ABSTRACT
Recent studies on biocatalysis in water-organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a(w)) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.
Subject(s)
Lipase/chemistry , Organic Chemicals/chemistry , Candida/classification , Candida/enzymology , Catalysis , Enzyme Stability , Esterification , Hydrophobic and Hydrophilic Interactions , Imidoesters/chemistry , Lipase/metabolism , Organic Chemicals/metabolism , Polyethylene Glycols/chemistry , Protein Engineering/methods , Solvents , Stereoisomerism , Substrate Specificity , Water/chemistryABSTRACT
Lipase from Candida rugosa was immobilized by entrapment on poly(N-vinyl- 2-pyrrolidone-co-2-hydroxyethyl methacrylate) (poly[VP-co-HEMA]) hydrogel, and divinylbenzene was the crosslinking agent. The immobilized enzymes were used in the esterification reaction of oleic acid and butanol in hexane. The activities of the immobilized enzymes and the leaching ability of the enzyme from the support with respect to the different compositions of the hydrogels were investigated. The thermal, solvent, and storage stability of the immobilized lipases was also determined. Increasing the percentage of composition of VP from 0 to 90, which corresponds to the increase in the hydrophilicity of the hydrogels, increased the activity of the immobilized enzyme. Lipase immobilized on VP(%):HEMA(%) 90:10 exhibited the highest activity. Lipase immobilized on VP(%):HEMA(%) 50:50 showed the highest thermal, solvent, storage, and operational stability compared to lipase immobilized on other compositions of hydrogels as well as the native lipase.
Subject(s)
Candida/enzymology , Lipase/metabolism , Alkylation , Stereoisomerism , Substrate SpecificitySubject(s)
2,4-Dichlorophenoxyacetic Acid/analysis , Carbofuran/analysis , Fruit/chemistry , Parathion/analysis , Soil Pollutants/analysis , Vegetables/chemistry , Water Pollutants, Chemical/analysis , Animals , Enzyme-Linked Immunosorbent Assay/methods , Haptens , Herbicides/analysis , Immunoglobulin G , Insecticides/analysis , Rabbits , Sensitivity and SpecificitySubject(s)
Candida/enzymology , Lipase/chemistry , Water/chemistry , Alkylation , Fungal Proteins/chemistry , Hexanes/chemistry , SolventsABSTRACT
A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40 degrees C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which are affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p-nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non-polar polymers.
Subject(s)
Enzymes, Immobilized , Lipase , Candida/enzymology , Enzymes, Immobilized/chemistry , Ion Exchange Resins , Lipase/chemistry , Microspheres , PolymersABSTRACT
Two strains ofRhizopus rhizopodiformis that produced lipases in broth culture were isolated. Maximum lipase production (23 U/ml) was obtained after 72 h culture. Both the crude lipases were stable at 50°C for 30 min and at 45°C for 24 h. Maltose was the best carbon source and peptone the best nitrogen source for the production of lipases. Only glycerol and lecithin stimulated lipase production further.