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.

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Analysis of fifty sorghum [Sorghum bicolor (L.) Moench] varieties used in Burkina Faso showed that they have different contents of phenolic compounds, peroxidase (POX), and polyphenol oxidase (PPO). Most of the varieties (82%) had a tannin content less than 0.25% (w/w). POX specific activity was higher than the monophenolase and o-diphenolase specific activities of PPO. For POX, there was a diversity of isoforms among varieties. No clear correlation could be made between the quantitative composition of the grain in phenolics, PPO, and POX, and resistance of plant to pathogens. In general, varieties good for a thick porridge preparation ("tô") had low phenolic compounds content and a medium POX activity. From the red varieties, those used for local beer ("dolo") had a high content in phenolic compounds and PPO, and a low POX activity. The variety considered good for couscous had a low POX content. The characteristics might be useful as selection markers for breeding for specific applications.

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.

Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol is further oxidized to vanillin. Catalysis is limited by the formation of an abortive complex between enzyme-bound flavin and creosol. Moreover, in the second step of the process, the conversion of vanillyl alcohol is inhibited by the competitive binding of creosol. The VAO-catalyzed conversion of vanillylamine proceeds efficiently at alkaline pH values. Vanillylamine is initially converted to a vanillylimine intermediate product, which is hydrolyzed nonenzymatically to vanillin. This route to vanillin has biotechnological potential as the widely available principle of red pepper, capsaicin, can be hydrolyzed enzymatically to vanillylamine.

1. Evidence is presented that the direct depressing effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii is due to inhibition of the electron transport system to nitrogenase. Furthermore, we were able to confirm the observation [Houwaard, F. (1979) Appl. Environ. Microbiol. in the press] that ammonium chloride has no short-term effect on nitrogen fixation by isolated bacteroids of Rhizobium leguminosarum.2. By means of the flow dialysis technique it could be demonstrated that in A . vinelandii ammonium is taken up as a cation in response to the A $ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the A $ across the cytoplasmic membrane. In A . vinelandii, like in bacteroids, the generation of reducing equivalents at a potential low enough to reduce nitrogenase was found to be extremely sensitive towards changes in A$. At A$ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a d $ value of 110 mV nitrogenase is supplied optimally with reducing equivalents. The nature of the ammonium transport system in A. vinelandii and its significance as a regulator for the rapid 'switch off/switch on' of nitrogenase activity is discussed.3. Bacteroids of R. leguminosarum did not accumulate ammonium and no effect of ammonium on A $ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the A pH.It has been firmly established that the process of nitrogen fixation by free-living bacteria [l-41 as well as by symbiotic associations [5,6] is inhibited in the presence of fixed nitrogen, especially ammonium. In the free-living nitrogen-fixer Azotobacter two effects of ammonium can be distinguished. Firstly, the energetically expensive nitrogenase reaction is rapidly switched off (short-term effect) [l]. Secondly, the synthesis of nitrogenase is repressed, resulting in a decline of total activity (long-term effect) [2,7]. On the other hand, fixed nitrogen does not affect the nitrogenase activity of isolated bacteroids of Rhizobium leguminosarum [8]. However, when these bacteroids are in symbiosis with the plant host cell, fixed nitrogen affects the nitrogenase activity [5,6].Little is known about the short-term effect of am- enough energy in the form of ATP and reducing equivalents. Since cell-free nitrogenases are insensitive to ammonium concentrations that completely inhibit the nitrogenase activity in whole cells [9,10], it has been suggested that ammonium in Azotobacter depresses the flow of reducing equivalents and/or the supply of ATP to nitrogenase [l, 1 I].In the present paper we investigated the shortterm effect of ammonium chloride on nitrogen fixation by the free-living organism Azotobacter vinelandii and the symbiotic nitrogen-fixer R. leguminosarum. Evidence is presented that electron transfer to nitrogenase in A. vinelandii, like in R. leguminosarum bacteroids [12], is regulated exclusively by the electrical component (A$) of the protonmotive for...

20P-Hydroxysteroid dehydrogenase was enclosed in reversed micellar media consisting of cetyltrimethylammonium bromide, hexanol, organic solvent and Hepes buffer. The influence of the composition of these media on the enzymatic reduction of the apolar steroids progesterone and prednisone was investigated by varying the water content, concentration of hexanol and type of organic solvent. By changing the water content and the type of organic solvent, the hexanol to cetyltrimethylammonium bromide ratio in the interphase can be varied. This ratio was determined by phase boundary titrations. It was found that the higher this ratio, the higher the rate of steroid conversion. From variations of the hexanol content it was concluded that the rate of steroid conversion is determined by the hydrophobicity of the steroid relative to the hydrophobicity of the continuous phase and the hydrophobicity of the interphase. The hydrophobicity of the phases was expressed in log P-values. Log P i s defined as the logarithm of the partition coefficient in an octanol-water two-phase system. This enabled us to derive the following relations between the hydrophobicity values for the substrate (log PJ, for the interphase (log PJ and for the continuous phase (log Pcph): (log P,-log P J must be minimal to ensure a high steroid concentration in the interphase and llog P c p h -log PSI must be large to keep the steroid concentration in the continuous phase low. With these considerations, for any given apolar compound, a medium can be composed that gives optimal enzymatic conversion.Interest in the application of enzymes for the conversion of water-insoluble compounds in apolar media is growing. To date, several methods have been described, most of which deal with enzymes in mixtures of water and water-miscible organic solvents. Such systems can never have a very apolar character and are therefore, less suitable for the conversion of highly water-insoluble compounds. Furthermore, enzyme activity in such systems is usually lost and substrate specificity disappears when the percentage of water-miscible organic solvent exceeds 10 -40 % . Hence systems are preferred that consist for a major proportion of a non water-miscible organic solvent [l 1.A relatively new method of enzyme immobilization in organic media is the entrapment of enzymes in reversed micelles [2 -41. A reversed micellar medium is a multi-phase system consisting of tiny water droplets stabilized by surfactants in a bulk, water-immiscible, organic solvent. As was shown by several authors, reversed micelles produce a unique microenvironment for enzymes which protects them against denaturation by the surrounding organic solvent and enables enzymes to convert non water-soluble: or poorly water-soluble compounds present in the bulk organic phase. Such a reversed micellar medium possesses an enormous interfacial area (z 100 m2/ml) and the distance between the interface and the Abbreviations and Symbols. CTAB, cetyltrimethylammonium bromide; HSDH, 20 j-hydroxysteroid debydrogenase; a,, mol...

Interactions between fluorescent horse heart cytochrome c derivatives (e. g. porphyrin cytochrome c and Znporphyrin cytochrome c) with surfactant interfaces in reversed micellar solutions have been studied, using different spectroscopic techniques. Anionic [sodium bis(2-ethylhexyl)sulfosuccinate, AOT] and cationic (cetyltrimethylammonium bromide, CTAB) surfactant solutions have been used in order to investigate the effects of charge interactions between proteins and interfaces.Circular dichroism reveals that much of the protein secondary structure is lost in AOT-reversed micelles, especially when the molar water/surfactant ratio, wo, is high (w, = 40), whereas in CTAB-reversed micelles secondary structure seems to be preserved.Time-resolved fluorescence measurements of the porphyrin in the cytochrome c molecule yields information about the changes in structure and the dynamics of the protein upon interaction with surfactant assemblies both in aqueous and in hydrocarbon solutions. With AOT as surfactant a strong interaction between protein and interface can be observed. The effects found in aqueous AOT solution are of the same kind as in hydrocarbon solution. In the CTAB systems the interactions between protein and surfactant are much less pronounced. The measured effects on the fluorescence properties of the proteins are different in aqueous and hydrocarbon solutions.In general, the observations can be explained by an electrostatic attraction between the overall positively charged protein molecules and the anionic AOT interface. Electrostatic attraction can also occur between the cytochrome c derivatives and CTAB because there is a negatively charged zone on the surface of the proteins. From the fluorescence anisotropy decays it can be concluded that in the CTAB-reversed micellar system these interactions are not important, whereas in an aqueous CTAB solution the proteins interact with surfactant molecules.Surfactant assemblies in organic media, called reversed micelles or, more general, water-in-oil microemulsions, have been investigated extensively over the past years. Many studies have been carried out towards the elucidation of structure and dynamics of reversed micelles [I -41 but also on the (bio)chemical and (bio)technological applications of these systems [2, 3, 5-91. In our laboratory an important line of research consists of the incorporation of proteins in reversed micelles in order to perform bioconversions of apolar compounds or to isolate proteins [lo -121. At present, the effects of protein solubilization on both protein and micelle structure and dynamics are not very clear. Because reversed micellar solutions are optically transparent, spectroscopic and ultracentrifugation techniques can be applied to study these effects. Several systems have been the subject of this kind of investigation, some containing cytoplasmatic, others containing membrane proteins [5, 7-91. Horse heart cytochrome c is a protein which in vivo is strongly associated via electrostatic interactions with the inner Correspo...