Functional characterization of hormone sensitive-like lipase from Bacillus halodurans: synthesis and recovery of pNP-laurate with high yields

Abstract

The HSL-like lipase encoding gene (Blip) from the polyextremophile Bacillus halodurans C-125 has been heterologously expressed in E.coli BL21(DE3). The enzyme is a monomer of ~42 kDa. It has extremely high thermal stability with a t of 35 min at 100 °C. Thermal denaturation/renaturation studies by CD and fluorescence analysis revealed complete refolding of the protein back to its native conformation even after 30 min at 90 °C. Blip prefers substrates with mid to long chain fatty acids. It has a higher catalytic efficiency on para-nitrophenyl fatty acyl esters as opposed to triacylglycerides (k/K with pNP-palmitate as a substrate was 2.52 × 10 mM min while that with glyceryl tripalmitin was 4.06 × 10 mM min, respectively). The enzyme also has a unique selectivity for hydrolysis of unsaturated fatty acyl esters. The enzyme catalyses the synthesis of pNP-laurate with an optimized conversion of 95.94 ± 0.24%. A simple procedure for purification of the product has been developed that led to 89.91 ± 0.33% product recovery.

Bacterial Hormone-Sensitive Lipases (bHSLs): Emerging Enzymes for Biotechnological Applications

“…CR-53 [38], Rheinheimera sp. [39], Lactobacillus plantarum WCFS1 [40,41], and Bacillus halodurans [45]. Furthermore, many bHSL genes were identified through metagenomic approaches directly from environmental DNA samples [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64].…”

“…Most of the bHSL family members function optimally at mesophilic temperatures, although a systematic approach was carried out to identify HSL genes from glacier soil [95]. However, Blip from Bacillus halodurans C-125 showed high thermal stability with a t 1 / 2 of 35 min at 100°C, and complete refolding to its native conformation after 30 min at 90°C was observed [45]. In addition, EstE1 and EST2 were active at high temperatures of up to 95°C [96,97].…”

Identification and characterization of a novel esterase from Thauera sp.

“…While using triacylglycerides olive oil as a substrate, no significant activity could be detected for TLip enzyme, suggesting that TLip corresponds to esterase. As many microbial esterases display broad substrate selectivity (aminolysis, acylation) and high specificity (steroselectivity, regioselectivity, and enantioselectivity) , further detailed work on the investigation of substrate specificity, chiral‐ and regio‐selectivity of TLip would be much interesting. Michaelis–Menten kinetics analysis of TLip using pNPD (C 12 ) as a substrate revealed the catalytic efficiency ( K cat / K m ) was 1.19±0.04 mM −1 Min −1 .…”

A Valuable Product of Microbial Cell Factories: Microbial Lipase

“…R02, was activated by urea, but inhibited by Triton X-100 and SDS, with 50% maximum activity observed at an SDS concentration of 0.5% (w/v); PMSF had no significant impact on the enzyme activity ( Castilla et al, 2016 ). Finally, a lipase derived from Bacillus halodurans remains relatively stable in commercial detergents and is not affected by EDTA, Triton X-100 and Tween 80; however it is activated by para-Chloromethyl benzoic acid (pCMBA)and 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB) and inhibited by diethylpyrocarbonate (DEPC), PMSF, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC)and Woodward’s Reagent K (WRK; Dua and Gupta, 2017 ).…”