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Molecular cloning and host optimization study for enhanced expression of thermostable protease to meet the commercial demand

Jatavathu. Madhavi , Jatavathu. Srilakshmi, M.V. Raghavendra Rao, K. Krishna Satya, KRS Sambasiva Rao

The present study was designed to explore potential of thermostable protease in leather processing and further host optimization studies to produce enzymes to fulfill commercial demand. Thermostable protease gene from Geobacillus stearothermophilus was cloned in pET28a vector and expressed in E. coli BL21 (DE3). The new generation expression host systems were used in the current study to enhance expression fold and we have used E. coli C41 (DE3) and E. coli Rosetta and achieved more than three time expression of conventional host system. An average molecular weight 60 kDa thermostable protease was produced by recombinant DNA technology and the enzyme has shown tremendous scope in leather processing especially dehairing. The expressed thermostable protease was stable and active in different range of temperature (20-900C) and pH (5-13). The protease inhibitors have shown minimal inhibition on protease activity and stability. The expressed thermostable protease was reported significant kinetics parameters Km (0.9mM) and Vamx (0.084 mM/Sec) and maximum enzymatic activity was reported 0.18U/ml (E. coli rosetta) and 0.17 U/ml (E. coli C41) at pH 8 and 650C. The expressed protease was analyzed for proteolytic activity, dehairing activity and shown tremendous scope for leather processing.