Screening, Culture Optimization And Enzymatic Characterization Of A Trypsin-producing Strain

Trypsin is an important protease which is of great value in medicine, leather processing, food industry and agriculture. The main materials for producing this enzyme are from animals and the applications are contrained with some problems such as high costs, the risk of animal virus and regilous problems. Microbial trypsin can effectively solve the above problems, and microbial fermentation for trypsin can have low production costs, high yield and enzyme activity, and easy for preparation, which is gaining more and more attention in the recent years.In this paper, samples were collected from a tannery and were used for screening and isolating trypsin-producing strains. In the first screening, skimmed milk powder was used as the carbon and nitrogen sources in order to isolate the strains which can produce proteases. In the second screening, the protease-producing strains were taken into fermentation to detect the trypsin activity. Using the specific trysin detection method, 8 trypsin-producing strains were isolated and one strain named DMN6 which its trypsin activity was 20 U·m L-1 was chosen for the next research. According to the methods of physiological and biochemical identification and 16 S r DNA sequencing, the strain was identified as Bacillus licheniformis.The fermentation conditions were optimized via the method of single factor optimization. The optimized conditions were as follows: corn flour 15 g·L-1, soy peptone 15 g·L-1, potassium phosphate dibasic 7 g·L-1, potassium dihydrogen phosphate 3 g·L-1, ferric chloride 5 mmol·L-1, magnesium chloride 15 mmol·L-1, 1% inoculum, cultured 84 h under 37°C. As a results, the fermentation activity was up to 140 U·m L-1 and 7 times higher than the original enzyme activity.Trypsin in the fermentation liquid was purified using DEAE Sepharose FF anion-exchange chromatography and Superdex 75 gel filtration chromatography. The yield of purification was 24.5% and the purification fold was 8.5 which the specific activity was 350.0 U · mg-1. Its molecular weight was determined to be 44 k Da by SDS-PAGE analysis.The purified trypsin was detected for enzyme properties and the results showed that the optimum temperature of the enzyme was 65°C, the optimum p H was 9.0 and had a good range of temperature and p H adaptation. The enzme can have up to 85% of the highest activity in 45°C and had a good stability in this temperature, which indicated that the enzyme had a good prospect in industrial applications of lowe temperatures. Ba2+ at low concentration can increase trypsin activity significantly, while the majority of the metal ions showed some kind of inhibition. Serine protease inhibitor PMSF can strongly inhibit enzyme activity, which indicated that the enzyme is a serine protease. Surfactants in some degrees can decrease enzyme activity. The enzymatic reaction kinetic parameters were detected using BAEE as the substrate and the results showed Km and Vmax were 0.75 mmol·L-1 and 184 U·m L-1 respectively.