The Optimal Culturing Conditions Of MR 137-3, Characterization And Immobolization Of Its Proteinase

Abstract: The optimal culturing conditions of high proteinase-producing Mucor racemosus 137-3 and the catalytic effect ,the security and the immobolization of MR 137-3 proteinase were studied. MR 137-3 proteinase was isolated and purified ,and properties of the enzymcthe influence of metal ionics and modification reagents on the proteinase catalytic activity were also studied.Mucor racemosus 137 was isolated from fermented bean curd and brought to mutation by radiation of CO2 laser. The duration for radiation was 40s with distance of 22cm, and the diameter of laser bundle was 0.4cm. High proteinase-producing mutant MR137-3 was obtained after screening cultures. The MR137-3 was found to have stable proteinase-producing activity in descendant cultures. The optimal culturing could be obtained at 30癈, pH 6.0, culturing duration of 72h, with water content of 65% of culture medium.NaCl solution of 0.3mol/L was adopted to extract the crude protein from solid-fermentationed material at 40℃ and then the crude protein was concentrated with hollow cellulose DC-30 column. Proteinase of MR137-3 has high safety in application. And its catalytic effect on milk protein hydroysis is better than papain on the market.This paper deals with the immobilization of MR 137-3 Proteinase onchitosan(as a support) by glutaraldehyde(as a cross-linking reagent). The best conditions of immobilization were 0.5 g dry chitosan ,0.4% glutaraldehyde and 0.3g proteinase,treating for 6 hours.So 62.3% of the enzyme activity could be reserved . Optimum pH of immobilized proteinase descended . Enzyme activity was hardly effected by pH in the range from pH 6.5 to pH 7.5 and was high in the range from 55℃ to 70℃. The activity of free enzyme reached to its maximum at 2mol/L urea,but descended with the increase of the urea concentration.On the contrary, the activity of immobilized enzyme at 2mol/L and 5 mol/L urea was higher than at other urea concentration. Both free enzyme and immobilized enzyme almost lost equal activity after treated by Zn2+. Ca2+ had little influence on the activity of free enzyme but led to 60% loss of the activity of immobilized enzyme. After dealed with Cu2+, Mn2+ Mg2+, the activity of free enzyme decreased but the activity of immobilized enzyme increased strikingly. The heat resistance of enzyme was enhanced by immobilization. At 80℃ and during 10 minutes, 17% of the activity of immobilized enzyme was reserved but only 6% of the activity of free enzyme did. During 20 minutes, 7% of the activity of immobilized enzyme was reserved but almost all the activity of free enzyme lost. The organic solvents resistance of enzyme was enhanced by immobilization too .except the resistance to acetone.The purified proteinase was obtained by sulfate fractionation, chromatography with DEAE-Sephadex A-50.CM-Sephadex C-50 and Sephadex G-100.The purified enzyme showed a single electrophoretic band on SDS-PAGE. The specific activity was 117490.4 U/mg and the purification multiple was 118.8 . Its subunit weight was 33.9 kDa. Isoelectric focusing study showed that pi of the enzyme was 8.4. The optimum pH value for the enzyme was 9.0 and the optimum temperature was 45℃.The enzyme was stable under 50℃ and in the range from pH6.0to pH 11.0 - especially at pHlO.O.The Michaelis-Menten constant (Km) was 5.35g/ml. The enzyme was activated by Mg2+and Fe2+ strikingly, while inhibited by Ag+ and Cu2+. EDTA had little effect on the activity of the enzyme. DFP and PMSF could inhibit almost all the activity . NBS had notable inhibitation on the activity while pCMB didn't at all.