Screening, Identification, Optimization Of Fermentation Conditions And Stability Of Enzyme Activity

The objective of this paper is to isolation of protease strains to hydrolyse rice dreg protein and whole-cell of the screened strain is directly employed to hydrolyse rice dreg protein, which can advance green exploitation and full utilization of rice dreg protein. The advantages of this method is possessing benefit of enzymatic hydrolysis, reducing production cost, circulation utilization, simplicity and conveniency compared to chemical and commercial enzymic methods. Its researchful content contains isolation and identification of isolation to hydrolyse rice protein, optimization of the conditions of protease-producing isolate for hydrolysis rice dreg protein and discussion of stability of enzymic activity.A total of 116 protease isolates were screened from 23 samples rich protein by casein method. These protease strains were incubated with rice protein as exclusive carbon and nitrogen source. Two protease-producing isolates, that are HP60 and HP90 were gained from fermented samples by analysis of proteases of their fermentation liquors via further screenings. Enzymic activities of the HP60 and HP90 were reached 263±7 U/mL and 354±1.5U/mL, and degree of hydrolysis of rice dreg protein were achieved 15.63% and 19.83%, severally. The HP60 and HP90 strains were identified as Bacillus amyloliquefaciens HP60 and Bacillus subtilis HP90, respectively, and identified methods were by different morphological, physiological and biochemical characteristics(API 20 NE, Biolog GP; API 20 NE and API 50 CH) and analysis of their 16 S rRNA and recA sequence.On the basis of univariate analysis, fermentation conditions of hydrolysis of rice dreg protein were optimized based Orthogonal Design and Response Surface method for the strain HP60(CCTCC NO. M 2014200) and HP90(CCTCC No. M 2014201), respectively. And the optimal parameters of the strain HP60 and HP90 for hydrolysis of rice dreg protein were pH 7.0, fermentation time 66 h, inoculum amount 9% and concentration of rice dreg protein 1.0g/100 mL and pH 7.35, fermentation time 60 h, inoculum amount 7% and concentration of rice dreg protein 0.97g/100 mL, severally. The degree of hydrolysis of rice dreg protein was improved 23% and 42 % compared with before optimization condition by the strain HP60 and HP90, respectively.It was discussed that pH, temperature, and metal ions such as Ca2+、Mg2+、Cu2+ had an effect on stability of protease activities of fermentation liquors from the isolate HP60 and HP90. The results showed that the HP60 and the HP90 strain had a good stability of protease activity at temperature ranges 20°C~40°C and p H ranges 6~8, respectively. And 5mM Ca2+ and Mg2+ can play an activated role in protease activity of fermentation broth from the strain HP60 and HP90 but Cu2+ can play an inhibited role in that.