Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.This work was partially supported by grants from the Spanish Ministry of Industry, Commerce and TourismPeer reviewe

Hydrolysis of animal fats by immobilized Candida rugosa lipase

Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa was immobilized by adsorption on a commercially available microporous polypropylene support of 200- to 400-μm particle size. A contact period of 90 min allowed the highest degrees of hydrolysis to be achieved, particularly in the second and third hydrolysis reactions. The optimal hydrolysis conditions were 0.10 kg enzyme per kilogram fat, 50% (w/v) fat, and 40°C for 24 h. The immobilized enzyme can be repeatedly used and hydrolysis degrees of 90% or higher can be achieved. Of the three animal fats studied, edible pork lard consistently yielded the highest degrees of hydrolysis (95%) in the first hydrolysis reaction and inedible beef tallow the lowest (65%). The immobilized enzyme lost its activity above 45°C. The support could be easily recovered and reused up to 5 times.This work was partially supported by grants from the Spanish Ministry of Industry (CDTI) and the Department of Industry of the Basque Government.Peer reviewe

Kinetic properties of soluble and immobilized Candida rugosa lipase

Immobilized lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene and used for the batch hydrolysis of different animal fats. The effect of the reaction products at concentrations similar to those obtained at 90% hydrolysis, both on soluble and immobilized lipase, was studied. Glycerol showed low inhibitory effect but oleic acid caused 50% inhibition. A mixture of free fatty acids present in the complete hydrolysis of beef tallow inhibited lipase activity more than 70%. The stability of the enzyme (both soluble and immobilized) was highest in the presence of 20% isooctane. The apparent Michaelis constant for each substrate for the soluble enzyme did not change on immobilization.Peer reviewe