Cloning And Expression Of Acidic Protease Gene

Aspartic proteases, commonly known as acidic proteases, are the endopeptidases that depend on aspartic acid residues for their catalytic activity. The family of aspartic proteases are active at acidic or nearly neutral pH. These are proteases can function as either degradative or processing type proteases. They typically prefer to cleave between two hydrophobic amino acids. The members of the pepsin family have a bilobal structure with the active-site cleft located between the lobes. The active-site aspartic acid residue is situated within the motif Asp-Xaa-Gly.Frist, rennin gene was amplified from Mucor pusillus and sequenced .The nucleic acid sequence and amino-acid sequence encoded by the gene were compared using DNAman biological software. Results showed that amplified fragment is a novel rennin gene(mcp). Recombinant MK₃ was constructed successfully by resistant screening (G418). Initial fermation experiments were carried out and methanol was used as sole carbon source. The rennet activity of recombinant MK₃ was 5.4 U/ml in above condition and the proteolytic activity was 3 U/ml.By sequence alignment, a fragment of 1182bp encoding a peptide was found in the whole genome sequence of N. crassa similar to the precursors of acidic protease from Botryotinia fuckeliana, Aspergillus oryzae. The peptide was found to possess the conserved sequence motif Asp-Xaa-Gly, which was the enzyme activity site of microbial acidic protease. pPIC9K-ap was constructed successfully and subsequently electrotransformed into P. pastoris GS115. Initial fermation experiments were carried out and methanol was used as sole carbon source. The enzyme activity of recombinant NA₃ was 6.8 U/ml in above condition.P_GPD gene was amplified from Saccharomyces cerevisiae W303A-1. Yeast integration type plasmid pPIC9K-P_GPD-ap was constructed successfully and obtained a integrated recombinant APS by resistant screening (G418). Initial fermation experiments were carried out and the acidic protease activity of APS was 6.5 U/ml. Ethanol fermentation experiments were carried out and it proved that rate of the ethanol fermentation of the APS is faster than Saccharomyces cerevisiae 825.