Cytoplasmic Expression of Papain Isozymes in Engineered Escherichia coli for Substrate Selectivity and Computational Insights

Abstract Challenges in isolating individual papain isozymes from papaya latex and in producing full-length, correctly folded, and soluble recombinant papain isozymes in Escherichia coli have limited detailed investigations into their distinct enzymatic properties and substrate specificities. In this study, we report the successful high-yield cytoplasmic expression of papain, protease omega, proteinase IV (pro-IV), and chymopapains I, II, and III in engineered E. coli SHuffle strains and a comprehensive investigation of their distinct biochemical and computational mechanistic insights. Building upon our previous work, we employed an engineered SHuffle strain using two synthetic constructs. In the first, each isozyme is translationally fused with maltose-binding protein (MBP) and expressed under engineered T7 promoters. In the second, each isozyme was co-expressed with thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI) pairs, yielding ∼349 mg/L soluble recombinant papain. We then hypothesized that by using this strain with the above two synthetic constructs, each isozyme would be successfully expressed as soluble proteases. Indeed, the engineered E. coli SHuffle strain produced purified papain and isozymes in yields ranging from 340 to 355 mg/L in batch fermentation. Each isozyme was kinetically characterized using two synthetic substrates, benzyloxycarbonyl-Phe-Arg-pNA and acetyl-Phe-Gly-pNA, revealing catalytic efficiencies spanning from 9.4 × 102 to 2.52 × 105 M–1 s–1 and marked differences in substrate selectivity. The optimum temperature, pH activity profiles, thermostability, and responses to metal ions were determined, revealing significant functional diversity. Structural modeling and docking studies were carried out, revealing nonconserved residues in proteinase IV that distort the active-site geometry. Site-directed mutagenesis of the conserved residues (E23G/R65G) in proteinase IV improved its catalytic function, demonstrating that active-site remodeling contributes to enhanced proteolytic activity. Together, this platform opens the door to engineering papain isozymes and variants with tailored properties for synthesizing valuable peptides, food processing, and pharmaceutical manufacturing, where enzyme purity, specificity, and optimum conditions are critical.