The Screened Production Of High-temperature Protease Bacteria And Its Enzymatic Properties

The thermophilic bacteria strains which produce thermophilic protease were isolated from the hot spring samples from Nanjing, Tangshan using media designed. After secondary screening, four strains TSl, TS2, TS3 and TS4 which produce thermophilic protease were obtained. The output of thermophilic protease produced by TSl is more than others .Based on morphological and physiological properties, strain TS1 was determined as GeobaciiJus. By multiplex test, the influence of various factors on enzyme production of strain TS1 was investigated .The suitable fermentation condition of strain TSl was obtained. The medium was as follows: pH7.5, glucose0.1% , casein 1.5%, lactose 3%, yeast 5% , sodium chloride0.5% , zinc sulfate0.001% and incubation for 9 hours at 50℃. In these factors, casein , lactose and yeast were the highly marked influences and temperature was also of important influence. The activity of 36.5 u/mL in fermented culture could be obtained under the suitable condition.The purification procedures included centrifugation, ammonium sulfate precipitation, dialysis and column chromatography with Sephadex-G100 and acetone deposition, the activity of the purified enzyme was 89.2u/mL.The protease has two subunits and the subunits' molecule weights are about 20.72kD, 18.14kD respectively.The optimum pH value and temperature were pH7.5 and 60℃. The enzyme was stable when incubated at 50℃ for 9 hours. The activity of the thermophilic protease was inhibited by EDTA and some ions such as Fe²⁺,Mg²⁺, Cu²⁺. But The activity of the thermophilic protease was accelerated by Zn²⁺and the ions of Ca²⁺ had no influence on the activity.