Study On The Regulation Of L - Lactate - Related Enzymes Produced By Lb.paracasei HD1.7

L-lactic acid is a kind of important organic acids which is used widely as the raw material in food processing,pharmaceutical industries and chemical engineering. Meanwhile,it is the precurosor of polylactic acid（PLA）which can be used as the raw material of biodegradable plastics.PLA has been recognized as one of the ideal materials to replace the traditional plastics,attributing to its biodegradeability,well usability and reproducibility of its material.Increasing the yield and the optical purity of the L-lactic acid and lowering the cost of production have become the hot topic in recent years.We isolated a strain of bacteria named Lb.paracasei HD1.7,which produced L-lactic acid with optical purity.The ability to produce L-lactic acid and metabolic characteristics of Lb.paracasei HD1.7 were studied by optimized cultural conditions and analyzed metabolic flux.If the metabolic properties of producing lactic acid can be analyzed on the level of enzyme activity and the production of the L-lactic acid can be improved by increased the metabolic flux,the ability to produce lactic acid will be raised,which has significant theoretically guiding effect on the metabolic control of the L-lactic acid fermentation.This paper studied the enzymes which related to the metabolism of producing lactic acid of Lb.paracasei HD1.7.The relation between the enzymes activity and the L-lactic acid fermentation have been analyzed through controled the nutritional and environmental factor to six metabolic related enzymes PFK,PK,LDH,PDH,PDC and PC.Then the sequences of 1dhL and 1dhD have been cloned and analyzed,which are the genes of key enzymes of producing lactic acid.The research results showed:1.When the concentration of glucose was lower than 45g/L,cell density was comparatively low,strain growthed well,the enzyme activity and the yield of the L-lactic acid was low. When the concentration of glucose was between 60g/L and 90g/L,enzyme activity was highest. But the yield of the L-lactic acid was not highest,because the activity of PDH,PDC and PC which are the enzymes in metabolic shunt pathways of pyruvate were increased simultaneously.When the concentration of glucose was 105g/L,the yield of the L-lactic acid was highest,that was 80.2%,24.1%higher than when the concentration of glucose was 15 g/L.Then,determined 105g/L was the optimal concentration of glucose for the fermentation of producing L-lactic acid.2.When the concentration of peptone was 5g/L,the enzyme activity,the cell density and the yield of L-lactic acid were all the lowest;when the concentration of peptone was 15g/L, the enzyme activity was the highest;when the concentration of peptone was 20g/L,the cell density was highest;when the concentration of peptone was 10g/L,the yield of the L-lactic acid was highest,that was 81.3%,15.5%higher than when the concentration of peptone was 5g/L.Then,determined 10g/L was the optimal concentration of peptone for the fermentation of producing L-lactic acid.3.When the concentration of MgSO₄·7H₂O,ZnSO₄·7H₂O and MnSO₄·H₂O was 0.15g/L,0.25g/L and 0.40g/L respectively,the activation to PFK and PK was the highest.When the concentration of MgSO₄·7H₂O,ZnSO₄·7H₂O and MnSO₄·H₂O was 0.20g/L,0.25g/L and 0.40g/L respectively,the activation to LDH was the highest.But under the above two conditions,the activity of PDH,PDC and PC also increased greatly,which was not beneficial to increasing the yield of the L-lactic acid.When the concentration of MgSO₄·7H₂O,ZnSO₄·7H₂O and MnSO₄·H₂O was 0.10g/L,0.25g/L and 0.40g/L respectively,the cell density was the highest,the yield of the L-lactic acid was also highest,that was 81.9%,21.9%higher than when the concentration of MgSO₄·7H₂O,ZnSO₄·7H₂O and MnSO₄·H₂O was 0.10g/L,0.25g/L and 0.20g/L respectively.So the best mixed concentration of Mg²⁺,Zn²⁺ and Mn²⁺ was 0.10g/L,0.25g/L and 0.40g/L respectively.4.When 0.075mg/L of vitamin B₁ was added into the culture media,the activation to PFK and LDH was the highest.When the concentration of the vitamin B₃ was 4.0mg/L,the activation to PDC was the highest.When the concentration of the vitamin B₇ was 0.1mg/L,the activation to PC was the highest.When the concentration of the vitamin B₇ was 0.075mg/L,the activation to PK,the cell density and the yield of L-lactic acid were all the highest,the yield of L-lactic acid was 84.5%,4.8%higher than blank. So added 0.075mg/L vitamin B₇ into the culture media was beneficial to improving the production of the L-lactic acid.5.When pH of fermentation was controlled between 4.5～5.0,the cell density,the enzyme activity and the yield of L-lactic acid were all the lowest;when pH was 6.5～7.0, the cell density and the enzyme activity were the highest;when pH was 5.5～6.0,the yield of the L-lactic acid was the highest,that was 81.2%.As a result,5.5～6.0 was the optimal pH of the fermentation of producing L-lactic acid by Lb.paracasei HD1.7.6.When cultural temperature was 30℃,the enzyme activity,the cell density and the yield of L-lactic acid were all the lowest;when cultural temperature rose to 42℃, before 12h,the activation to the enzymes was the highest.But in later period of fermentation,the activation fell dramatically,so was the yield of L-lactic acid.When cultural temperature was 37℃,the yield of L-lactic acid was highest,that was 81.6%. As a result,37℃was the optimal temperature of fermentation of producing L-lactic acid by Lb.paracasei HD1.7.7.By the template of genome DNA of Lb.paracasei HD1.7,1dhL sequence of 981bp and 1dhD sequence of 1002bp were obtained by cloning.Bioinformatics analysis showed:two sequences were 100%similar to nucleotide sequence of 1dhL of Lactobacillus casei BL23 and 99%similar to that of ldhD of Lactobacillus casei BL23; they belonged to the family of NAD dependant dehydrogenase;no obvious hydrophilic or hydrophobic property;no signal peptide;there was a obvious transmembrane domain in 1dhL sequence of 981bp,but there was no transmembrane domain in ldhD sequence of 1002bp;by secondary structure analyzed,it was certain thatα-helix was the primary constructional element of the protein.