Preparation Of The Lactate Oxidase Immobilized Catalytic Dl-lactic Acid Production Of Pyruvic Acid Research

The optimal culture medium and fermentation conditions for LactateOxidase production by Mycobacterium Smegmatis, extract technology ofLactate Oxidase, enzymology properties and preparative production ofpyruvate from DL-lactate by immobilized Lactate Oxidase on sodium al-ginate were investigated.The results were as follows: 3%glycrin, 0.4%peptone, l%DL-lactate,0.005 % VB₁, 0.05 % KH₂PO₄·H₂O, 0.05% MgSO_a·7H2O, 0.01%(NH₄)₂Fe(SO₄)₂ could enhance the yield of Lactate Oxidase. The optimiz-ed fermentation conditions were 50 mL medium in 250 mL conical flask,initial pH of the medium7.2, incubation temperature 37℃, fermentationtime 4d. Under these conditions the average activity of Lactate Oxidasewas 65.4 U/ml.Discussed two normal methods of extracting Lactate Oxidase fromMycobacterium Smegmatis by the Uniform Design. The optimal extracti-ng conditions of ultrasonic processing were as follows : the output powerof ultrasonic 100 W, the totalt reatment time of ultrasonic 8 min, the pHof phosphate buffer 7.8. The optimal extracting conditions of freezing andthawing method were as follows: the freezing and thawing times 4, thethawing time25min, the thawing temperature37℃, the pH of phosphatebuffer 7.4. The ultrasonic processing was more suitable to extract LactateOxidase than the freezing and thawing.Through proliferating, sonification and high speed refrigeratedcentrifugation, crude enzyme extracts were extracted from MycobacterimSmegmatis AS1.0562, and the enzyme was purified after 30%～70%saturation ammonium sulfate and dialysis, Then studies on theenzymology properties. The results showed that the optimal temperatureand pH for catalyzed of DL-lactate were 37℃and 7.4 respectively. TheLactate Oxidase was stable at the range ofpH 6.5～8.0, 40% activity waspreserved at 55℃for 60 min, and was denaturalized at 65℃. The enzymewas activated by low concentration of Na⁺ and Mn²⁺, Co²⁺, but Ca²⁺,Mg²⁺,Zn²⁺,Al³⁺,Fe³⁺,Cd²⁺ had significant inhibition on enzyme activity.The thermostability of Lactate Oxidase was enhanced in solution of SDS, Tween20 and Tween60. The kinetic analysis of enzyme showed that itcatalyzed the DL-lactate with a Michaelis constant 9.24x10^-33mol/L and aVmax2.42μmol/(min·mg).The Lactate Oxidase was immobilized on sodium alginate bycross-link with glutaraldehyde The immobilization conditions andpartial properties of immobilized enzyme were investigated, 1mL crudeenzyme and 1mL 4% sodium alginate were mixed and the mixture wasadded dropwise into a 20mL 0.2mol/L CaCl₂ solution solidification for 2hat the temperature of 25℃. Then cross-linked in the solution of 0.2%glutaraldehyde for 2h at the temperature of 25℃, after filtration anddrying the immobilized enzyme beads were gained. The thermal stabilityof the immobilized enzyme was enhanced prominence. The free enzymelost all its activity when heated at 65℃for 1h but the immobilizedenzyme still kept 86% of the original activity. The optimal reactiontemperature of the immobilized enzyme was 55℃compared to 37℃ofthe free Lactate Oxidase, the optimal reaction pH of the immobilizedenzyme was 7.4 as same as the free enzyme. Storaged at the temperatureof 4℃for 50 days without any protection by reagent, the free enzymeonly kept 40% of the original activity but the immobilized enzyme stillretained 80% of the activity. Preparative production of pyruvate from DL-Lactate by immobilized Lactate Oxidase, the highest yield of pyruvatewas 75% after 3h, and the immobilized enzyme maintained 85% of theactivity after consecutive use 5 times.