Journal of Biotechnology & Biomaterials
Open Access

Abstract

Numerous enzymes have been synthesized as a zymogen in different stage and activated time-specific manner in many insect pests for its growth and development. Particularly, 37kDa serine protease was synthesized as a zymogen precursor in the midgut and activated upon pupation of B. mori. 37 kDa serine protease encodes a 37 kDa composed of 329 amino acid which is playing a critical role in the process of digestion of food and physiological process including apoptosis and tissue remodelling in B. mori. To purify and characterize the 37 kDa serine protease, it was over expressed by using PET30a expression vector in Escherichia coli (BL21 DE3) with N-terminal His6-tag. The over expressed His-tag protein was successfully purified using Ni- NTA and observed as a single band with a molecular mass of 42.5 kDa on SDS-PAGE. The pI of the purified protein was found to be three major spots between the pI of 6.2 to 6.8 with MW 45kDa were determined by using 2D-PAGE. The resulted protein spots were also confirmed by MALDI-TOF-MS analysis. Circular Dichroism showed that 37 kDa serine protease zymogen was consistent over the range of 6-7 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The composition of purified p37k serine protease was analysed using HPLC. Computational prediction of cleavage site for signal peptide and propeptide of 37 kDa serine protease between positions of 18-19, 35-66 aa respectively were determined through Signal P-4.1 tool. This result revealed that pI/Mw of active protease of 37kDa serine protease after removal of pro-peptide showed 5.91 / 28007.38. This overall results revealed that the complex structure of this protease reasoned for inactive zymogen and slightly acidic pH at (>5) plays a major role in activation 37 kDa serine protease in the midgut for tissue remodelling during pupation or anti-feeding stage. In future, the identification of zymogen (pro-protein) processing enzyme will be much helpful for the development of novel drug/pesticide for the control of insect pests.

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