Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Abstract

Lipases are ubiquitous enzymes which catalyze the hydrolysis of fats into fatty acids and glycerol at the waterlipid interface and reversing the reaction in non-aqueous media. Lipases occupy a place of prominence among biocatalysts owing to their novel and multifold applications in oleochemistry, organic synthesis, detergent formulation and nutrition. Lipase was extracted and isolated from the alimentary canal and digestive gut of Catla catla (catla). The tissue was homogenized in a ratio of 1:3 with starting buffer (0.01 M TrisHCl, pH 7.2). The crude extract thus obtained was precipitated using ammonium sulphate (20-80%). Excess salt was removed by Dialysis and the resultant dialysate (Desalted Enzyme) was subjected to DEAE-Cellulose column for Ion Exchange Chromatography at a flow rate of 0.5 ml/min. Elution was carried out by a step gradient of NaCl (100-800 mM) in the starting buffer. Active fractions were pooled as Purified Fraction (PF) and were used for physical characterization of pH, temperature and effect of calcium on the enzyme activity, structural characterization, molecular characterization and for kinetic studies. The Purified Fraction (PF) showed final specific activity of 1438.72 U/mg. The optimum pH was 7.8 and the optimum temperature was found to be 20˚C. Melting Temperature (T m) value was 42˚C and the activation energy of purified lipase was 34.82 KJ/mol/K. Thermal stability of lipase was found to be at 20˚C. Lipase activity retained up to 3 h of incubation with 10 mM and 20 mM of CaCl 2 in starting buffer. This shows that Calcium has enhancing property from the denaturation of enzyme. Michaelis-Menten constant (K m) of lipase from Indian major carp, catla for the hydrolysis of pNPP was 6.695 mM. Turnover number (k cat) of lipase (catla) was 0.0022 s-1. Catalytic efficiency (k cat /K m) of lipase was 0.0003412 s-1 mM-1. SDS-PAGE of purified lipase (PF) revealed a homogenous single band with molecular mass of 70 kDa. Secondary structural arrangement of α helices and β strands of purified lipase results 48.51% and 9.74%, respectively using Circular Dichroism.

Cloning, Expression, and Biochemical Characterization of an Enantioselective Lipase, YLIP9, from Yarrowia lipolytica MSR80

“…K m and V max were calculated using the Lineweaver-Burk plot and Michaelis-Menten equation. The turnover rate (k cat ) and the catalytic efficiency of ZZ-YLIP9 were calculated using the following formulae [12]:…”

Euthynnus affinis viscera-an alternative source for protease and lipase enzymes: Characteristic and potential application as destainer agent

“…SDS-PAGE and zymography analysis forecasted the expected molecular mass of lipase was about 32 kDa (Figures 1.A and 1.B). This finding was supported by Sharma et al (2014) who characterized lipase from the alimentary canal and digestive gut of Catla catla (catla) with 30 and 67 kDa in dialysate fraction of SDS-PAGE. Similarly, Kurtovic et al (2009) reported that a partially purified lipase from the hepatopancreas of neo flying squid (Ommastrephes bartramii) showed a molecular weight of 33 kDa, lower compared to the pancreatic lipase from mammalian species.…”