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bioRxiv · 10.1101/2025.01.11.632526

Improvement in the enzymatic efficiency of a zinc metalloprotease through random mutagenesis approach: Studies on the enzymatic and structural properties of the mutant

Srivastava, N.·Khare, S. K.
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Abstract

Microbial proteases are significant enzymes acquiring 60% of the enzyme market. The heterologous expression provides higher enzymatic yield, yet such proteases possess less stability and low catalytic efficiency. A directed evolution strategy could improve proteases properties to meet industrial demands. This study attempted the directed evolution of a recombinant metalloprotease (rsep) gene by a single round of error-prone PCR, wherein four hundred PCR were performed for random mutant gene library preparation. Eighteen amplicons (1230 bp) originated from modified PCR conditions were selected, cloned into pET22b+ plasmid, and overexpressed in Escherichia coli BL21(DE3). The recombinants showing soluble expression were screened, and one of the mutants (rsepA1) showed an 8.04-fold enhancement in the relative protease activity. It was purified to homogeneity with 92kDa molecular size. Its enzymatic properties like pH, temperature optima, and stability were comparable to rsep(WT). Furthermore, it exhibited improved enzymatic efficiency ([~] 4.2-fold towards casein) and better substrate affinity. The structural elucidation studies through bioinformatic tools, sequencing data, and comparative biophysical studies (Fluorescence-CD spectra-DSC) of the wild and mutant rsep showed increased histidine in the mutant. This probably resulted in enhanced substrate affinity and catalytic efficiency of rsepA1, which were well correlated with the ICP-MS analysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/632526v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@cfdb5borg.highwire.dtl.DTLVardef@4a4016org.highwire.dtl.DTLVardef@b5a106org.highwire.dtl.DTLVardef@5e9354_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Srivastava, N., Khare, S. K.. 2025-01-12. Improvement in the enzymatic efficiency of a zinc metalloprotease through random mutagenesis approach: Studies on the enzymatic and structural properties of the mutant. https://doi.org/10.1101/2025.01.11.632526

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