Alan David Peilow

Bio: Alan David Peilow is an academic researcher. The author has contributed to research in topics: Lipase & Rhizomucor miehei. The author has an hindex of 7, co-authored 8 publications receiving 473 citations.

Lipases from different sources vary widely in dependence of catalytic activity on water activity.

Abstract: We have measured the rates of esterification in hexane catalysed by suspended immobilised lipases (triacylglycerol acylhydrolase, EC 3.1.1.3), with pre-equilibration to known thermodynamic water activity (a(w)). There were important differences between the enzymes from five different microbes in their retention of activity at low a(w). That from Rhizomucor miehei showed over 40% maximal activity at an a(w) of 0.12, and that from Rhizopus niveus was also fairly active at low a(w). Lipases from other sources required higher a(w) values to show good activity, increasing in the sequence Humicola sp., Candida rugosa and Pseudomonas cepacia. The behaviour was generally similar to two very different support materials, anion-exchange resin and macroporous polypropylene. Comparison of the sequences of the homologous enzymes from Rh. miehei, Rh. niveus and Humicola sp. suggests that changes in charged residues in the 'hinge and lid' region of the structure may be significant in low a(w) tolerance.

Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading

Abstract: Rhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost-effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency-loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.

Relationship between water activity and catalytic activity of lipases in organic media. Effects of supports, loading and enzyme preparation.

Abstract: The rates of synthesis of dodecyl decanoate in hexane have been measured as a function of water activity (aw), for various immobilised preparations of the lipases from Rhizomucor miehei and Candida rugosa. Only very large changes in the amount of enzyme adsorbed to the support affect the shape of the rate/aw profile; at the highest loadings the profiles tend to become somewhat flatter. A similar levelling can be obtained by pre-adsorbing an inert protein. The effect is probably due to adjacent protein molecules effectively replacing water; it does not simply reflect mass transfer or interfacial area limitation. The activity/aw profile was essentially the same with most supports tested: polypropylene, anion-exchange resin, celite, anion-exchange modified silica. A hydrophobic porous glass support reduced the rate somewhat at intermediate aw values with both enzymes; a polyamide material had this effect only with the lipase from Rh. miehei. The shape of the activity/aw profile was not affected by large differences in purity of the lipase preparation, but did differ between forms that probably differ in glycosylation. Overall, relatively few manipulations of the system can significantly affect the shape of the rate/aw profiles, which seem to be mainly an intrinsic property of the enzyme molecules used.

Polyethylene glycol-modified subtilisin forms microparticulate suspensions in organic solvents

Abstract: Mono-methoxy polyethylene glycol chains may be attached to subtilisin Carlsberg by reaction with their tresyl esters. The resulting PEG-subtilisin can be dispersed in toluene and 1,1,1-trichloroethane to give optically clear systems in which it is catalytically active. However, these contain large aggregates of the PEG-subtilisin monomers: dynamic light scattering indicates mean diameters of at least about 300 nm, while small-angle x-ray scattering and ultracentrifugation confirm sizes greater than 20 nm or so (in the latter method there is a tendency to neutral buoyancy). Hence it seems that this mainly organic reaction system is closer to the well-known suspensions of dried enzyme particles than to a molecular solution. Probably because of this aggregation, the precise dispersibility of the PEG-subtilisin is poorly reproducible, though a reversible temperature-dependent apparent solubility limit is sometimes observed. The UV spectrum of the enzyme in trichloroethane can be close to that in water, though circular dichroism suggest some structural change when dispersed in the solvent.

Process for the preparation of fatty acid esters

Abstract: In the preparation of fatty acid esters, reactants providing fatty acid and alcohol residues are reacted to produce said fatty acid esters in the presence of a lipase enzyme acting as catalyst. This reaction is conducted in a substantially non-aqueous homogeneous liquid phase, the lipase being physically attached by adsorption to a hydrophobic porous solid support material of average pore size not less than 50 nm. The support is preferably a highly porous polymer, e.g. vinyl-based.

Immobilisation and application of lipases in organic media.

Abstract: Different methods of preparing lipases for use in organic media are critically reviewed. Solid lipase preparations can be made by typical immobilisation methods such as adsorption, entrapment, covalent coupling or cross-linking. Immobilisation is especially attractive for lipases because, in addition to the normal benefits of enzyme immobilisation, it can also lead to a considerable increase in catalytic activity, probably caused by conformational changes in the lipase molecules. Activation can be achieved, for example, using hydrophobic support materials or surfactants during the immobilisation procedure. Surfactants can also be used to solubilise lipases in organic media via the formation of hydrophobic ion pairs, surfactant-coated lipase or reversed micelles. Lipase preparation methods are discussed with regard to potential lipase inactivation and activation effects, mass transfer limitations, lipase stability and other features important for applications. The practical applications of lipases in organic media reviewed include ester synthesis, modification of triacylglycerols and phospholipids, fatty acid enrichment, enantiomer resolution, biodiesel production and acylation of carbohydrates and bioactive compounds.

One Biocatalyst–Many Applications: The Use of Candida Antarctica B-Lipase in Organic Synthesis

Abstract: The application of the B-component lipase from the yeast Candida antarctica in organic synthesis is reviewed. This enzyme has been found to be a particularly efficient and robust lipase catalyzing a surprising diversity of reactions including many different regio- and enantio-selec-tive syntheses. Furthermore, the C. antarctica B-lipase is an example of an enzyme for which its specificity has been predicted based on the crystal structure and modeling of the active site region. This prediction is compared to experimental observations and a very close correlation is found.

Thermodynamic predictions for biocatalysis in nonconventional media: theory, tests, and recommendations for experimental design and analysis.

Abstract: This article discusses the application of thermodynamic and related analysis to reaction systems for enzymic or whole cell catalysis, in which there are high proportions of organic liquid, gas, or supercritical fluid. A variety of predictions may be made, especially based on the partitioning of components between the different phases normally present. In many cases, observed behavior can be explained without invoking any direct molecular effects on the biocatalyst. The predictable changes should always be allowed for before seeking explanations for the residual effects, which are often very different from the crude observations. A summary of the general thermodynamics of multiphase systems is presented, and then the main classes of component that distribute between the phases are discussed in turn. Thermodynamic water activity (aW) determines the mass action effects of water on hydrolytic equilibria. It also describes the distribution of water between the various phases that can compete in binding water. Because catalytic activity is very sensitive to the hydration of the enzyme molecules, aW often predicts an unchanging optimum as other aspects of the system are changed. Hence the aW should be measured and/or controlled in these systems, whether the primary aim is to study the effects of water or of other changes. The methods available for measurement and control of aW are discussed. Adverse effects of organic solvents or similar nonpolar species partly reflect their tendency to partition into the relatively polar phase around the biocatalyst, especially when this is dilute aqueous. The well-established log P parameter is a measure of this. But other mechanisms of inactivation can occur: directly through contact of the biocatalyst with the phase interface, or indirectly via hydration changes. In these cases the molecular property log P is probably not the best solvent parameter. In low-water systems the biocatalyst remains in a separate phase even when water-miscible solvents are used. Hence, the categorization of solvents in terms of miscibility becomes less relevant. This accounts for the “two peak” dependence of catalytic activity on water content in some miscible systems. Differential solvation of reactants and products, as the bulk phase is altered, causes changes in concentration-based equilibrium constants and yields. These changes in solvation may be monitored through partition coefficient or solubility measurements. Reactant solvation can also account for differences in biocatalyst kinetics, whether or not partitioning into a dilute aqueous phase is involved. These predictable effects should be allowed for when studying effects of solvent or similar changes on activity or specificity. Acidic or basic species (such as reactants) can partition into the microenvironment of the enzyme molecules and adversely affect their protonation state. If a dilute aqueous phase is present, these effects may be analyzed in terms of a pH value, and the problem is simply one of measurement (where the phase is microscopic); some methods are available. At low aW, it may be more useful to think in terms of direct reaction with protein groups.

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil.

Abstract: Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.

Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches

Abstract: Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol. Besides this, they are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Therefore, those enzymes are nowadays extensively studied for their potential industrial applications. Examples in the literature are numerous concerning their use in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and detergency applications. However, the drawbacks of the extensive use of lipases (and biocatalysts in general) compared to classical chemical catalysts can be found in the relatively low stability of enzyme in their native state as well as their prohibitive cost. Consequently, there is a great interest in methods trying to develop competitive biocatalysts for industrial applications by improvement of their catalytic properties such as activity, stability (pH or temperature range) or recycling capacity. Such improvement can be carried out by chemical, physical or genetical modifications of the native enzyme. The present review will survey the different procedures that have been developed to enhance the properties of lipases. It will first focus on the physical modifications of the biocatalysts by adsorption on a carrier material, entrapment or microencapsulation. Chemical modifications and methods such as modification of amino acids residues, covalent coupling to a water-insoluble material, or formation of cross-linked lipase matrix, will also be reviewed. Finally, new and promising methods of lipases modifications by genetic engineering will be discussed.