Investigation of The Activity of Lipase Variants on Different 4-Nitrophenyl Esters by Spectrophotometric Assay

Abstract

Microbial enzymes are important tools that are frequently used in the field of biotechnology. These microbial enzymes, which play a role in intracellular biological reactions, are used in many different industries. Lipase, proteases and amylases are important members of hydrolytic enzymes. Lipase enzyme, which has the most common usage area among hydrolytic enzymes, is an enzyme that catalyzes the hydrolysis of ester bonds between lipid molecules. The activity of lipase enzyme is commonly measured by spectrophotometric method. P-nitrophenol esters are commonly preferred for kinetic analysis. In the spectrophotometric analysis method, the colored product is measured as a result of the hydrolysis of p-nitrophenol ester substrates by the lipase enzyme. In this study, p-nitrophenyl acetate (Acetic acid 4-nitrophenyl ester), p-nitrophenyl butyrate (Butyric acid 4-nitrophenyl ester), p-nitrophenyl octanoate (Octanoic acid 4-nitrophenyl ester, 4 -Nitrophenyl caprylate), p-nitrophenyl dodecanoate (Dodecanoic acid 4-nitrophenyl ester), p-nitrophenyl palmitate (p-Nitrophenyl palmitate, Hexadecanoic acid 4-nitrophenyl ester) substrates were used. The products formed as a result of incubation of substrates with different carbon lengths with lipase enzyme periods were measured spectrophotometrically. Trials were carried out at 25°C between 5min-120min. As a result of the experiments carried out in four repetitions, it was determined that the activity of the lipase enzyme varies according to the length of the carbon chain of the substrates. Vmax values of wild lipase enzyme were calculated as 0.42 U/mg protein, 0.95 U/mg protein, 1.1 U/mg protein, 0.78 U/mg protein, 0.18 U/mg protein for pNP-acetate, pNP-buritate, pNP-octanoate, pNP-dodecanoate, pNP-palmitate, respectively. It was determined that the activity of lipase enzyme on p-nitrophenyl palmitate was very low. It was determined that the activity of wild lipase enzyme on the eight-carbon chain pNP-octanoate substrate was higher than the other substrates.

Keywords

• microbial enzyme
• lipase
• protein engineering
• spectrophotometry
• 4-nitrophenol

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