Study On Cold-adapted Protease Produced By Psychrotrophic Bacillus Cereus SYP-A3-2

Several cold-adapted microorganisms were screened from the NO.1 Glacier Mountain inChina by our laboratory. The strain SYP-A3-2, identified as Bacillus cereus, could secretecold-adapted protease. The fermentation conditions and characterization of the protease werestudied in this paper.The strain SYP-A3-2, which could grow in the temperature range from 0 to 38℃, was atypical psychrotrophile. The optimal temperature for production cold-adapted protease was 15℃. The optimal carbon and nitrogen sources were soluble amylum and casein, the optimalconcentration of them were 10g/L and 8g/L respectively. The strain couldn't grow well at thelow level of carbon and nitrogen sources, but the function of synthesis and secretecold-adapted protease could be suppressed at the high level. The protease secreted from cellcould be more stable by adding the Ca2+ to fermentation broth.The cold-adapted protease, stable at 15℃, could be secreted in large quantity by the strainof SYP-A3-2. When the culture temperature was changed from 15℃ to 20℃, the strain grewfaster while the enzyme yield was decreased. In order to shorten fermentation cycle, thefermentation process could be carried out in different phases by controlling the temperature.After fermenting for 8hr at 20℃, the temperature was declined at the rate of -1.25℃/h tillto 15℃, at which the fermentation process was continued to the end. During this course theenzyme activity was not affected and the fermentation cycle was shortened from 48hr to 36hr.The higher casein concentration, the more cell and lower protease production. Feeding caseinto broth for the control of nitrogen source was adopted. Started with 8g/L casein initially, after36hr the 50g/L casein liquor was added by 50mL per 4 hours, the enzyme activity was 8.13percent higher than the control. The two control methods were worth applying in the industry.The purification of the cold-adapted protease was also studied in this paper. The 36 kDenzyme protein was obtained by chromatograph of DEAE and Superdex-G75. It's optimalreaction pH,temperature were 7.0~8.5, and 42℃ respectively. The cold-adapted enzymeactivity could be maintained well at 20~30℃, and the enzyme activity at 0℃ was 6.3 percentof that at 42℃. The enzyme activity could be activated by Mn2+ while could be suppressed byCu2+ As the enzyme activity was suppressed by EDTA greatly, not by serine protease inhibitor(PMSF), the enzyme was supposed to be one of metallproteases.