryzae fermentation, directed-screening of L-lactic acid high-producing strain and the construction of "engineered strain". At the same time, the research can also act as a reference for carbon metabolism regulation of other microorganisms.