In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

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.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.     

Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.     

Two benzaldehyde dehydrogenases in bacterium N.C.I.B. 8250. Distinguishing properties and regulation

Evidence is presented for the existence in bacterium N.C.I.B. 8250 of two inducible NAD⁺-linked benzaldehyde dehydrogenases. They may be distinguished in crude extracts by their different thermal stabilities at high pH values, benzaldehyde dehydrogenase I being much more heat-stable than benzaldehyde dehydrogenase II. Only benzaldehyde dehydrogenase I is activated by K⁺ and certain other univalent cations. Gel-filtration experiments indicate that both enzymes have molecular weights of about 180000. Both enzymes are induced by growth on l-mandelate or phenylglyoxylate; only benzaldehyde dehydrogenase I is gratuitously induced by thiophenoxyacetate and only benzaldehyde dehydrogenase II is induced by benzyl alcohol, by benzaldehyde, and by a number of heterocyclic compounds which do not support growth. Mutants have been isolated that lack either benzaldehyde dehydrogenase II or benzyl alcohol dehydrogenase, or both of the enzymes. Results obtained in induction experiments with the wild-type bacterium N.C.I.B. 8250 and with the mutants show that benzaldehyde dehydrogenase II and benzyl alcohol dehydrogenase are co-ordinately regulated. Overall, the results suggest that benzaldehyde dehydrogenase I is associated with the metabolism of l-mandelate whereas benzaldehyde dehydrogenase II is associated with the metabolism of benzyl alcohol.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme.     

Production of L-phenylacetyl carbinol by biotransformation: product and by-product formation and activities of the key enzymes in wild-type and ADH isoenzyme mutants of Saccharomyces cerevisiae

The capacities of yeast wild-type and mutants strains known to lack specific ADH isoenzymes to produce L-phenylacetyl carbinol (PAC) and benzyl alcohol in biotransformation trials were also investigated. Pyruvate decarboxylase activity, responsible for PAC formation and ADH activity, which can participate in reduction of benzaldehyde to benzyl alcohol, was also determined in each strain. In addition, the capacity of each strain to produce ethanol was investigated. Mutant strains lacking all of the isoenzymes, ADH-I, ADH-II, and ADH-III, still exhibited some ADH activity and were capable of production of benzyl alcohol and ethanol.     

Production of aryl metabolites in solid-state fermentations of the white-rot fungus Bjerkandera adusta

Bjerkandera adusta produced aromatic compounds such as benzaldehyde (bitter almond aroma), benzyl alcohol and benzoic acid from L-phenylalanine (3 g kg^–1). Two supports for the fungus, wheat bran (organic support) and Perlite (mineral support), gave optimal production with water contents of 66% and 60%, respectively. Benzyl alcohol (4.53 g kg^–1) and benzaldehyde (1.56 g kg^–1) were produced after 4 days on wheat bran respectively with 20 and 30 g L-phenylalanine kg^–1. Aryl alcohol oxidase activity, which oxidises benzyl alcohol to benzaldehyde, was only detected when the fungus was grown on wheat bran, the support which promotes the most benzaldehyde production. Results are compared with those obtained in submerged liquid cultures.     

Bioproduction of benzaldehyde in a solid–liquid two-phase partitioning bioreactor using <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-phase partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single phase biotransformation. The two-phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.     

Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans Gpo1. Expression of xylene monooxygenase genes could efficiently be controlled by n-octane and dicyclopropylketone. Xylene monooxygenase was found to catalyze the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes. For all three transformations (18)O incorporation provided stong evidence for a monooxygenation type of reaction, with gem-diols as the most likely reaction intermediates during the oxygenation of benzyl alcohols to benzaldehydes. To investigate the role of benzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes, xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB may prevent the formation of high concentrations of the particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribute to benzaldehyde formation via the energetically favorable dehydrogenation of benzyl alcohols. The results presented here characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes.     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.

Toluene and related aromatic compounds can be mineralized to CO₂ under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD⁺), benzaldehyde dehydrogenase (NADP⁺), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.Dedicated to Prof. Achim Trebst     

Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel<Emphasis Type="Italic"> Citrobacter freundii</Emphasis> strain

Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH₄, CO₂ and NH₄ ⁺, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO₂, H₂ and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.     

Benzyl alcohol metabolism by Pseudomonas putida: a paradox resolved

Evidence is presented for the existence in Pseudomonas putida of two NAD-linked dehydrogenases that function sequentially to oxidize benzyl alcohol. Induction of muconate lactonizing enzyme, a 3-oxoadipate pathway enzyme, indicated that P. putida oxidized benzyl alcohol to benzoate. Polyacrylamide gel electrophoresis with activity staining and enzymatic assays for an NAD-dependent dehydrogenase both showed that cells contained a single, constitutive alcohol dehydrogenase capable of oxidizing benzyl alcohol. This enzyme was shown to have the same specificity in extracts of glucose-grown as in benzy alcoholgrown cells. An NAD-aldehyde dehydrogenase oxidized benzaldehyde but was most active with normal alkyl aldehydes. This aldehyde dehydrogenase was shown to be induced, by enzymatic assays and by activity staining of polyacrylamide gel electropherograms, not only in cells grown on benzyl alcohol, but also in cells grown on ethanol. These experiments suggested that the aldehyde dehydrogenase was induced by the alcohol being oxidized rather than the substrate aldehyde.In sum, the evidence from enzyme assays and polyacrylamide gel electrophoresis of extracts indicates that Pseudomonas putida catabolizes benzyl alcohol slowly when it is the sole carbon and energy source, by the action of a constitutive, nonspecific, alcohol dehydrogenase and an alcohol-induced, nonspecific aldehyde dehydrogenase to yield benzoate, which is further metabolized via the 3-oxoadipate (beta-ketoadipate) pathway.In memory of R. Y. Stanier     

漆酶催化苯甲醇氧化制备苯甲醛

以自制的高活性漆酶为催化剂,考察漆酶催化苯甲醇制备苯甲醛的工艺条件(底物浓度、介质体系、溶剂体系、氢受体、酶的用量、通氧方式等)对氧化反应的影响。结果发现:优化反应条件为以2,2,6,6-四甲基哌啶-1-氧基(TEMPO)为介质体系且TEMPO与苯甲醇的摩尔比为1∶4、60 mmol/L的丙酮为氢受体、漆酶比酶活80 U/mL、60mmol/L的苯甲醇,反应体系通O20.5 h后密闭反应36 h,苯甲醛的产率达98%。     

Regulation of the synthesis of aryl metabolites by phospholipid sources in the white-rot fungus Bjerkandera adusta

The white-rot basidiomycete Bjerkandera adusta was cultivated in a liquid medium enriched with l-phenylalanine and various phospholipid sources (lecithin, egg yolk and asolectin). Three aromatic metabolites (benzaldehyde, benzyl alcohol and benzoic acid) were produced under these culture conditions. High concentrations of benzaldehyde (404 mg l^–1) were obtained when the cultures were supplemented with 10 g lecithin l^–1. Benzyl alcohol production was promoted when the strain was grown with 5 or 10 g lecithin l^–1. In the absence of or with a low concentration of lecithin (2.5 g l^–1), benzoic acid was the major aryl metabolite synthesized. The results presented here indicate that aryl alcohol oxidase, an extracellular enzyme catalyzing the oxidation of benzyl alcohol into benzaldehyde, was maximally detected when significant amounts of benzaldehyde were produced. Aryl alcohol oxidase activity was significantly enhanced in the presence of elevated concentrations of phospholipid sources. Together with lignin peroxidase, methoxylated and hydroxylated aryl metabolites were also synthesized under these culture conditions. The possible involvement of phospholipids in the synthesis of aryl metabolites is discussed. Received: 7 August 1998 / Accepted: 30 November 1998     

Enzymes of the mandelate pathway in bacterium N.C.I.B. 8250

1. Bacterium N.C.I.B. 8250 was grown on dl-mandelate, benzyl alcohol, benzoyl-formate, benzaldehyde and benzoate and also on 2-hydroxy, 4-hydroxy, 3,4-dihydroxy and 4-hydroxy-3-methoxy analogues of these compounds. The enzymic complements of the cells were determined and the specificities of some of the enzymes examined. 2. Growth on mandelate or benzoylformate induces l-mandelate dehydrogenase, benzoylformate decarboxylase, benzyl alcohol dehydrogenase and a heat-stable as well as a heat-labile benzaldehyde dehydrogenase. Growth on benzyl alcohol or benzaldehyde induces benzyl alcohol dehydrogenase and the heat-labile benzaldehyde dehydrogenase. 3. The enzymes of the mandelate-to-benzoate pathway are non-specifically active on, and induced by, all the substituted analogues that support growth. 4. Benzoate oxidase is induced by growth on benzoate or on 2-hydroxybenzoate. 2-Hydroxybenzoate hydroxylase, 4-hydroxybenzoate hydroxylase and 4-hydroxy-3-methoxybenzoate O-demethylase are induced only by growth on homologous substrates. 5. The results of the investigation are discussed with regard to the possible regulation of the enzyme systems.     

Effect of organic solvent on biotransformation of benzaldehyde to benzyl alcohol by free and silicone-alginate entrapped cells

Summary Biotransformations of benzaldehyde to benzyl alcohol by whole cells of baker's yeast immobilised in a 50%:50% silicone-alginate mixed hydrophobic/hydrophilic matrix were successfully carried out in apolar solvents. In more polar solvents, the biotransformation was ineffective when either free or entrapped cells were used.