Efficient Purification and Characterization of Neem Oil Hydrolysing Lipase from <i>Aspergillus aculeatus </i>for Enrichment of Immunomodulators

Abstract

Neem oil hydrolysing lipase from <i>Aspergillus aeuleatus </i>was purified to homogeneity with very high yield, i.e, 64.0 per cent with 12.8-fold purification using octyl-sepharose affinity column. The specific activity of enzyme significantly increased from 125 U/mg protein to 1600 U/mg protein. The molecular weight of purified lipase was 42 ± 2 kD as determined by native

and SDS-PAGE. This lipase was highly active towards neem oil (350 per cent relative activity) and exhibited 1,3 regiospecificity. In general, this lipase showed high activity towards diglyceride to triglyceride of palmatic series. This novel lipase showed wide temperature tolerance (10-70°C) with maximum activity at 37°C. Enzyme undergo thermal inactivation at 60°C, whereas it looses its 90 per cent activity after 60 min. The enzyme showed pH tolerance (5.0-10.0) with maximal activity at 8.0. At the concentration of 10μm, this lipase inhibited by some of the serine inhibitors such as diethylparanitrophenyl

phosphate and aprotenin, whereas there is no effect of perfobolac and leupeptine. Mg⁺ and Ca⁺⁺ are neutral to this lipase, whereas Zn⁺⁺, CU⁺⁺ and Hg⁺⁺ caused inhibtion.. Enzyme highly stable in water immisible organic

<span style="font-size:14.0pt;line-height:115%;font-family:" times="" new="" roman";="" mso-fareast-font-family:"times="" roman";color:black;mso-ansi-language:en-in;="" mso-fareast-language:en-in;mso-bidi-language:hi"="" lang="EN-IN">solvents, whereas water missible organic solvents generally caused inhibition. As neem oil contains varieties of intact highly hydrophobic immunomodulators the purification and enrichment of these molecules in oil using lipase will be a novel approach.</span>