Serratia sp. TKU019所生產蛋白酶及幾丁聚醣酶之純化與定性

Abstract

The protease and chitosanase producing strain, Serratia sp. TKU019, was isolated from the soil in Taiwan. The optimized condition for protease and chitosanase production were found when the culture was shaken at 37℃ for 3 days in 100mL of medium contain 0.5% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH7). One chitosanase (C1) and one protease (P1) were purified by chromatography procedures of DEAE-Sepharose, Macro-prep DEAE Cartridge, and Sephacryl S-100. The molecular mass of the chitosanase (C1) and the protease (P1) determined by SDS-PAGE was approximately 36 kDa and 57 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of C1 were ( pH 11, 60℃, pH 7-10 and <40℃),(using colloidal chitin as substrate) and ( pH7, 60℃, pH 4-7 and <40℃) ,(using chitosan as substrate).Those of P1 were pH 10, 50℃, pH 5-9 and <40℃,respectively. Both of C1 and P1 were inactivated by Mn2+ and EDTA, but only P1 activated by 2% (v/v) Tween 40. The result of the effects of SPP and SSP on reducing sugars show that, the culture supernatant of SPP (1.5%, 5th day) and SSP (1%, 3rd day) had the highest concentration of reducing sugars ( 900 μg/ml and 270 μg/ml, respectively). To analyze antioxidant activity, it was found the culture supernatant of SPP (0.5%, 2 nd day) and SSP (1.5%, 1st day) had the best results of the DPPH free radical scavenging activity.