The Preliminary Study Of Purification And Immobilization About D-hydantoinase

D-hydantoinase was separated and purified from Burkholderic cepecia 1003 following the steps of the crude separation(the cellular fragmentation ,the precipitation of (NH₄)₂SO₄), the purification of chromatography(Phenyl CL-4B HIC,DEAE Sepharose Fast Flow negative IEX) in this experiment. The purification multiple was 25.11 and the recycle yield of enzymatic activity was 11.87%. The result of SDS-PAGE electrophoresis was the relative molecular weight of the subunit was about 33 KD. After above some steps were optimized, the result was shown that the conditions of the ultrasonic cellular fragmentation were fragmentized time 20s,interval time 15s,power 600W,temperature 25℃,total 10min, and the conditions of the classifiable precipitation of (NH₄)₂SO₄ were the solid (NH₄)₂SO₄ was added to 40% saturation, placed for some time, the topper clarify liquid was collected after high speed centrifugal effect, then the solid (NH₄)₂SO₄ was added to 60% saturation in above liquid, placed for some time, the precipitation was collected after high speed centrifugal effect. The research on the characters of D-hydantoinase was shown that the condign concentration of substance in catalyzed reaction was 1.7mg/mL, the optimal pH was 9.0, the optimal temperature was 40℃, the range of pH stability was pH8.0～9.0, the range of temperature stability was 25℃～40℃, the catalyzed kinetic constants were km=13.1732mmol.L^-1,rmax=0.1744mmol.L^-1.min^-1. Eupergit C,Eupergit C_250L and the amino resins were selected and used as the carriers of the immobilized D-hydantoinase. We discussed the influences of pH of enzyme,concentration of enzyme,the immobilized time and the usage of EDC on the immobilized effects. The result was shown that the optimal immobilized conditions of two epoxy resins(Eupergit C,Eupergit C_250L) were pH of enzyme 7.0,concentration of enzyme 0.4mg/mL,the immobilized time 20h, and the optimal immobilized conditions of two amino resins(Eupergit C(-NH₂),Eupergit C_250L(-NH₂)) were pH of enzyme 8.0,concentration of enzyme 0.4mg/mL,the immobilized time 15h, the usage of EDC was Eupergit C(-NH2) 50μL,Eupergit C250L(-NH2) 150μL. Under the above optimal conditions of four immobilized D-hydantoinase, the recycle yield of enzymatic activity and the immobilized yield of protein for Eupergit C were 73.05%,81.67%, the recycle yield of enzymatic activity and the immobilized yield of protein for Eupergit C250L were 61.84%,86.85%, the recycle yield of enzymatic activity and the immobilized yield of protein for Eupergit C(-NH2) were 94.25%,44.24%, the recycle yield of enzymatic activity and the immobilized yield of protein for Eupergit C250L(-NH2) were 145.36%,72.2%. Based on above results, the basic characters of the immobilized D-hydantoinase prepared by four resins were studied primarily. The result was shown that the optimal pH of the immobilized D-hydantoinase used Eupergit C,Eupergit C250L and Eupergit C(-NH2) was 9.0, the optimal pH of the immobilized D-hydantoinase used Eupergit C250L(-NH2) was 8.0, the optimal temperature of four immobilized enzyme was all 60℃, the kinetic constants of the immobilized D-hydantoinase used Eupergit C were kmapp=111.6449mmol.L-1, rmax app=0.0229mmol.L-1.min-1, the kinetic constants of the immobilized D-hydantoinase used Eupergit C250L were kmapp=43.8111mmol.L-1, rmax app=0.0161mmol.L-1.min-1. The stability result of the immobilized D-hydantoinase was shown that after stored for about 50 days, the enzymatic activity of above four immobilized enzyme was lost in different degrees, but remained upwards a half at least.