Lactate Dehydrogenase Preparation And Immobilized

The optimal culture medium and fermentation conditions for Lactate Dehydrogenase production by Saccharomyces cerevisiae,extract technology of Lactate Dehydrogenase,enzymology properties and preparative production of L-lactate from pyruvate by immobilized Lactate Dehydrogenase on differenet carder were investigated.The results were as follows:glucose 50 g/L,peptone+yeast 5 g/L, VB₁ 0.1 g/L,MgSO₄ 0.5 g/L,KH₂PO₄ 1 g/L,NaCl 0.5 g/L could enhance the yield of Lactate Dehydrogenase.The optimized fermentation conditions were 50 ml medium in 250ml conical flask,initial pH of the medium 6.5,incubation temperature 35℃,fermentation time 28 h. Through five experiments under these conditions,the value of RSD is 3.9 %,so the optimal technical condition has high stabilization.Discussed two normal methods of extracting Lactate Dehydrogenase from Saccharomyces cerevisiae by the Uniform Design.The optimal extracting conditions of freezing and thawing method were as follows: the freezing and thawing times 6,the thawing time 35 min,the thawing temperature 40℃,the pH of phosphate buffer 5.8,the average activity of Lactate Dehydrogenase was 241.7 U/ml.The optimal extracting conditions of ultrasonic processing were as follows:the output power of ultrasonic 400 W,the totalt reatment time of ultrasonic 10 min,the pH of phosphate buffer 5.8,the average activity of Lactate Dehydrogenase was 286.8 U/ml.The ultrasonic processing was more suitable to extract Lactate Dehydrogenase than the freezing and thawing.Through proliferating,sonification and high speed refrigerated centrifugation, crude enzyme extracts were extracted from Saccharomyces cerevisiae,and the enzyme was purified after 30%-70%saturation ammonium sulfate and dialysis,Then studies on the enzymology properties.The results showed that the optimal temperature and pH for catalyzed of pyruvate were 35℃and 6.8 respectively.The Lactate Dehydrogenase was stable at the range of pH 6.2-7.4,50%activity was preserved at 55℃for 60 min,and was denaturalized at 65℃.The enzyme was activated by low concentration of Na⁺,Mn²⁺and Co²⁺,but Ca²⁺,Fe³⁺,Zn²⁺,Ba²⁺,Cu²⁺,Mg²⁺and Al³⁺had significant inhibition on enzyme activity.The thermostability of Lactate Dehydrogenase was enhanced in solution of SDS,Tween20 and Tween60.The kinetic analysis of enzyme showed that it catalyzed the pyruvate with a Michaelis constant 2.58 mmol/L and a V_max306.75 mmol/min.Magnetic chitosan microspheres(M-CS)were prepared and used for Lactate Dehydrogenase immobilization.The optimum technology and the properties of immobilized enzyme were studied.The optimal immobilization conditions for Lactate Dehydrogenase were:20 ml of 0.25 mg/ml of Lactate Dehydrogenase in phosphate buffer(0.05 mol/L,pH 7.0). reacted with 50 mg of magnetic M-CS at 4℃for 2 h.The microspheres were characterized by transmission electron microscopy,the results showed that M-CS were regular sphere with an average size of 30 nm and had magnetic response characteristic.The M-CS suspended in H₂O solution was easily precipitated and separated by magnetic field. Mechanical strength and crosslinking degree of M-CS were influenced by the ratio of carrier and free enzyme,ion concentration in phosphate buffer and pH of the solution.The immobilization was slightly influenced by the reaction temperature.The optimal reaction temperature of the immobilized enzyme was 40℃compared to 30℃of the free enzyme,the optimal reaction pH of the immobilized enzyme was 6.8 as same as one of the free enzyme.The immobilization would improve its thermal,basic resistant and acid resistant stability.The K_m value of immobilized enzyme for pyruvate was 2.58 mmol/L compared to 3.31 mmol/L of the free enzyme,it would reduce its appetency for the substrate.The immobilized enzyme maintained 70%of the activity after circular use 5 times. Storaged at the temperature of 4℃for 30 days without any protection by reagent,the free enzyme only kept 20%of the original activity but the immobilized enzyme still retained 60%of the activity.The Lactate Dehydrogenase was immobilized on camcium alginate by cross-link with glutaraldehyde.The immobilization conditions and partial properties of immobilized enzyme were investigated.1 ml crude enzyme and 1 ml 4%sodium alginate were mixed and the mixture was added dropwise into a 20 ml 2%CaCl₂ solution solidification for 4 h at the temperature of 25℃.Then cross-linked in the solution of 0.5% glutaraldehyde for 4h at the temperature of 35℃,after filtration and drying the immobilized enzyme beads were gained.The thermal stability of the immobilized enzyme was enhanced prominence.The free enzyme lost all its activity when heated at 65℃for lh but the immobilized enzyme still kept 86%of the original activity.The optimal reaction temperature of the immobilized enzyme was 55℃compared to 35℃of the free enzyme,the optimal reaction pH of the immobilized enzyme was 5.8 compared to 6.8 of the free enzyme.Preparative production of L-Lactate from pyruvate by immobilized Lactate Dehydrogenase,the highest yield of L-Lactate was 75%after 3 h,and the immobilized enzyme maintained 85%of the activity after consecutive use 5 times. Storaged at the temperature of 4℃for 30 days without any protection by reagent,the free enzyme only kept 40%of the original activity but the immobilized enzyme still retained 80%of the activity.