Srivastava M, Ghazi IA, Kaushal RK, Joshi GK and Kanwar SS*.

Department of Biotechnology, Himachal Pradesh University,

Summer-Hill, Shimla-171 005. India.

Phone: 91-177-231948 (0ffice); 91-177-250254 (Residence);

Fax: 91-177-230775.

E-mail: kanwarss@satyam.net.in

Dr. S. S. Kanwar, Reader, Department of Biotechnology,

Himachal Pradesh University, Summer-Hill, Shimla-171 005, India.

Phone: 91-177-231948 (Office), 91-177-250254 (Residence),

Fax: 91-0177-230775.

E-mail: kanwarss@satyam.net.in; sdichpu@jla.vsnl.net.in;

Abstract

Lipase (EC 3.1.1.3) is a triacylglyceroester-hydrolase, which catalyses the hydrolysis of tri-, di-, and monoacylglycerols to glycerol and fatty acids. The present study evaluates the methods of immobilization of lipase obtained from a Gram-negative bacterial isolate BTG1-99. The effect of pH, temperature, detergents, substrates, alcohols, organic solvent etc. on the stability of immobilized enzyme was evaluated. The enzyme was immobilized on native and activated (alkylated) matrices i.e. silica and celite. Exposure to either glutaraldehyde or formaldehyde, at 1% and 2% concentration (v/v) activated each of the matrices. The enzyme was adsorbed very rapidly on to the activated silica than activated celite. The incubation of the immobilized lipase markedly affected the activity of enzyme. The immobilized lipase showed optimal activity at 50⁰C following one-hour incubation. The lipase was specifically more hydrolytic to medium C-length ester (p-nitrophenyl caprylate than p-nitrophenyl laurate). The immobilization/entrapment enhanced the stability of the lipase at a relatively higher temperature (50^oC) and also promoted enzyme activity at an acidic pH (pH-5.5). Moreover, the immobilized lipase was quite resistant to the denaturing effect of SDS.

Key words: Bacterial lipase, Immobilization.

Lipases (triacylglycerol ester hydrolase, EC 3.1.1.3) are enzymes whose biological function is to catalyse the hydrolysis of triacylglycerols. These reactions usually proceed with high regio- and or enantioselectivity, making lipases an important group of biocatalysts in organic chemistry. Microbial lipases have enormous biotechnological potential because they are stable in organic solvents, don’t require cofactors, possess broad substrate specificity and also exhibit a high enantioselectivity. The industrial use of lipases as enantioselective catalysts depends on their efficient immobilization. In low water systems, lipase catalyses reactions such as ester synthesis, alcoholysis, acidolysis and inter-esterification.

Immobilization of lipase has been previously performed using different methods viz. covalent attachment, adsorption, entrapment and cross-linking on numerous supports (Malcata et al., 1990). An easy and efficient method of immobilization of lipase is adsorption on more or less hydrophobic supports (Brandy et al., 1988; Norin et al., 1988; Lie and Molin, 1991; Cho and Rhee, 1993; Ampon et al., 1994; Reyes et al. 1994; Basri et al., 1995 and Al Duri, 1995). Immobilization of lipases on solid supports offers several advantages including simple recovery, allowing repeated use of the catalyst, easy separation of the enzyme from product, possibility of continuous operation and improvement of enzyme stability. These advantages are well known for enzyme utilization in aqueous media, (Chibata et al., 1978). However, it is currently difficult to assess the relative merits of different approaches (Rubin et al., 1997 and Farrell et al., 1997).

A promising approach to enzyme entrapment is to use inorganic matrices such as silica gel or celite (Shtelzer et al., 1992). In entrapped form many enzymes tend to display activities 30-100% relative to those of the natural non-entrapped state (Shtelzer et al., 1992). Unfortunately, most of the attempts to extend this methodology to lipase have been intriguingly disappointing (Reetz et al., 1995). It is known that lipase are active at the lipid-water interface (Reetz et al., 1996). The silica or celite modified by alkylation (exposure to glutaraldehyde or formaldehyde) resulted in a rapid adsorption of lipase. The immobilization/adsorption might provide a changed microenvironment that causes lipase to trigger a phenomenon similar to classical interfacial activation (Reetz et al., 1996). In the present study, the activity of lipase immobilized on celite and silica matrices has been evaluated.