A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1T (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 (α/β)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and α/β-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 °C (150 rev min-1). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80-85 °C, and revealed great thermal (30-85 °C) 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 Ca2+, Mn2+, and Mg2+ 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). Km, Vmax, kcat, Vmax Km-1 and kcat Km-1 of TpS9 with pNPP were 0.421 mM, 4015 µmol 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.
Keywords: Detergents; Hyperstable; Lipase; Peptidase; Thermotoga petrophila; α/β-Hydrolase.
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