Molecular Characterization of Thermo Stable Proteases from Thermophilic Bacterial Strains

Thermophilic microorganisms have gained world-wide importance due to their tremendous potential to produce thermostable enzymes that have wide applications in pharmaceuticals and industries. Proteases are such enzymes which account for nearly 60% of the total world-wide enzyme sales. Thermostable proteases are of greater advantage in applications because they do not only denature at high temperatures, but they also remain active at such temperatures. This project explores the production and biochemical and molecular characterization of the thermostable proteases from thermophilic bacterial strains. The growth conditions of Bacillus subtilis strains in submerged cultivation have been investigated to understand fermentation behavior of the microorganism and the production pattern of the thermostable enzyme. The optimum protease production (178 U/mL) was observed with 1% casein substrate after 48 h of cultivation at 60°C from Bacillus subtilis BSP in shake flasks study. The optimization of various factors affecting the protease production was statistically determined by Box Behnken design based on response surface methodology (RSM). The simultaneous effects of four test interacting factors on the protease production were monitored and conditions were optimized for maximum enzyme production. Optimized cultivation media formulation included: initial pH 8, casein concentration 2% (w/v) and inoculum density 1% when fermentation was carried out for 48 h. The enzyme was 9.8 fold purified in a 2-step procedure involving ammonium sulfate precipitation, DEAE cellulose and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a molecular weight of 36 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was most active at 50°C, pH 8, with casein as substrate. This enzyme was 100% stable at 50°C and retained its 82% activity at 60°C after 30 minutes of incubation. The enzyme became completely inactivated after incubation at 80°C for 3h. It was strongly activated by metal ions such as Ca2+ and Mn+2. Enzyme activity was inhibited strongly by ethylene diamine tetra acetic acid (EDTA) confirming its identity as a metalloprotease. Protease was not only stable in the presence of organic solvents and surfactants studied but it also exhibited a higher activity than in the absence of some surfactants. Consequently, the DNA encoding the thermostable protease of Bacillus subtilis BSP has been identified. The gene contained an open reading frame of 1638 bp and encoded for a mature peptide sequence of 318 amino acid residues. The protease gene was cloned and expressed in E.coli expression system and the recombinant protease was purified. Sequence analysis showed that this protease has close homology with thermolysin class of proteases. Primary structure analysis of thermostable protease showed 35% of its content to be alpha helix making it stable for three dimensional structure modeling. Homology model of thermolysin has been constructed using swiss model as the workspace. The model was validated by ProSA and RMSD. The results showed the final refined model is reliable. It has 0.06 Å as RMSD and has -2.19 as Z-score. For expression in Bacillus subtilis 1A751 two integration vectors (pDR111 and pSG1154) were compared in order to get the higher extracellular protease yield. 1A751/pDR-BSP-MprT transformant was found to be secreted higher amount of thermostable protease in the culture medium when compared to 1A751/pSG-BSP-MprT strain. The recombinant enzyme was undergone for further studies to check its stability under native enzyme conditions and it was found to be as stable as native protease BSP-MprT. The thermostability of Bacillus subtilis BSP protease was improved by two rounds of error prone PCR mutagenesis. A random mutant library of Bacillus subtilis BSP protease was generated by ep-PCR. The generated mutants have been successfully expressed in E. coli. The mutant proteases obtained in this mutant library were screened for increased protease thermostability. The SDS-PAGE pattern of enzyme was exhibiting a well-defined band (36 kDa). In this study, the thermostability was enhanced from 60°C to 80°C by the single mutation Gly347Cys which has a stabilizing effect on the irreversible thermal inactivation. The BSPmutant has exhibited increase in thermal stability and 2.7 folds half-life at 80°C when compared to wild type protease. Sequence analysis and comparison of both wt-BSPMprT and Mutant-BSP-MprT showed that wt-BSP-MprT had only one cysteine residue at Cys518 position while another cysteine has substituted Gly347 in MutantBSP-MprT. Introduction of another cysteine resulted in the formation of disulfide bridge between the two cysteine residues which play important role in the stability of protein tertiary structure. The inter subunit disulfide bond was estimated to be one of the factors for thermal stability. Another putative alkaline serine thermostable protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.