| Microbe proteases are robust enzymes with considerable industrial potential in detergents, leather processing, silver recovery, medical purposes, food processing, feeds, and chemical industries, as well as waste treatment. These enzymes contribute to the development of high value-added applications or products by using enzyme-aided (partial) digestion. A strain NSB-2of bacteria produce protease was isolated from NanHai nearsea water(stored by lab). After purification culture, the strain was identified by physiological, biochemical and genetic characterization as Bacillus. In order to improve the yield of the protease BP-1we optimized the fermentation conditions of marine NCB-2. And then the protease BP-1was preliminary purified and characterized. Meanwhile we screened a series of factors to keep the stability of the liquid enzyme. All the work we do lay the foundation for the industrial production of protease and its application in the future.According to morphology and biochemistry characteristic result, and16S rDNA sequencing analyses, NSB-2is identified as a Bacillus. The strain is gram-positive bacteria, length of about2to3microns. The growth pH ranges from5.0to11.0, and can be well growth in4℃to50℃temperature.The fermentation conditions of marine NCB-2were optimized use one-single factor and central composite design. The single factor was used to screen these seven factors:the nitrogen source, carbon source, inorganic salt, initial pH, inoculation volume, broth content, fermentation temperature and rotating speed. The best result carried out of each single factor was as follows:wheat bran50g/L, pulse flour40g/L,1.5g/L of magnesium sulfate,5g/L of sodium chloride, inoculation quantity of4%, initial pH7.0,40ml fluid volume, temperature30℃, speed180r/min. Then, the central composite design (CCD) and the analysis by Design expert software were adopted to determine the optimal level of the three main factors. The most suitable variables were identified as follows:wheat bran53.2g/L, pulse flour38.2g/L,3g/L of sodium chloride, respectively. The protease activity was predicted to be6466.8U/ml at the very conditions. The result of verification experiment under the optimum conditions showed that6346U/ml was the maximum activity. These results suggested that the predicted model was reliable and available for the optimization of protease fermentation conditions. Therefore, the yield of protease was raised obviously nearly7times compared with the basal medium fermentation conditions.Using ammonium sulfate precipitation and membrane separation purify the enzyme. The study of the preliminary purified protease characteristics indicating that might belong to the serine protease family. The enzyme had pH and temperature optima of10.0and50℃, of which condition the stability of the protease is very good. The metal ions of Ca2+, Mg2+,Ba+,K+,Co2+had no influence on the enzyme activity. Al3+,Li+,Fe2+,EDTA could inhibit the protease activity to some degree. And the activity was strongly inhibited by Fe3+,Zn2+,Ag+,Cu2+. The EDTA and usual surfactant tolerance such as SDS, TritonX-100,Tween-40,Tween-60shows that the enzyme has good application prospect as washing additive.The paper studied the effects of sugars(mannose, cane sugar, glucose and dextrin)and polyols (1,2propanediol, glycol, glycerol,D-sorbitol and mannitol) on the thermostability of BP-1were studied. Three additives (sucrose, chitosan and sorbitol)showed good performance to improve the stability of protease BP-1. The optimal combination of additive was obtained using the uniform design DPS. The liquid enzyme was under37℃at a calorstat. Real-time detection to the change of enzyme activity was carried out in7days. The optimal results showed to be glucose2%, glycerin7%and calcium chloride0.1%. After the test the enzyme activity retained38%higher than the blank group. |
PDF Full Text Request