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P J Halling

Publications and source records attributed to P J Halling.

At least 37 records · Page 2Linked to original sources
PubMed1994

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

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.

Candida↗
PubMed1994

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

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotechnology↗
PubMed1992

Effect of thermodynamic water activity on amino-acid ester synthesis catalyzed by agarose-chymotrypsin in 3-pentanone.

Chymotrypsin linked to agarose beads by multi-point covalent attachment catalyzes synthesis of Ac-Trp-OEt in 3-pentanone even when the thermodynamic water activity (aw) of the system is reduced to as low as 0.4. If fully hydrated catalyst is added to the reaction mixture before removal of water, product is formed linearly once aw has stabilized. The initial rate is reduced from that if aw is kept close to 1 (0.47 mmol s-1 (kg enzyme)-1), to 50% (aw 0.9), 25% (aw 0.4) and < 1% (aw 0.25). The large drop between aw of 1 and 0.9 probably reflects the effects of water removal on the agarose gel structure. Catalyst partly dried (even only to aw 0.86) before adding to the organic phase is inactive. At reduced aw, the equilibrium (when reached) is shifted in favor of the ester, as expected.

Amino Acids↗
PubMed1992

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

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.

Adsorption↗
PubMed1992

Rhizomucor miehei lipase remains highly active at water activity below 0.0001.

The lipase from Rhizomucor miehei adsorbed on polymer beads retains substantial catalytic activity even after exhaustive drying, and the use of dry box procedures to prevent entry of atmospheric water. Rates of esterification and transesterification (alcoholysis) were measured while stirred in hexane pre-dried to similar low water activity (aw). The rate of dodecyl decanoate synthesis was over 30% of that at the optimum (aw 0.55) after drying with anhydrous CuSO4 (aw less than 10(-3)) or MgO (aw less than 10(-4). Freshly reactivated molecular sieve could cause a further reduction in, but not elimination of, activity.

Catalysis↗
PubMed1992

Reaction rate with suspended lipase catalyst shows similar dependence on water activity in different organic solvents.

We have studied the effect of thermodynamic water activity (a W) on the initial rate of esterification catalysed by an immobilised lipase (Lipozyme) suspended in an organic reaction mixture. The catalyst and the organic phase were separately pre-equilibrated to the same aw value. The rate shows similar dependence on aw in reaction mixtures based on five different organic solvents ranging in polarity from pentan-3-one to hexane, and in a liquid reactant mixture. There is a maximum at aw about 0.5, with a decline to 30-70% at aw of either 0.9 or less than 0.01. When the rates are presented in terms of water concentration in the organic phase (or total water content of the system), the maxima for the various solvents come at very different positions, reflecting the widely varying solubilities of water in the organic phase.

Catalysis↗
PubMed1991

Salt hydrates buffer water activity during chymotrypsin-catalysed peptide synthesis.

Chymotrypsin (EC 3.4.21.1) powder suspended in hexane in the presence of Na2CO3.10H2O is a good catalyst for peptide synthesis. The salt hydrate releases water to fix the thermodynamic water activity of the system in accord with its dissociation pressure. Salt hydrates can be useful to buffer water activity in mainly organic enzyme reaction mixtures at a value permitting activity of the catalyst while minimising hydrolytic side reactions.

Buffers↗
PubMed1991

Effect of thermodynamic water activity on protease-catalyzed peptide synthesis in mainly organic media.

The role of water in mainly organic reaction mixtures for biocatalysis is best analysed in terms of the thermodynamic water activity. This determines water mass action effects on the equilibria of protease-catalysed peptide or ester synthesis. It can also be useful to predict the amount of water bound by the enzyme, and hence its catalytic activity, as other factors are changed.

Catalysis↗
PubMed1990

High-affinity binding of water by proteins is similar in air and in organic solvents.

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.

Adsorption↗
PubMed1990

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay.

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.

Bicarbonates↗
PubMed1990

Protease-catalysed peptide synthesis in aqueous-organic two-phase systems: reactant precipitation and interfacial inactivation.

Peptides can be efficiently synthesized by the proteases thermolysin and pepsin acting in aqueous-organic two-phase systems similar to those reported previously. However, a number of problems can prevent successful reactions in these systems, with these and other proteases: (1) Complexes between amino acid reactants that are sparingly soluble in both phases may be precipitated, though their solubility behavior is complex; (2) the enzyme may be subject to rapid inactivation by contact with the phase interface, and the rate of this is unusually dependent on the nature of the solvent; and (3) the reaction rate is usually slow.

Amino Acids↗
PubMed1987

Biocatalysis in multi-phase reaction mixtures containing organic liquids.

A wide range of enzymes and whole microbial cells will act as catalysts in reaction mixtures that contain 2 or more phases, one of which is an organic liquid (either a reactant or including water-immiscible organic solvents). These "biphasic" systems have a variety of structures, knowledge of which aids predictions about biocatalyst activity and stability. There is often a dilute aqueous solution phase (containing the biocatalyst), which may be emulsified with the organic phase, or "trapped" within catalyst particles; sometimes however there may only be traces of water adsorbed to the enzyme or cells. These reaction systems offer several advantages for industrial applications, notably the higher solubilities of many reactants of interest, and the ability of readily available hydrolytic enzymes to catalyse syntheses. The most non-polar organic liquids are least likely to inactivate biocatalysts, though many do remain active with relatively polar solvents. Modification of the biocatalyst may stabilise against inactivation, especially where this is due to direct contact with the phase interface. The mass transfer processes required in these systems remain poorly understood, particularly because the interfacial area is often unknown. Attractive continuous reactors may be operated using a packed bed of catalyst with a trapped aqueous phase.

Journal Article↗
PubMed1981

Protein-stabilized foams and emulsions.

This review is a survey of studies on protein-stabilized foams and emulsions in relatively simple, well-defined systems (rather than in food products per se). The emphasis is on the extent of basic understanding developed, particularly in terms of the physicochemical properties involved. The stability of the system, its formation, and its rheology are covered. Unfortunately, much work is of limited fundamental value because of poorly designed experimental approaches and the failure to measure key parameters. Reasonable generalization can be made concerning the effects of a number of variables, particularly protein solubility, pH, protein structure and the presence of small molecule surfactants. However, several possible explanations of these effects in terms of surface properties remain tenable. A number of observations can fairly confidently be ascribed to the influence of rheology on flow processes within foams and emulsions.

Emulsions↗
PubMed1979

Improved nonporous magnetic supports for immobilized enzymes.

Ni powders coated by deposition of TiO2 or controlled oxidation to NiO develop substantial resistance to corrosion. Chymotrypsin immobilized to these coated Ni supports shows very high stability of activity on storage. Chymotrypsin immobilized by adsorption and glutaraldehyde crosslinking was fairly rapidly eluted under operational conditions in the presence of substrate. If 3-aminopropyltriethoxysilane (APS) was used to produce a covalent linkage, desorption of enzyme still occurred because of relatively unstable bonding of the silane to the oxide surface. A more stable attachment was produced by joining together many silane links with a layer of polyglutaraldehyde. The mechanism of action of APS as a coupling agent under these conditions is discussed. gamma-Fe2O3, and particularly a Mn-Zn ferrite, are suitable magnetic support materials available with smaller particle sizes. Particles below 1 mum give the expected higher specific activities of immobilized enzymes.

Adsorption↗