The present study reports production, partial purification, and media optimization for alkaline protease using Bacillus cereus PW3A. A profiling study for protease production indicates maximum enzyme activity (17.22 U/ml) was observed after 48 h of incubation. The studies also showed that the enzyme activity increased with the decrease in carbon content indicating the growth associated with nature protease production. Partial purification of protease was done using ammonium sulfate precipitation and dialysis. Further studies were conducted to assess significant media ingredients influencing protease production using the one-factor-at-a-time approach and Plackett-Burman design. Fructose and yeast extract were identified as the most significant variables. Response surface methodology was applied to optimize the factors for maximizing protease production. The results showed that the production increased from 17.22 U/ml to 47.43 U/ml indicating a three-fold augment in enzyme activity. Characterization of protease showed that the highest enzyme activity was shown at pH 8.0 and temperature 50°C; however, significant enzyme activity was retained till pH 10 and temperature 60°C. Using casein as substrate, the enzyme showed maximum activity V max 39 U/ml and K m 18 μM. The activity was enhanced by MgCl 2 and CuSO 4 and inhibited by HgCl 2 . Since the enzyme has both pH and temperature stability with greater substrate affinity, this protease finds many useful industrial applications.
The study highlights the production of chitinase from Bacillus cereus BSH-4, which was isolated from a marine soil source and identified by 16S rRNA gene sequencing. After 96 hours of incubation in shake flask conditions profiling studies showed maximum enzyme activity and growth. The ammonium sulfate precipitation method was used at 80% saturation for partial enzyme purification with further purification done by dialysis wherein the purification fold increased to 1.75. The sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and silver staining methods were employed to assess the enzyme molecular weight observed to be 60 kDa. The enzyme activity and stability process parameters effects like temperature and pH were studied. At a pH value of 7.0, the greatest enzyme activity was noted which was relatively retained at 80% till pH 9.0. Similarly, higher enzyme activity was observed at a temperature of 30 o C and retained 70% relative activity till 60 o C, indicating the thermostable nature of the enzyme. Studies on the influence of metal ions showed that Zn 2+ , Cu 2+ , Ca 2+ , Mg 2+ , Mn 2+, and Fe 2+ inhibited the enzyme by 20% to 80%. An investigation was also done on the influence of organic solvents like methanol, ethanol, acetone, and isopropyl alcohol on enzyme stability. The results showed that the enzyme was stable in all the solvents, exhibiting 43% to 70% relative activity. The enzyme kinetic studies indicated Km and Vmax values of 4.54 mg ml -1 and 76.92 UmL -1, respectively. The enzyme also demonstrated antifungal activity against Aspergillus niger NCIM 1207 using the agar well diffusion method.
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