Barry D. Moore

Biocatalyst behaviour in low-water systems

An improving understanding of the parameters that affect biocatalyst activity, specificity and stability in low-water non-aqueous media make reliable predictions about the behaviour of such systems increasingly feasible. Here, we discuss some of the key factors, such as control of water activity, and the effects of solvent on K m and protein-ionization state, that must be addressed in order to obtain predictable results.

Activity and mobility of subtilisin in low water organic media: hydration is more important than solvent dielectric.

The relationship between hydration, catalytic activity and protein dynamics was investigated for subtilisin Carlsberg in organic solvents with low water content. The organic media were cyclohexane, dichloromethane or acetonitrile, with controlled thermodynamic water activity (aw). Catalytic rate profiles showed the same dependence on aw for the three different solvents. The structural mobility of the enzyme in air and organic media was probed by proton solid-state NMR relaxation measurements. Both spin-lattice relaxation time (T1 ) and line width at half height (apparent spin-spin relaxation time (T2)) were determined for protein which was exchanged and hydrated with D2O. We found NMR relaxation was much more dependent on aw than medium identity (despite very different dielectrics) showing that enzyme hydration is the primary determinant of mobility. Results suggest that initial hydration up to aw 0.22 causes rigidification of part of the protein structure. As aw is increased further, enzyme mobility is found to increase. Above aw 0.44, a large increase in the proportion of more mobile protons coincides with a steep rise in catalytic activity for the enzyme in each of the solvents studied.

Practical route to high activity enzyme preparations for synthesis in organic media

A single pot method to rapidly prepare immobilised subtilisin Carlsberg and α-chymotrypsin gives 1000-fold greater catalytic activities in polar organic solvents than freeze-dried powders.

Optical spectroscopic methods for probing the conformational stability of immobilised enzymes

We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm(-1) to 1632 cm(-1), indicating a shift from alpha-helical structure to beta-sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to beta-sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

‘Organic phase buffers’ control biocatalyst activity independent of initial aqueous pH

Combinations of triisooctylamine with its hydrochloride, or of triphenylacetic acid with its Na+ salt, can function as buffers for use during biocatalysis in organic media. They can control the pH of an adjacent aqueous phase, even though both forms of each buffer remain in the organic phase. With 0.1 M aqueous NaCl, the mid-point pH values obtained with the two buffer systems are around pH 4.5 and 7.0, respectively. The activity of an immobilized subtilisin Carlsberg shows a strong dependence on the ratio of the two forms of the triphenylacetic acid buffer system. Without the buffer, the rate shows the normal dependence on the pH of the aqueous solution before drying; however, this is almost eliminated if the buffer is used. The amine buffer system can similarly affect the activity of an immobilized Rhizomucor miehei lipase.

Enzyme-coated micro-crystals: a 1-step method for high activity biocatalyst preparation

A rapid, inexpensive method for producing water-soluble enzyme-coated micro-crystals which exhibit dramatically enhanced catalytic activity and stability in non-aqueous media and can be re-dissolved easily in aqueous solution is described.

Predicting when precipitation-driven synthesis is feasible: application to biocatalysis.

Precipitation-driven synthesis offers the possibility of obtaining high reaction yields using very low volume reactors and is finding increasing applications in biocatalysis. Here, a model that allows straightforward prediction of when such a precipitation-driven reaction will be thermodynamically feasible is presented. This requires comparison of the equilibrium constant, Keq, with the saturated mass action ratio, Zsat, defined as the ratio of product solubilities to reactant solubilities. A hypothetical thermodynamic cycle that can be used to accurately predict Zsat, in water is described. The cycle involves three main processes: fusion of a solid to a supercooled liquid, ideal mixing of the liquid with octanol, and partitioning from octanol to water. To obtain the saturated mass action ratio using this cycle, only the melting points of the reactants and products, and in certain cases the pKa of ionisable groups, are required as input parameters. The model was tested on a range of enzyme-catalysed peptide syntheses from the literature and found to predict accurately when precipitation-driven reaction was possible. The methodology employed is quite general and the model is therefore expected to be applicable to a wide range of other (bio)-catalysed reactions.

α-Chymotrypsin stability in aqueous-acetonitrile mixtures: is the native enzyme thermodynamically or kinetically stable under low water conditions?

Abstract Like many proteins, α-chymotrypsin is denatured in 50% volume aqueous-acetonitrile mixtures. However, it also shows high catalytic activity in 70% or more acetonitrile. Good activity in two different aqueous organic composition ranges has been described for several other enzymes. The stability of the native protein under low water conditions is generally believed to be a kinetic phenomenon, though there are also arguments for thermodynamic stability. We have distinguished between these possibilities by studying the effects of changing medium composition at different times. In preliminary experiments, we found catalytic activity could be recovered by adding neat acetonitrile to chymotrypsin in a 50% mixture, suggesting that the enzyme could renature under these conditions. However, in the 50% mixture, the true initial activity at 30°C is not zero, as the literature suggests. Instead, there is an initial burst of product formation over a few minutes, after which the enzyme becomes inactivated. By pre-incubating a 50% aqueous-acetonitrile mixture at 30°C prior to enzyme addition, the product burst could be eliminated. Activity could not then be recovered by slow addition of acetonitrile to the denatured enzyme. In contrast, it was possible to renature by dilution with aqueous buffer so that regeneration of catalytic activity was achieved. Thus, the good practical performance at high acetonitrile concentrations almost certainly results from a high kinetic barrier towards denaturation. The kinetics of enzyme denaturation in 50% and 70% acetonitrile were also investigated both at 30 and 20°C. Loss of catalytic activity was faster at higher temperature and at lower acetonitrile concentrations.

Enzymatic synthesis of beta-lactam antibiotics via direct condensation

In this paper, the feasibility of precipitation driven synthesis of acidic and zwitterionic beta-lactam antibiotics is studied. As an example of the first type, penicillin G was produced in good yield (160 mmol kg(-1)) directly from the free acid and amine aqueous substrate suspension, where the synthesis product precipitated. Such a precipitation driven synthesis via direct reversal of the hydrolytic reaction is thermodynamically unfavourable for zwitterionic beta-lactam antibiotics, such as amoxicillin. In this paper, a novel method is suggested to help favour precipitation of (poorly soluble) product salts by deliberate addition of certain counter-ions. After screening a number of different counter-ions, it was found that the amoxicillin anion forms a poorly soluble salt with Zn(2+). Despite increased beta-lactam degradation due to the presence of zinc ions, in a synthetic reaction with 0.1 M ZnSO(4) present the synthetic yield could be increased at least 30-fold.

Acid-Base Control for Biocatalysis in Organic Media: New Solid-State Proton/Cation Buffers and an Indicator

Although great care is generally taken to buffer aqueous enzyme reactions, active control of acid-base conditions for biocatalysis in low-water media is rarely considered. Here we describe a new class of solid-state acid-base buffers suitable for use in organic media. The buffers, composed of a zwitterion and its sodium salt, are able to set and maintain the ionisation state of an enzyme by the exchange of H+ and Na+ ions. Surprisingly, equilibrium is established between the different solid components quickly enough to provide a practical means of controlling acid-base conditions during biocatalysed reactions. We developed an organosoluble chromoionophore indicator to screen the behaviour of possible buffer pairs and quantify their relative H+/Na- exchange potential. The transesterification activity of an immobilised protease, subtilisin Carlsberg, was measured in toluene in the presence of a range of buffers. The large observed difference in rates showed good correlation with that expected from the measured exchange potentials. The maximum water activities accessible without formation of hydrates or solutions of the buffers are reported here. The indicator was also used to monitor, for the first time in situ, changes in the acid-base conditions of an enzyme-catalysed transesterification reaction in toluene. We found that even very minor amounts of an acidic by-product of hydrolysis were leading to protonation of the enzyme, resulting in rapid loss of activity. Addition of solid-state buffer was able to prevent this process, shortening reaction times and improving yields. Solid-state buffers offer a general and inexpensive way of precisely controlling acid-base conditions in organic solvents and thus also have potential applications outside of biocatalysis.