Akram_2024_Bioorg.Chem_151_107658

A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1(T) (TpS9) was over-expressed as an active, soluble and hyperstable lipolytic enzyme in the mesophilic host system. The sequence analysis demonstrated, TpS9 is an esterase/lipase-like protein belongs to alpha/beta (alpha/beta)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and alpha/beta-hydrolase fold. Various approaches (induction and cultivation) were employed to enrich TpS9 production, 6.04- and 7.26-fold increment was observed with IPTG (0.4 mM) and lactose (200 mM) in the modified 4ZB medium (pH 7.0), but with IPTG-independent auto-induction strategy 9.02-fold augmentation was achieved after 16 h incubation at 24 degreesC (150 rev min(-1)). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80-85 degreesC, and revealed great thermal (30-85 degreesC) and pH (6.0-9.0) for 8 h. No obvious constraint was perceived with various metal ions, surfactants, commercial laundry detergents, and chemical modulators. Whereas, TpS9 activity was improved with Ca(2+), Mn(2+), and Mg(2+) by 210 %, 142.5 %, and 134.3 %, respectively. With 2.5 M NaCl (215 %), 50 % (v/v) methanol (140 %), 50 % (v/v) ethanol (126.6 %), 50 % (v/v) n-butanol (122.3 %), 50 % (v/v) isopropanol (120.4 %), 50 % (v/v) acetone (118.6 %) and 50 % (v/v) glycerol (113.2 %) TpS9 activity was also enriched. TpS9 demonstrated great affinity toward natural oils and p-nitrophenyl ester substrates, but showed peak activity with p-nitrophenyl palmitate (3160 U mg(-1)). K(m), V(max), k(cat), V(max) K(m)(-1) and k(cat) K(m)(-1) of TpS9 with pNPP were 0.421 mM, 4015 micromol mg(-1) min(-1), 906.4 s(-1), 9536.8 min(-1), and 2152.96 mM(-1) s(-1), respectively. Moreover, TPS9 has notable ability to clean stains (5 min) and degrade the animals' fat (3 h). Hence, TpS9 is a favorable candidate as cleaning bio-additive in detergent formulation, fat degradation and various other applications.