Akram_2024_Int.J.Biol.Macromol_279_135107

A hyperstable lipase from Thermotoga naphthophila (TnLip) was cloned and overexpressed as a soluble and active monomeric protein in an effectual mesophilic host system. Sequence study revealed that TnLip is a peptidase S9 prolyl oligopeptidase domain (acetyl esterase/lipase-like protein), belongs to alpha/beta (alpha/beta)-hydrolase superfamily containing a well-conserved alpha/beta-hydrolase fold and penta-peptide (GLSAG) motif. Various cultivation and induction strategies were applied to improve the heterologous expression and bacterial biomass, but TnLip intracellular activity was enhanced by 14.25- fold with IPTG-independent auto-induction approach after 16 h (26 degreesC, 150 rev min(-1)) incubation. Purified TnLip (35 kDa) showed peak activity at 85 degreesC in McIlvaine buffer (pH 7.0-8.0), and has great stability over a broad range of pH (5.0-10.0), and temperature (40-85 degreesC) for 8 h. TnLip exhibited prodigious resistance toward various commercial detergents, chemical additives, and salt. TnLip activity was improved by 170.51 %, 130.67 %, 127.42 %, 126.54 %, 126.61 %, 120.32 %, and 116.31 % with 50 % (v/v) of methanol, ethanol, n-butanol, isopropanol, acetone, glycerol, and acetic acid, respectively. Moreover, with 3.0 M of NaCl, and 10 mM of Ca(2+), Mn(2+), and Mg(2+) TnLip activity was augmented by 210 %, 185.64 %, 152.03 %, and 116.26 %, respectively. TnLip has an affinity with various substrates (p-nitrophenyl ester and natural oils) but maximal hydrolytic activity was perceived with p-nitrophenyl palmitate (pNPP, 3600 U mg(-1)) and olive oil (1182.05 U mg(-1)). The values of K(m) (0.576 mM), V(max) (4216 micromol mg(-1) min(-1)), V(max)K(m)(-1) (7319.44 min(-1)), k(cat) (1106.74 s(-1)), and k(cat)K(m)(-1) (1921.42 mM(-1) s(-1)) were calculated using pNPP substrate. Additionally, TnLip degraded animals' fats and removed oil stains within 3 h and 5 min, respectively. All these features make halo-alkali-thermophilic TnLip as an auspicious contender for laundry detergents (cleaning bio-additive), fat degradation, wastewater treatment and endorse eco-friendly stewardship along with various other biotechnological applications.