Abstract
The lipase enzyme converts long chain acyltriglycerides into di- and monoglycerides, glycerol and fatty acids. The catalytic site in lipase is situated deep inside the molecule. It is connected through a tunnel to the surface of the molecule. In the unbound state under aqueous conditions, the tunnel remains closed. The tunnel can be opened when the enzyme is exposed to a lipid bilayer or a detergent or many hydrophobic/hydrophilic surfaces. In the present study, the lipase was subjected to three-phase partitioning (TPP) which consisted of mixing in tert-butanol and ammonium sulphate to the solution of lipase in the aqueous buffer. The enzyme formed an interfacial precipitate between the tert-butanol rich and water rich phases. The stability of the enzyme subjected to TPP was found to be higher (Tm of 80 °C) than the untreated enzyme (Tm of 77 °C). The activity of the enzyme subjected to TPP (3.3 U/mg) was nearly half of that of the untreated one (5.8 U/mg). However, the activity of the treated enzyme was higher (17.8 U/mg) than the untreated one (8.6 U/mg) when a detergent was incorporated in the assay buffer. The structure determination showed that the substrate binding site in the treated enzyme was more tightly closed than that of the untreated protein.
Highlights
The lipase enzyme converts long chain acyltriglycerides into di- and monoglycerides, glycerol and fatty acids
We report the protein structures based upon X-ray crystallographic studies of TLL including (1) native untreated form (TLL), (2) TLL after being subjected to three-phase partitioning (TPP-TLL)
As indicated in the introduction, TLL subjected to three phase partitioning (TPP) under optimized conditions showed an increase in the initial rates of hydrolytic activity from 8.6 to 17.8 U/mg protein (Table 1)
Summary
The lipase enzyme converts long chain acyltriglycerides into di- and monoglycerides, glycerol and fatty acids. The catalytic site in lipase is situated deep inside the molecule. It is connected through a tunnel to the surface of the molecule. In the unbound state under aqueous conditions, the tunnel remains closed. The tunnel can be opened when the enzyme is exposed to a lipid bilayer or a detergent or many hydrophobic/hydrophilic surfaces. Enzymes offer a more sustainable option for catalyzing chemical processes [1]. Enhancing their stability and activity is a worthwhile goal. The present work describes efforts to understand how a simple process called three phase partitioning (TPP) resulted in altering both stability and activity of a lipase from Thermomyces lanuginosus (TLL). The structure of TLL was reported earlier in the native state which showed that
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