C. N. A. Razak

Enzymic synthesis of fatty esters by hydrophobic lipase derivatives immobilized on organic polymer beads

Lipase fromCandida rugosa was modified with several hydrophobic modifiers before being adsorbed onto organic polymer beads. The effects of different enzyme modifiers, supports, solvents, reaction temperatures, fatty acids, and alcohols on the activity of the immobilized enzyme were investigated. The immobilized lipases were good biocatalysts for esterification reactions in organic solvents. They exhibited high activities in all solvents tested, including polar solvents. The activity seemed to depend on the type of support rather than on the modifier of the enzyme. The medium polar support, XAD7, appeared to be the best for the modified lipases. The immobilized lipase favored the medium-chain fatty acids rather than the long-chain fatty acids as acyl donors. The alcohol selectivity of the enzyme was unchanged upon immobilization. The native and immobilized lipases favored the short-chain and terpene alcohols as nucleophiles.

Immobilization of lipase from Candida rugosa on synthetic polymer beads for use in the synthesis of fatty esters

Lipase from Candida rugosa was immobilized on three different supports, i.e. Amberlite XAD7, poly(methylmethacrylate) (PMMA) and celite. With the conditions tested, maximum adsorption can be achieved after 30 min. The activities of the immobilized lipases were determined by the esterification reaction of oleic acid and butanol. The immobilized lipases were found to be very effective in the esterification reaction. The immobilized activities generally were high in apolar organic solvents with log P values from 2·0 to 4·0. The preference for fatty acids as acyl donors differed in all cases of immobilized lipases. Lipase immobilized on XAD7 and PMMA exhibited high preference of acyl donors (fatty acids) with chain lengths 12–18 and 8–18, respectively. Lipase immobilized on celite, however, showed high activity in all cases of fatty acids. The nucleophile (alcohol) selectivity studies showed that lipase immobilized on XAD7 and celite was more accessible to alcohols of chain lengths 3–12. However, lipase immobilized on PMMA showed a significant preference towards alcohols of chain lengths from 3 to 10.

Amidination of lipase with hydrophobic imidoesters

Lipase fromCandida rugosa was chemically modified by amidination with imidoester hydrochlorides of different hydrophobicity. The modified enzyme showed a higher ester synthesis activity but a lower ester hydrolysis activity compared with the native enzyme. The maximum specific activity of the modified enzyme depended on its degree of derivatization. Benzene was found to be the best solvent for the synthesis reaction. The optimal temperature for the reaction was not affected by modification of the lipase. The modified lipase was more thermostable and solvent-stable than the native enzyme. When fatty acids of different carbon chainlength were tested as substrates in the synthesis of esters with the modified lipase, the highest activity was observed with myristic acid and propanol.

Reductive alkylation of lipase

Candida rugosa lipase was modified via reductive alkylation to increase its hydrophobicity to work better in organic solvents. The free amino group of lysines was alkylated using propionaldehyde with different degrees of modification obtained (49 and 86%). Far-ultraviolet circular dichroism (CD) spectroscopy of the lipase in aqueous solvent showed that such chemical modifications at the enzyme surface caused a loss in secondary and tertiary structure that is attributed to the enzyme unfolding. Using molecular modeling, we propose that in an aqueous environment the loss in protein structure of the modified lipase is owing to disruption of stabilizing salt bridges, particularly of surface lysines. Indeed, molecular modeling and simulation of a salt bridge formed by Lys-75 to Asp-79, in a nonpolar environment, suggests the adoption of a more flexible alkylated lysine that may explain higher lipase activity in organic solvents on alkylation.

MODIFICATION OF LIPASE BY POLYETHYLENE GLYCOL

Lipase was modified using polyethylene glycol activated by p-nitrochloroformate. The hydrolytic activity of the polyethylene glycol-derivatised lipase (PEG-lipase) was relatively low compared with that of the unmodified enzyme in aqueous system. The esterification activity, however, was enhanced following the modification. The rate of esterification of butyric acid was higher than that of oleic acid. Benzene was the best solvent for the esterification reaction.

Sugar esterification catalysed by alkylated trypsin in dimethylformamide

SummaryReductive alkylation of porcine pancreatic trypsin with acetaldehyde, propionaldehyde, octaldehyde and benzaldehyde resulted in about 5 to 6 folds increase in the sugar esterification activities of the enzyme in DMF. The optimum activities of the modified enzymes depend on the degrees of their modification with the respective aldehydes. These alkylated trypsins were more stable in DMF compared to the native unmodified enzyme at temperatures between 26 and 60°C.

Modified enzymes for reactions in organic solvents

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 (aw) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.

Poly(Methyl Methacrylate) as a matrix for immobilization of lipase

Poly(methyl methacrylate) (PMMA) was found to be suitable for the immobilization of lipase fromCandida rugosa. The best result based on hydrolytic activity was obtained by adsorption of the purified unbuffered enzyme solution onto PMMA beads without any modification of the beads. Prolonged exposure of the protein to the beads increased its adsorption but the expressed activity decreased after 1 h of exposure. The magnitude of the immobilized activity also varied with the size of the beads. Immobilization of the lipase shifted its optimal reaction temperature from 37 to 45°C. The immobilized enzyme is also more stable than the free enzyme in solution. The operational half-life of the immobilized lipase packed in a column and assayed in a closed system is 40 d.

The Effect of Attachment of Hydrophobic Modifiers on the Catalytic Activities of Lipase and Trypsin

Abstract Several hydrophobic groups of different sizes and steric hindrance have been covalently attached to lipase and trypsin. Attachments of polyethylene glycol and chemical modification by reductive alkylation and amidination with hydrophobic aldehydes and imidoesters, respectively, increased the ester synthesis activities of these enzymes by 2–6 folds compared to the native unmodified enzymes. The maximal esterification activities of the alkylated enzymes appeared to depend on the particular aldehyde and on the degree of modification.For example, lipase required a lower degree of modification with the more bulky and hydrophobic aldehydes (eg. dodecyldehyde) to attain its maximal activity compared with that which was modified with acetaldehyde. Excessive modifications result in a decrease in the activity. Trypsins which were derivatized to their maximal activities with different aldehydes, appeared to have very similar hydrophobicity. Most of the alkylated enzymes thus prepared were active and thermostable in the anhydrous organic solvents used, particularly in dimethyformamide (DMF) and pyridine. Modifications with polyethylene glycol and hydrophobic imidoesters gave results very similar to those obtained with hydrophobic aldehydes.

Catalytic Activity of Lipase Modified with Hydrophobic Imidoesters

The alteration and improvement of the native properties of enzymes for biotechnological applications can be achieved by chemical modifications.Iv2 Modification of protein by amidination with imidoesters offers several advantages over other approaches to amino group derivatization. The reaction conditions are relatively mild, the reaction is specific for the epsilon-amino-lysil and alpha-amino residues of proteins, the amidine linkages formed are stable over a wide range of experimental condition^,^ and the derivatized protein retains the positive charges of the amino