PAL的活性

Received 7 May 1996; revised 21 October 1996; accepted 29 October 1996
Abstract
L-Phenylalanine ammonia-lyase ŽEC 4.3.1.5., ŽPAL. was shown to be active in a monophasic non-aqueous medium for the first time. Ultraviolet absorbance spectra of trans-cinnamic acid were shown to be similar in both water and n-octanol. High catalytic rates were observed only when the enzyme was placed in solvents containing high concentrations of water. PAL forward reaction was observed only when the water concentration in n-octanol exceeded 2.0% Žvrv., which corresponds to a value of 0.8 in thermodynamic water activity Ž aw . terms. In n-octanol containing either 2.0 or 3.5% Žvrv. H 2O Žand 2 mM L-phenylalanine., lyophilized and aw s 0.113 pre-equilibrated PAL powder exhibited catalytic rates 0.02 and 1.75% of the value observed in aqueous solution respectively. A freshly lyophilized Žnon-equilibrated. PAL preparation incubated in water-saturated n-octanol Žmeasured wH 2Ox s 3.6% Žvrv., L-phenylalanine concentration f 6.8 mM. gave catalytic activity values 17% of those observed in aqueous solution. This is the first demonstration of catalytic activity of an amino acid ammonia-lyase in monophasic organic solvent.
in a monophasic, organic reaction medium. This enzyme has several features that render it suitable for more fundamental studies: the PAL-catalysed reaction is only single-substrate ŽL-phenylalanine. and neither consumes or produces water, the reaction catalysed in solvents is identical to that catalysed in aqueous solution and the enzyme requires no coenzymes or cofactors.
Biochimica et Biophysica Acta 1338 Ž1997. 121–126
Activity of L-phenylalanine ammonia-lyase in rganic solvents
D. Gareth Rees 1, D. Hugh Jones )
Molecular Biology Research Group, School of Biological Sciences, UniÕersity of Wales Swansea, Swansea SA2 8PP, UK
) Corresponding author. Fax: q44 1792 295447. 1 Present address: Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, G1 1XW, United Kingdom.
Keywords: Enzyme activity; Water activity; Organic solvent; Ammonia-lyase; Ž Rhodosporidium.
1. Introduction
An emerging field of enzymology which has important implications for applied biocatalysis is the use of enzymes in reaction media consisting of low-water, monophasic organic solvents. Enzymes suspended in these reaction media can exhibit novel properties including stabilization towards heating, changes in enantioselectivity and substrate specificity, molecular imprinting and inhibitor-induced enzyme activation w1,2x.
2. Materials and methods
2.1. Enzyme and solÕent preparation
Unless otherwise indicated partially purified and lyophilized PAL in microfuge tubes Žprepared as described previously w9x. was equilibrated over a saturated LiCl P 1H 20 solution Ž aw s 0.113 at 308C w10x.. The time required for equilibration of solid enzyme powders was taken from published recommendations w4x. Solvents were dried with 3A molecular sieves prior to addition of water andror substrate.
2.2. Spectrophotometric determination of trans-cinnamic acid in n-octanol
ate times tubes were withdrawn from the bath, briefly centrifuged Ž13 000 r.p.m.. and the supernatants analyzed by taking a complete ultraviolet absorbance spectrum. This process took less than one minute to complete and absorbance spectra of supernatants did not change on standing, indicating that this step was sufficient to remove all the Žsolid. enzyme from the liquid phase before analysis. Reference solutions for the spectrophotometric baseline consisted of the solvent containing the same amount of water as the experimental tubes. Separate experiments conducted in water-saturated n-octanol and amyl acetate showed that, as expected w11x, PAL protein powder is insoluble in the organic solvents studied.
0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 6 . 0 0 1 9 5 - 1
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D.G. Rees, D.H. Jones r Biochimica et Biophysica Acta 1338 (1997) 121–126
One of the most important aspects of non-aqueous enzymology is the effects on catalysis of the small quantities of water distributed amongst the various phases in the reaction system w3–7x. It has been demonstrated that these systems are best studied at fixed water activity Ž aw., for example, it has been shown that the maximal reaction rate of suspended lipase catalyst occurred at similar aw values in solvents of differing polarity w4x. No such consistency was observed when only the water content in the organic phase or the total water content of the system are considered.