Investigation Of The Organic Solvent Tolerant Protease Produced By Serratia Marcescens SYBC YH

Protease is one kind of importantly industrial enzyme, which has been used extensively in food industry ,leather processing and laundry detergents. So screening of microorganisms with unique catalytic properties and investigation of its fermentation character have strongly theoretical significance and potential appliedvalue.In this research, one kind of strain which can produce protease had been screened from decayed blue-green algae. Based on the analysis of colonial morphology, physiological-biochemiscal characteristic, 16S rRNA and fatty acid composition, this strain was identified to be Serratia marcescens and was named as Serratia marcescens SYBC YH.(1) Research about the influence of fermentation conditions on the production of protease. Carbon source and nitrogen source have been investigated in the liquid state fermentation. Optimized fermentation conditions: carbon source feather powder; nitrogen source corn steep powder; fermentation temperature 30; initial pH 5.0; inoculum level (v/m) 18%; rotation speed 200 rpm. Optimized medium composition (g/L): feather powder 21; corn steep powder9; NaCl 9. Under this optimized fermentation conditions and medium composition, the protease activity (537.6 U/mL)was 5.12 folds of that before optimization(105 U/mL). This train could make blue-green algae as sole nitrogen source to produce protease.(2) Resarch about the purification of protease. The electrophoretically pure protease KYH was purifiered via ammonium sulfate fractionation, ion exchange chromatography using DEAE-cellulose-52 column and gel filtration using Sephadex G-100 column. More than 7.59-fold purified protease was obtained and the recovery was 13.80%. The specific activity and molecular weight were 1004.12 U/mg and 48.5 kDa, respectively.(3) Research about enzymatic characteristics of purified protease K YH. The optimized reaction temperature and pH were 40°C and 5.0, respectively. Na+ has an some activition and Fe2+ has obious inbibition on KYH. Moreover, Protease has organic solvent tolerance. Km and Vmax of K YH are 1.5 mmol/L, 1107.9 U/mg, respectively.