Title
Functional characterization of hormone sensitive-like lipase from Bacillus halodurans: synthesis and recovery of pNP-laurate with high yields
Author
Ashima Dua, Rani Gupta
Year
2017
Journal
Extremophiles
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 1/2 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 cat/K m with pNP-palmitate as a substrate was 2.52 × 105 mM−1 min−1 while that with glyceryl tripalmitin was 4.06 × 102mM−1 min−1, 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.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Thermal stability, Chemical stability, Biochemistry