Studies On The Production Of Alkaline Protease From Pseudoalteromonas Sp. Strain CHS

217 bacterium strains were isolated from seawater, fishes, shells and sediment in Lianyungang sea area, from which 23 strains with high production of alkaline protease were selected. Through physiological and biochemistry experiments and 16SrDNA sequence analysis, the two marine bacteria strains CHS and SY, were further identified as Pseudoalteromonas. The fermentation conditions of the alkaline protease production of CHS and SY were studied. And the alkaline protease produced by CHS was purified and its properties were studied as well.The optimal fermentation conditions for alkaline protease secretion of CHS and SY were: 15℃, 24hours, 2.5% inoculation amount, 180 r/min of shaking flask culture; The optimal initial pH for CHS was 9.0 and 8.0 for SY. The optimum carbon source was glucose and peptone as the optimum nitrogen source.The alkaline protease from strain CHS was purified from the culture supernatant by centrifugation, homogeneity by ammonium sulfate precipitation, dialysis, ion-exchange chromatography and gel filtration chromatography. The specific activity of the enzyme was raised from 59.4U.mg-1 to 860.8U.mg-1, which was 14.5 times that of the culture supernatant with a yield of 3.24%. The purified enzyme showed one protein band on SDS-PAGE and its molecular weight was estimated to be about 56.9 kDa..The optimum temperature for its casein hydrolysis was 40℃ and 10.0 as its optimum pH. It keeps stable between pH 8.0-11.0. When incubated at 40℃ for 30min, the enzyme activity decreased about 50%, which showed its low thermostability. The activity of the protease were inhibited strongly by PMSF, but was not inhibited by EDTA and IAA. Ions Ca2+ and Cu2+ were its activator for the activity of the enzyme, while the enzyme was inhibited intensely by Ag+. The enzyme was fairly stable in the presence of ethanol and urea, and resistant to Tween-20, either. Furthermore SDS inhibited its activity.