Kandasamy Selvam, Muthusamy Govarthanan, Balakrishnan Senthilkumar, Duraisamy Senbagam, Thangasamy Selvankumar, Seralathan Kamala-Kannan, Chinnappan Sudhakar
Centre for Biotechnology, Muthayammal College of Arts and Science, Rasipuram, Namakkal, Tamil Nadu, 637 408, India.
Department of Applied Sciences, College of Environmental Technology, Muroran Institute of Technology, 27-1 Mizumoto, Muroran, Hokkaido, 050-8585, Japan.
3 Biotech. 2016 Dec;6(2):167. doi: 10.1007/s13205-016-0481-z. Epub 2016 Aug 12.
The aim of the study was to identify new sources of substrate from agro-industrial waste for protease production using Bacillus sp., a local bacteria isolated from an agro-waste dumping site. The strain was identified as Bacillus sp. BT MASC 3 by 16S rRNA sequence followed by phylogenic analysis. Response surface methodology-based Box-Behnken design (BBD) was used to optimize the variables such as pH, incubation time, coffee pulp waste (CPW) and corncob (CC) substrate concentration. The BBD design showed a reasonable adjustment of the quadratic model with the experimental data. Statistics-based contour and 3-D plots were generated to evaluate the changes in the response surface and understand the relationship between the culture conditions and the enzyme yield. The maximum yield of protease production (920 U/mL) was achieved after 60 h of incubation with 3.0 g/L of CPW and 2.0 g/L of CC at pH 8 and temperature 37 °C in this study. The molecular mass of the purified enzyme was 46 kDa. The highest activity was obtained at 50 °C and pH 9 for the purified enzymes.
本研究的目的是利用从农业废弃物倾倒场分离出的本地细菌芽孢杆菌属，确定用于生产蛋白酶的农业工业废弃物新底物来源。通过16S rRNA序列鉴定该菌株为芽孢杆菌属BT MASC 3，随后进行系统发育分析。基于响应面法的Box-Behnken设计（BBD）用于优化pH、培养时间、咖啡渣废弃物（CPW）和玉米芯（CC）底物浓度等变量。BBD设计显示二次模型与实验数据有合理的拟合。生成了基于统计的等高线图和三维图，以评估响应面的变化，并了解培养条件与酶产量之间的关系。在本研究中，在pH 8、温度37°C下，用3.0 g/L的CPW和2.0 g/L的CC培养60小时后，蛋白酶产量达到最大值（920 U/mL）。纯化酶的分子量为46 kDa。纯化酶在50°C和pH 9时活性最高。
