General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.

The substrate specificity of the flavoprotein vanillyl-alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl-alcohol oxidase catalyzes besides the oxidation of 4-hydroxybenzyl alcohols, the oxidative deamination of 4-hydroxybenzylamines and the oxidative demethylation of 4-(methoxymethy1)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed.Vanillyl-alcohol oxidase weakly interacts with 4-hydroxyphenylglycoIs and a series of catecholamines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epinephrine are substrates for vanillyl-alcohol oxidase, but the R isomer is preferred.Vanillyl-alcohol oxidase is most active with chavicol and eugenol. These 4-allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the Cy atom of the side chain is derived from water. The 4-hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation.The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein-bound FAD. pH-dependent binding studies suggest that vanillyl-alcohol oxidase preferentially binds the phenolate form of isoeugenol (pK,<6, 25°C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a p-quinone methide intermediate is proposed for the vanillyl-alcoholoxidase-catalyzed conversion of 4-allylphenols.Keywords: 4-allylphenols ; aromatic alcohol oxidase; covalently bound flavin; substrate specificity ; vanillin.Several fungi produce aryl-alcohol oxidases that are involved in the biodegradation of lignin, the most abundant aromatic biopolymer Abbreviations. Adrenalone, 3',4'-dihydroxy-2-methylaminoacetophenone; 4-allylanisole, 4-methoxy-allylbenzene; anisyl alcohol, 4-methoxybenzyl alcohol ; chavicol, 4-allylphenol ; cinnamyl alcohol, 3-phenyl-2-propene-1-01; coniferyl alcohol, 4-hydroxy-3-methoxycinnamyl alcohol ; coumaryl alcohol, 4-hydroxycinnamyl alcohol; epinephrine, 3,4-dihydroxy-u-(methylaminomethyl)benzyl alcohol (adrenalin); eugenol, 4-allyl-2-methoxyphenol ; homovanillyl alcohol, 4-hydroxy-3-methoxyphenethyl alcohol ; isoeugenol, 2-methoxy-4-propenylphenol; isosafrole, 1,2-(methylenedioxy)-4-propenylbenzene; metanephrine, 4-hydroxy-3-methoxy-a-(methylaminomethyl)benzyl alcohol; norepinephrine, a-(aminomethyl)-3,4-dihydroxybenzyl alcohol (noradrenalin); normetanephrine, n-(aminomethyl)-4-hydroxy-3-methoxybenzyl alcohol; octopamine, a-(aminornethyl)-4-hydroxybenzyl alcohol; safrole, 4-allyl-1,2-(methylenedioxy)benzene; synephrine, 4-hydroxy-a-(methylaminomethy1)benzyl alcohol; vanillyl alcohol, 4-hydroxy-3-methoxybenzyl alcohol ; vanillylamine, 4-hydroxy-3-methoxybenzylarnine ; vanillin, 4-hydroxy-3-methoxybenzaldehyde; veratryl alcohol, 3,4-dirnethoxybenzyl alcohol.

No abstract

The periplasmic Fe-hydrogenase from Desulfovibrio vulguris (Hildenborough) contains three ironsulfur prosthetic groups : two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two Fclusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing revmIs the existence of multiple charge conformers with PI in the range 5 7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H,-production activity determined under standardized conditions (z 7000 Ujmg). The enzyme exists in two different catalytic forms: as isdated the protein is 'resting' and 02-insensitive; upon reduction the protein becomes active and 02-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equiiibrium potentials are undefined, and previously reported apparent The iron-sulfur protein hydrogenase catalyzes the reversible activation of molecular hydrogen, a process involving the transfer of two electrons. Most presently known hydrogenases are also nickel proteins. The nickel ion is generally assumed to be the redox-active catalytic center [l, 21. A small subclass is formed by the Fe-hydrogenases; these enzymes presumably contain no other potentially redox active transition metals than iron [3]. By exclusion, this implies that the H2 activation is located on an iron-sulfur cluster. Redox catalysis is not Correspondence to W.

No abstract