| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 88 === A protease producing bacterium was isolated and identified as Bacillus sp. strain P-6. The optimal condition for protease production was found, when the culture was shaken at 37℃ with 100 rpm for 30 hours in 250 ml Hinton flask containing 50 ml of medium consisted of 0.5 % disodium succinate ( 6 H2O ) , 0.2 % casamino acid, 0.2 % yeast extract, 0.14 % NaH2PO4, 0.44 % K2HPO4, 0.01 % CaCl2 and 0.02 % MgSO4. Under such conditions, protease activity of 342 U/ml was attained.
The protease was purified through ultrafiltration, ammonium sulfate fractionation, CM-Sepharose CL-6B ionic exchange chromatography, and Sephacryl S-200 gel filtration. A 9-fold purification of the enzyme over crude enzyme preparation and specific activity of 8066 U/mg was shown. And the recovery of activity was 14 %. The molecular weight of the enzyme was determined to be 35 kDa by SDS-PAGE. The N-terminal amino acid sequence of this protease is VTGTN(Y/V)VGTGIG , and showed a high degree of homology with other neutral proteases belonging to thermolysin superfamily .
The protease was found to have a pH optimum at 7.0, a temperature optimum at 60℃ as acting on casein. The protease was stable at ~55℃and pH 8.0-10.0. Among several natural substrates determined, casein was the best substrate. Limited substrate specificity of the protease was observed while the product of casein digested by the protease was analysed with SDS-PAGE.The activity of protease was strongly inhibited by EDTA. The EDTA-inhibited protease was partial reactivated by Co2+, Ca2+, Mg2+, Fe+2.
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