Changes of the structure and activity of candida rugosa lipase during the denaturation by heat and organic solvent

Abstract

Although there are many reaction systems to utilize the lipase more efficiently, the denaturation of the enzyme is one of the most serious problems to be solved in all systems tried to date. In order to maintain the enzyme activity it is important to understand the denaturation mechanisms. For the enzymatic production of fatty acids and the specialty lipids, properties of the lipase in organic solvent, which is necessary for solubilizing the water immiscible substrate, were studied. The Candida rugosa lipase was purified to above 97%. Its molecular weight and isoelectric point were 52000±800 and 4.16, respectively, and amino acid of N-terminal was proline. The kinetics of the lipase deactiviation was found to be a complex process, which consisted of the three regions; 1) initial linear region (of overall deactivation), 2) transient region, and 3) last linear region. The following techniques confirmed the above mentioned result; the ratio of absorption at 278 nm to absorption at 250 nm, the change in the accessibility of the neutral quencher to Trp region, and the pattern of ANS binding to nonpolar groups of the lipase surface. The kind of and/or density of surface charge of the lipase influenced its structure and activity, and Glu, Asp, and His were especially important in maintaining the native structure and in changing the structure during thermal treatment. This result was consistent with the change of the activity, UV spectrum, structure near Trp region, exposure of nonpolar amino acids, and the secondary structure of the Lipase during denaturation. The increase in the ionic strength, which changes the interction between water and enzyme, increased the deactivation rate of the lipase during thermal treatment, but it made the hydrophobic region (containing Phe), Trp region, exposure of the nonpolar groups, and the secondary structure of the lipase to be less sensitive to heat treatment. When less polar solvent was mixed with the lipase during heat treatm...