Catalytic Properties Of Lipases Research Articles

Investigation on the acyl chain length specificity of lipase by gas chromatography assay

Lipases are known for their high catalytic activity, unique substrate selectivity, and acyl chain length specificity. To investigate the acyl chain length specificity and catalytic properties of lipase, we developed a novel gas chromatography (GC) method by determining multiple substrates of saturated fatty acid ethyl esters with acyl chain lengths ranging from C4 to C18. The commercial immobilized lipase Novozym 435 (Candida antarctica lipase B) was selected as the model lipase. Acyl chain length specificity of Novozym 435 was determined with multiple substrates for three reactions (hydrolysis, esterification, and alcoholysis). Apparent Michaelis constant (Km) and Vmax for the reactions catalyzed by Novozym 435 were calculated, and the effect of organic solvent on lipase-catalyzed esterification and alcoholysis was further studied. The main advantages of this GC analytical method are using inexpensive reagents, simple detection operation, and a useful tool for simultaneously determining the acyl chain length specificity of lipase in catalytic hydrolysis, esterification, and alcoholysis reactions.

Catalytic properties of lipase from <i>Ficus trichopoda</i> and <i>Euphorbia unispina</i> latex: Study of their typoselectivity

Objective: The search for lipase with distinct features, from plant latex is of great importance for industrial applications. The catalytic properties of lipases from Ficus trichopoda and Euphorbia unispina latex were characterized.Methodology and Results: Fresh latex from Ficus trichopoda and Euphorbia unispina were collected and dried through solar. Dried latex was taken for complete proximate analysis and their activity was analysed by thin layer chromatography. The two lipases were optimally active at pH=5 and temperature of 35°C and 50°C for Ficus trichopoda and Euphorbia unispina latex, respectively. The presence of metal ions enhances the activity of Ficus trichopoda latex, while no significant enhancement was observed in the case of Euphorbia unispina latex. Both lipases were able to hydrolyze saturated esters, and showed typo-selectivity for this group. However, the lipases are weak selective for the hydrolysis of unsaturated esters, especially for 18:2 fatty acids.Conclusions and application of finding: The enzyme from Ficus trichopoda latex was able to attack specific oil to generate free fatty acids or ester as the major product. This understanding may help in devising efficient methods to produce valuable modified oils.Keywords: Latex; Lipase activity; esterification reaction; typo-selectivity; Ficus trichopoda; Euphorbia unispina

Catalytic properties of lipase adsorbed on nanocarbon-containing mesoporous silica in esterification and transesterification reactions

Nanocarbon-containing mesoporous silica covered with a varying amounts of nanostructured carbon of different morphologies were used as supports to immobilize Thermomyces lanuginosus lipase. The catalytic properties of the prepared biocatalysts were studied in both the transesterification of vegetable (linseed) oil in the presence of ethyl acetate and the esterification of the fatty acid (capric C10:0) in the presence of secondary (isopropyl or isoamyl) alcohols. The physico-chemical characteristics, such as the amount of adsorbed lipase, its specific activity, and the dependence of the activity and stability of the prepared biocatalysts on the support type were evaluated. The Michaelis-Menten kinetics was studied in the esterification of capric acid with isoamyl alcohol. The prepared biocatalysts were shown to retain up to 90% activity for >1000 h in the synthesis of isoamyl caprate. The half-time of the biocatalysts inactivation in the transesterification of linseed oil was found to be more than 700 h at 40°C.

Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations

Adsorption onto solid supports has proven to be an easy and effective way to improve the mechanical and catalytic properties of lipases. Covalent binding of lipases onto the support surface enhances the active lifetime of the immobilized biocatalysts. Our study indicates that mesoporous silica gels grafted with various functions are ideal supports for both adsorptive and covalent binding for lipase B from Candida antarctica (CaLB). Adsorption of CaLB on phenyl-functionalized silica gels improved in particular its specific activity, whereas adsorption on aminoalkyl-modified silica gels enabling covalent binding with the proper reagents resulted in only moderate specific activity. In addition, adsorption on silica gels modified by mixtures of phenyl- and aminoalkyl silanes significantly increased the productivity of CaLB. Furthermore, CaLB adsorbed onto a phenyl/aminoalkyl-modified surface and then treated with glutardialdehyde (GDA) as cross-linking agent provided a biocatalyst of enhanced durability. Adsorbed and cross-linked CaLB was resistant to detergent washing that would otherwise physically deactivate adsorbed CaLB preparations. The catalytic properties of our best immobilized CaLB variants, including temperature-dependent behavior were compared between 0 and 70°C with those of two commercial CaLB biocatalysts in the continuous-flow kinetic resolutions of racemic 1-phenylethanol rac-1a and 1-phenylethanamine rac-1b.

Immobilization and catalytic properties of lipase on chitosan for hydrolysis and esterification reactions

The objective of this study was to evaluate the immobilization of lipase on a chitosan support by physical adsorption, aiming at its application in hydrolytic and synthetic reactions. Two types of chitosan (flakes and porous) were used for immobilizing lipase from a microbial source (Candida rugosa) and animal cells (porcine pancreas). The best results for recovery of total activity after immobilization were obtained for microbial lipase and porous chitosan beads. This set was selected for further immobilization studies, including full characterization of the immobilized derivative in aqueous and organic media. In aqueous medium, the operational and thermal stabilities of this preparation were quantified. In organic medium, the direct synthesis of n-butyl butyrate in organic solvent was chosen as a model reaction. The influence of several parameters, such as temperature, initial butyric acid concentration and amount of enzyme in the reaction system, was analyzed. Production of n-butyl butyrate was optimized and an ester yield response equation was obtained, making it possible to predict ester yields from known values of the three main factors. Use of this immobilized preparation was extended to the direct esterification of a large range of carboxylic acids (from C2 to C12) with a variety of alcohols (from C2 to C10).

Pseudomonas fluorescens lipase adsorption and the kinetics of hydrolysis in a dynamic emulsion system

To elucidate the adsorption characteristics of lipases and to study the influence of the reaction conditions on the catalytic properties of lipases, the hydrolysis of decylchloroacetate by Pseudomonas fluorescens lipase in an emulsion reactor was studied as a model system. During the reaction the droplet size distribution of the emulsion was measured on-line using a particle sizer based on light scattering. Desorption experiments revealed that, at low surface coverage, the initial rate of reaction was not influenced by either the stirring speed or the organic volume fraction. Dilution of the reaction mixture during hydrolysis did not result in a decrease in activity. Based on these results, it is assumed that under the specified conditions adsorption of Pseudomonas fluorescens lipase is quantitative and probably irreversible. Based on activity measurements and assuming that only a monolayer of lipase is active, it is calculated that at saturation the emulsion interface is covered with 3 mg lipase per m 2. From these data the average interfacial area covered by one lipase molecule at saturation was calculated to be 1700–2100 Å 2 per molecule. The emulsion was shown to be dynamic, e.g., during hydrolysis a significant increase in interfacial area was observed as a result of a shift in droplet size distribution to smaller diameters. Experiments indicated that both the formation of decanol and the emulgating effect of the lipase account for these observations. The formation of decanol also resulted in a dramatic decrease in hydrolytic activity. Taking interfacial tension measurements into account, it is shown that decanol accumulates at the liquid-liquid interface. As a result of this high local decanol concentration at the interface, a reversed reaction, alcoholysis, occurred.