Characterization, crystallization and structure prediction of recombinant protease from Bacillus pumilus 115B

Proteases are widely used in industry as biocatalyst. Being able to synthesize peptide bonds in microaqueous environment while hydrolyse it in aqueous environment, this enzyme plays very important roles in food, pharmaceutical, detergent and leather industry. Protease from Bacillus pumilus 115B was capable to withstand in moderate temperature and proven to be stable in wide range of pH. To get better understanding of this enzyme as well as to enhance its productivity, molecular cloning, characterization and expression of the enzyme is compulsory. The full sequence of 1,149bp encoded a polypeptide of 383 amino acid residues from the organic solvent tolerant protease was successfully cloned into several vectors. Above all, pET 32b vector in BL21(DE3) host showed the highest expression. 115B protease was successfully purified and a single band of 33kDA mature protein was detected at the final step purification using ion exchange chromatography method. Further study on characterization, crystallization and structure prediction were carried out since the structure of this enzyme will give the new insight into organic solvent tolerant properties on a molecular level. The optimum temperature of 115B protease was found to be at 50°C and was stable in temperature range of 30°C to 45°C. The protease activity decrease rapidly at temperature higher than 55°C. 115B protease was stable in pH ranging from pH 7.0-pH 11.0 and the optimum pH was pH 8.0. The protease activity was 91% inhibited by PMSF suggesting that this protease belongs to the serine protease superfamily. Optimization on crystallization condition showed that 115B protease need the use of microseeding to kick start the nucleation process. Crystallization screening showed best crystal grew in formulation 22 from Molecular Dimension II. However, X-ray diffraction studies failed to give good diffraction spot that may result from the bad quality crystal. Homology modelling study helps to give a structural insight of the enzyme. 115B protease was modelled using crystallized structure of subtilisin BPN complex (PDB id: 1YJB) as template for homology modelling. The predicted structure gave the overall quality Z-score of -0.305 which considered a good quality of modelled structure. The model appeared to have an Aspartic, Serine and Histidine catalytic triad, like all the subtilisin family. The predicted structure shown that 115B protease contains a calcium binding site that located at similar place in the subtilisin BPN’ complex structure which is believe to attributes to the inefficient folding of subtilisin BPN’ mature enzyme. Experimental result showed that expression of 115B protease was improved compared from previous recombinant 115B/pQE30 by Mahammad (2007). 115B protease was successfully crystallized with the size of 80μm, however due to the size factor, X-ray diffraction failed to give sufficient data to construct the electron density map to build the structure. Hence, homology modelling was performed and successfully predicted the structure of 115B protease. This provides preliminary overview about the structure and function of the enzyme.

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