Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.     

Progesterone fate in rabbit cornea

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.     

Inducible and constitutive kynureninases. Control of the inducible enzyme activity by transamination and inhibition of the constitutive enzyme by 3-hydroxyanthranilate.

The inducible kynureninase from Neurospora crassa is inactivated by incubation with L-alanine or L-ornithine. The inactivated enzyme is resolved to the apoenzyme by dialysis. Reactivation of the apoenzyme is achieved by incubation with pyridoxamine 5'-phosphate plus pyruvate, as well as with pyridoxal 5'-phosphate. The kynurenine hydrolysis proceeds linearly in the presence of added pyridoxal 5'-phosphate, or pyridoxamine 5'-phosphate plus pyruvate. These findings indicate that the fungal inducible kynureninase can act as an amino-transferase to control the enzyme activity, and that the control mechanism is similar to that reported for the bacterial kynureninase (Moriguchi, M. & Soda, K. (1973) Biochemistry 12, 2974-2980). The ratio of kynureninase activity to aminotransferase activity was determined with bacterial and fungal enzymes. All the inducible kynureninases from various fungal species examined are also controlled by the transamination. In contrast, the pig liver kynureninase and the fungal constitutive enzymes are little or not at all affected by preincubation with amino acids. Thus, the present regulatory mechanism does not operate in these constitutive-type enzymes. The rate of hydrolysis of L-3-hydroxykynurenine by the pig liver enzyme decreases with increase in the incubation time; the enzyme is inhibited by 3-hydroxyanthranilate produced from L-3-hydroxykynurenine. The inhibition is found in all the constitutive-type enzymes, suggesting that 3-hydroxyanthranilate plays a regulatory role in NAD biosynthesis from tryptophan.     

Relationship between the Decomposition Process of Coarse Woody Debris and Fungal Community Structure as Detected by High-Throughput Sequencing in a Deciduous Broad-Leaved Forest in Japan

We examined the relationship between the community structure of wood-decaying fungi, detected by high-throughput sequencing, and the decomposition rate using 13 years of data from a forest dynamics plot. For molecular analysis and wood density measurements, drill dust samples were collected from logs and stumps of Fagus and Quercus in the plot. Regression using a negative exponential model between wood density and time since death revealed that the decomposition rate of Fagus was greater than that of Quercus. The residual between the expected value obtained from the regression curve and the observed wood density was used as a decomposition rate index. Principal component analysis showed that the fungal community compositions of both Fagus and Quercus changed with time since death. Principal component analysis axis scores were used as an index of fungal community composition. A structural equation model for each wood genus was used to assess the effect of fungal community structure traits on the decomposition rate and how the fungal community structure was determined by the traits of coarse woody debris. Results of the structural equation model suggested that the decomposition rate of Fagus was affected by two fungal community composition components: one that was affected by time since death and another that was not affected by the traits of coarse woody debris. In contrast, the decomposition rate of Quercus was not affected by coarse woody debris traits or fungal community structure. These findings suggest that, in the case of Fagus coarse woody debris, the fungal community structure is related to the decomposition process of its host substrate. Because fungal community structure is affected partly by the decay stage and wood density of its substrate, these factors influence each other. Further research on interactive effects is needed to improve our understanding of the relationship between fungal community structure and the woody debris decomposition process.     

A mechanistic investigation of the microbial chiral inversion of 2-phenylpropionic acid by Verticillium lecanii

Previous investigations have described the development of nongrowing suspension of Verticillium lecanii as a microbial model of the mammalian chiral inversion of the 2-arylpropionic acids (2-APAs). Mechanistic studies in mammals have shown that inversion involves loss of the α-methine proton but retention of the original atoms at the β-methyl position, and a mechanism has been proposed involving enzymatic epimerisation of acyl-CoA thioester derivatives of the substrate. Inversion of the 2-APAs by V. lecanii exhibits extensive intersubstrate variation in the presence, rate, extent, and direction of inversion, which are different from those observed in mammalian systems, possibly indicating differences in the mechanism of inversion between mammalian and microbial cells. This study involved the investigation of proton/deuterium exchange by ¹H-nuclear magnetic resonance following incubation of deuterated derivatives of 2-phenylpropionic acid (2-PPA), a model compound, in cell suspensions of V. lecanii and incubation of undeuterated 2-PPA in cell suspensions containing D₂O. The results indicated that the inversion of 2-PPA by V. lecanii also involved exchange of the α-methine proton but complete retention on the original atoms at the β-methyl position. No kinetic deuterium isotope effect was observed, indicating that loss of the α-methine proton is not the rate-limiting step of the inversion process. This suggests that the observed differences between microbial and mammalian systems probably involve the stereoselective acyl-CoA thioester formation step and not the subsequent epimerisation of the resultant diastereomers. Chirality 9:254–260, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of six copper-containing amine oxidases by the antidepressant drug tranylcypromine

Potential inhibitory effects of the clinically utilized monoamine oxidase inhibitor tranylcypromine (TCP) on mammalian, plant, bacterial, and fungal copper-containing amine oxidases have been examined. The following enzymes have been investigated: human kidney diamine oxidase (HKAO), bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris lysyl oxidase (PPLO). Only BPAO, EPAO, and AGAO were found to lose significant levels of activity when incubated with varying amounts of TCP. Inhibition of BPAO was completely reversible, with dialysis restoring full activity. TCP inhibition of AGAO was also found to be ultimately reversible; however, dialysis did not remove all bound compounds. Chemical displacement with either substrate or a substrate analogue successfully removed all bound TCP, indicating that this compound has a high affinity for the active site of AGAO. The notable lack of TCP inhibition on HKAO argues against the inhibition of diamine oxidase as a potential source for some of the deleterious side effects occurring in patients treated with this antidepressant. The marked differences observed in behavior among these enzymes speaks to the importance of intrinsic structural differences between the active sites of copper amine oxidases (CAO) which affect reactivity with a given inhibitor.     

Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

Effects of ruminal protozoa on cellulose degradation and the growth of an anaerobic ruminal fungus, Piromyces sp. strain OTS1, in vitro.

An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed, A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, and chitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids, notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formate was not detected at the end of the experiment and the amount of reducing sugars remained low throughout the incubation period. The fungal growth in the coincubations was negatively affected. While protozoal predation on zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulose degradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungal biomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more favorable environment.     

Production of alpha, omega-alkanediols using Escherichia coli expressing a cytochrome P450 from Acinetobacter sp. OC4

Our biotransformation using Escherichia coli expressing a cytochrome P450 (CYP) belonging to the CYP153A family from Acinetobacter sp. OC4 produced a great amount of 1-octanol (2,250 mg per liter) from n-octane after 24 h of incubation. This level of production is equivalent to the maximum level previously achieved in biotransformation experiments of alkanes. In addition, the initial production rate of 1-octanol was maintained throughout the entire incubation period. These results indicate that we have achieved the functional and stable expression of a CYP in E. coli for the first time. Further, our biotransformation system showed alpha,omega-diterminal oxidation activity of n-alkanes, and a large amount of 1,8-octanediol (722 mg per liter) was produced from 1-octanol after 24 h of incubation. This is the first report on the bioproduction of alpha,omega-alkanediols from n-alkanes or 1-alkanols.     

Dynamics of bacterial and fungal communities associated with eggshells during incubation

Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture‐based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival.     

Field and microcosm experiments to evaluate the effects of agricultural Cu treatment on the density and genetic structure of microbial communities in two different soils

The effects of Cu amendment on indigenous soil microorganisms were investigated in two soils, a calcareous silty clay (Ep) and a sandy soil (Au), by means of a 1-year field experiment and a two-month microcosm incubation. Cu was added as 'Bordeaux mixture' [CuSO(4), Ca(OH)(2)] at the standard rate used in viticulture (B1=16 kg Cu kg(-1) soil) and at a higher level of contamination (B3=48 kg Cu ha(-1) soil). More extractable Cu was observed in sandy soil (Au) than in silty soil (Ep). Furthermore, total Cu and Cu-EDTA declined with time in Au soil, whereas they remained stable in Ep soil. Quantitative modifications of the microflora were assessed by C-biomass measurements and qualitative modifications were assessed by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using B- and F-ARISA (bacterial and fungal automated ribosomal intergenic spacer analysis). In the field study, no significant modifications were observed in C-biomass whereas microcosm incubation showed a decrease in B3 contamination only. ARISA fingerprinting showed slight but significant modifications of bacterial and fungal communities in field and microcosm incubation. These modifications were transient in all cases, suggesting a short-term effect of Cu stress. Microcosm experiments detected the microbial community modifications with greater precision in the short-term, while field experiments showed that the biological effects of Cu contamination may be overcome or hidden by pedo-climatic variations.     

Substrate specificity for the 12beta-hydroxylation of bufadienolides by Alternaria alternata

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.     

Microbial models of drug metabolism: microbial transformations of Trimegestone (RU27987), a 3-keto-delta(4,9(10))-19-norsteroid drug

Screening microorganisms for the biotransformation of the 3-keto-delta(4,9(10))-19-norsteroid RU27987 (Trimegestone) resulted in the isolation of nine identified metabolites, some of them being selectively produced by different strains. Eight metabolites were found to be hydroxylated on various positions of the rings, and one was additionally epoxidized. These microbial metabolites could be used as chromatographic standards and two of them were found identical to the unknown major human metabolites. Moreover, most microbial metabolites were produced in sufficient amounts to be tested for their biological activities. All these features demonstrate the usefulness and versatility of microbial biotransformation systems as a tool for early identification and convenient production of potentially active mammalian and non-mammalian metabolites.     

Differential biotransformation of the enantiomers of isoidide dinitrate in isolated rat aorta

Previous studies have demonstrated that the D-enantiomer of isoidide dinitrate (IIDN) is 10-fold more potent than the L-enantiomer for relaxation and cyclic GMP accumulation in isolated rat aorta. To test whether preferential biotransformation of D-IIDN to a species that activates guanylate cyclase is the basis for this observed enantioselectivity, paired segments of rat aorta were exposed to D- and L-IIDN and the tissue accumulation of the parent compound and the formation of their respective metabolites (D- and L-isoidide mononitrate, IIMN) were determined. The extent of relaxation of rat aorta following exposure to 2 microM D-IIDN was greater than that by L-IIDN over a 5-minute time course, and this was associated with a higher rate of D-IIDN biotransformation to D-IIMN at all time points. In addition, the rate of D-IIDN biotransformation was greater than that of L-IIDN at most IIDN concentrations tested. By contrast, the amount of D- and L-IIDN in the tissue was the same at all time points and concentrations tested, indicating that selective uptake of D-IIDN into blood vessels did not occur. When tissues were made tolerant to organic nitrate-induced relaxation by treatment with a high concentration of glyceryl trinitrate, the biotransformation of both D- and L-IIDN was attenuated. This suggests that mechanism-based biotransformation may be affected during tolerance development. Furthermore, the association of preferential D-IIDN biotransformation with its greater potency for vasodilation and cyclic GMP accumulation suggests than an enantioselective site for biotransformation is an important component of organic nitrate-induced vasodilation.     

Systematic screening strategy for fungal laccase activity of endophytes from Otoba gracilipes with bioremediation potential

Fungal laccases are promising for biotechnological applications, including bioremediation and dye biotransformation, due to their high redox potential and broad substrate specificity. However, current bioprospecting methods for identifying laccase-producing fungi can be challenging and time-consuming. For early detection, it was developed a three-step, multi-criteria weighting system that evaluates fungal strains based on: First, the biotransformation capacity of three dyes (i.e., Congo red, brilliant blue G-250, and malachite green), at three different pH values, and with a relative weighting supported for the redox potential of each colorant. The relative decolorization coefficient (RDC), used as th2e first classification criterion, expressed their potential performance. Second, under the same conditions, laccase activity was estimated by observing the different degrees of oxidation of a given substrate. The selection criterion was the relative oxidation coefficient (ROC). Finally, laccase activity was quantified in submerged fermentations using three inducers (i.e., loofah sponge, Tween 80, and veratyl alcohol). This multicriteria screening strategy evaluated sixteen isolated endophytic fungal strains from Otoba gracilipes. The system identified Beltraniopsis sp. ET-17 (at pH values of 5.00 and 5.50) as a promising strain for dye biotransformation, and Phlebia floridensis as the best laccase producer, achieving a high activity of 116 μmol min⁻¹ L⁻¹ with loofah sponge as an inducer. In-vitro testing confirmed the efficacy of P. floridensis, with 53.61 % decolorization of a dye mixture (brilliant blue-Congo red. ratio 1:1) after 15 days of incubation. Thus, with the proposed screening strategy it was possible to highlight two species of interest at an early bioprospecting stage on a Colombian native tree poorly explored.     

Biopotency and nuclear binding of glucocorticoids

Nuclear binding abilities of 3 glucocorticoids, dexamethasone (Dex), prednisolone (Pred) and corticosterone (Cort), which exhibited different biopotencies were compared in vitro. cytosols labelled with 3H-Dex, 3H-Pred and 3H-Cort from the rat liver prepared by incubation at 0 degrees C for 16 hr were bound to isolated liver nuclei in rates of approximately 25%, 9% and 1% of added radioactivity, respectively. Nuclear binding rates observed were correlated with biopotencies of these steroids. Time course studies of the cytosol binding revealed that the difference in the nuclear binding ability of these ligands was attributable, at least in part, to the metabolic transformation of ligands during the incubation period. A significant portion of 3H-Pred and 3H-Cort was transformed to polar metabolite(s) even under the incubation conditions at 0 degrees C. Kd's of the cytosol binding to 3H-Dex which was metabolically stable were decreased with the length of incubation time, significantly lower Kd being observed in the cytosol incubated for 16 hr than in those incubated for 2 and 6 hr. Kd's and the number of maximum binding sites were erratic when the ligands received biotransformation during the course of incubation. Transformed 3H-Pred and 3H-Cort during the incubation still exhibited features of the protein bound state. Besides biotransformation of ligands, structure related difference in the nuclear binding ability of these glucocorticoids was also observed. These observations suggest that metabolic susceptibility as well as structure related ability of the nuclear binding may contribute to the biopotency of glucocorticoids.     

Biotransformation of the organochlorine pesticide trans-chlordane by wood-rot fungi

There is very limited information on the biotransformation of organochlorine pesticide chlordane by microorganisms, and no systematic study on the metabolic products and pathways for chlordane transformation by wood-rot fungi has been conducted. In this study, trans-chlordane was metabolized with the wood-rot fungi species Phlebia lindtneri, Phlebia brevispora and Phlebia aurea, which are capable of degrading polychlorinated dibenzo-p-dioxin and heptachlor epoxide. At the end of 42 days of incubation, over 50% of trans-chlordane was degraded by the fungal treatments in pure cultures. These fungi transformed trans-chlordane to at least eleven metabolites including a large amount of hydroxylated products such as 3-hydroxychlordane, chlordene chlorohydrin, heptachlor diol, monohydroxychlordene and dihydroxychlordene. P. lindtneri particularly can metabolize oxychlordane, a recalcitrant epoxide product of chlordane, into a hydroxylated product through substitution of chlorine atom by hydroxyl group. The present results suggest that hydroxylation reactions play an important role in the metabolism of trans-chlordane by these Phlebia species. Additionally, transformation of trans-chlordane and production of hydroxylated metabolites were efficiently inhibited by the addition of cytochrome P450 inhibitors, piperonyl butoxide and 1-aminobenzotriazole, demonstrating that fungal cytochrome P450 enzymes are involved in some steps of trans-chlordane metabolism, particularly in the hydroxylation process.     

Bacterial and fungal taxon changes in soil microbial community composition induced by short-term biochar amendment in red oxidized loam soil

To take full advantage of biochar as a soil amendment, the objective of this study was to investigate the effects of biochar addition on soil bacterial and fungal diversity and community composition. Incubation experiments with a forest soil (a red oxidized loam soil) with and without biochar amendment were conducted for 96 days. The culture-independent molecular method was utilized to analyze soil bacterial and fungal species after the incubation experiments. Results showed that bacteria and fungi responded differently to the biochar addition during the short-term soil incubation. Twenty four and 18 bacterial genara were observed in the biochar amended and unamended soils, respectively, whereas 11 and 8 fungal genera were observed in the biochar amended and unamended soils, respectively. Microbial taxa analysis indicated that the biochar amendment resulted in significant shifts in both bacterial and fungal taxa during the incubation period. The shift for bacteria occurred at the genus and phylum levels, while for fungi only at the genus level. Specific taxa, such as Actinobacteria of bacteria and Trichoderma and Paecilomyces of fungi, were enriched in the biochar amended soil. The results reveal a pronounced impact of biochar on soil microbial community composition and an enrichment of key bacterial and fungal taxa in the soil during the short time period.     

Effects of external calcium on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by <Emphasis Type="Italic">Paecilomyces bainier</Emphasis> 229-7

Calcium is a known signalling molecule in eukaryotic cells and plays a central role in the regulation of many cellular processes. In the following study, we report on the effect of external calcium treatments on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7. We observed that the intracellular calcium content of P. bainier 229-7 mycelia was increased in response to exposure to high external Ca²⁺ concentrations. Both ginsenoside Rd biotransformation and β-glucosidase activity were both found to be dependent on the external calcium concentration. At an optimal Ca²⁺ concentration of 45 mM, maximal ginsenoside Rd bioconversion rate of 92.44% was observed and maximal β-glucosidase activity of 0.1778 U was reached in a 72-h biotransformation. The Ca²⁺ channel blocker Verapamil blocked the trans-membrane influx of calcium and decreased ginsenoside Rd biotransformatiom. In addition, β-glucosidase activity and ginsenoside Rd content decreased by 36.0 and 29.2% respectively after a 72-h incubation in the presence of 0.05 mM Calmodulin (CaM) antagonist Perphenazine. These results suggest that both Ca²⁺ channels and CaM are involved in ginsenoside Rd biotransformation via regulation of β-glucosidase activity. This is the first report regarding the effects of calcium signal transduction on biotransformation and enzyme activity in fungi.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.