Gas phase biotransformation reaction catalyzed by baker's yeast

The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes. The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated. The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability.     

The catalytic serine of meta-cleavage product hydrolases is activated differently for C-O bond cleavage than for C-C bond cleavage

meta-Cleavage product (MCP) hydrolases catalyze C-C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl-enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, p-nitrophenyl benzoate (pNPB), with a k(cat) value (6.3 ± 0.5 s(-1)) similar to that of HOPDA (6.5 ± 0.5 s(-1)). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > n-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, k(cat)/K(m) values were independent of methanol concentration, while both k(cat) and K(m) values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, k(obs) values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C-C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl-enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism.     

A novel synthesis of carbocyclic C-nucleoside precursors by nonoxidative C-C bond fission

New carbocyclic c-nucleoside precursors have been synthesized in stereocontrolled manner by nonoxidative C-C bond fission (i.e., Grob fragmentation or retrograde aldol reaction) starting from Diels-Alder adducts of cyclopentadiene with 3-acetoxyacrylates or dimethyl 2-acetoxymethylenemalonate.     

C-C bond formation and cleavage in radical enzymes, a theoretical perspective

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C-C bond formation or cleavage reactions.     

C-C bond formation and cleavage in radical enzymes, a theoretical perspective

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C-C bond formation or cleavage reactions.     

Stereochemistry of internucleotidic bond formation by tRNA nucleotidyltransferase from baker's yeast.

Isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) is a substrate for tRNA nucleotidyltransferase from baker's yeast, whereas isomer B is a competitive inhibitor. The tRNA resulting from this reaction has a phosphorothioate instead of a phosphate diester linkage at the last internucleotidic linkage between cytidine and adenosine. On limited digestion of this tRNA with RNase A, one can isolate cytidine 2',3'-cyclic phosphorothioate which can be deaminated to uridine 2',3'-cyclic phosphorothioate. It can be shown that this compound is the endo isomer and that, therefore, the phosphorothioate diester bond in the tRNA must have had the R configuration. This result indicates that no racemization during the condensation of ATP alpha S, isomer A, onto the tRNA had occurred. Whether inversion or retention of configuration had taken place awaits elucidation of the absolute configuration of isomer A of ATP alpha S.     

Enantiofacial selective reduction of 2-allyl-2-carboethoxy-cyclopentanone mediated by baker's yeast

Enantioselective reduction of 2-allyl-2-carboethoxy-cyclopentanone (2) was accomplished in high enantiomeric excess ( > 99%), using baker's yeast in the presence of CuO, to obtain the (+)-2-allyl-2-carboethoxy-cyclopentanol derivative (6). This methodology also provides an entry to corresponding β-keto ester (-)-(2), representing an important strategy to prepare chiral functionalized 2-oxabicyclic[3.3.0]octane and 2-oxabicyclic[4.4.0]nonane derivatives, useful synthons to access new bioactive compounds. © 1996 Wiley-Liss, Inc.     

On a disturbance of the normal Pasteur reaction in baker's yeast

Resting cells of baker's yeast, suspended in phosphate buffer pH 5.0 with glucose give initially a normal Pasteur reaction, which means that fermentation is repressed under aerobic conditions by the respiratory process.However, after 1 to 2 hours fermentation a disturbance of the Pasteur reaction sets in, the aerobic fermentation rising to the anaerobic level or sometimes above this level without a corresponding decrease in respiration. It is demonstrated that this disturbance is closely related to an aerobic growth pattern in which the yeast in its final growth stage before harvesting obtains its energy exclusively from the respiratory process.The interrelation of fermentation and respiration is discussed. In this discussion the aerobic fermentation is defined as the metabolism of the excess of intermediates formed along the Embden-Meyerhof pathway and unable to enter the Krebs cycle due to the limited capacity of the electron transfer system.The author is greatly indebted to Prof. Dr. T. O. Wikén for his interest in this study and for offering him the opportunity to conduct the investigations in his laboratory.     

A vanadium-promoted C-N bond cleavage

A C-N bond in one arm of the mixed-valence V^III-V^IV complex bpbp(VOCl₂)(VCl₂), bpbpH = 2,6-bis((N,N-bis-(2-picolyl)amino)methyl)-4-tertbutylphenol, is cleaved in wet acetonitrile solution to give bpa(VOCl₂), bpa = bis(2-methypyridyl)amine, and 2-((N,N-bis-(2-picolyl)amino)methyl)-6-hydroxymethyl-4-tertbutylphenol. The reaction corresponds overall to hydrolysis of a tertiary amine to form a secondary amine and a primary alcohol. The structure of bpa(VOCl₂) was established by X-ray diffraction while 2-((N,N-bis-(2-picolyl)amino)methyl)-6-hydroxymethyl-4-tertbutylphenol was detected by ESI mass spectrometry. The phenol oxygen atom in bpbp(VOCl₂)(VCl₂) is proposed to be non-bridging and this asymmetry is likely to be important for the C-N bond cleavage reaction. A related asymmetrical V^IV complex, [bpbpH(VO)(H₂O)](ClO₄)₂ ·  H₂O, containing bpbp⁻ bound to only one metal ion, has also been characterized by X-ray diffraction. In slightly more basic solution, bpbp(VOCl₂)(VCl₂) is oxidized to the V^IV-V^IV complex [bpbp(VOCl)₂]⁺ and C-N bond cleavage is suppressed.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Mutarotase in galactose-induced baker's yeast

Cell-free extracts of baker's yeast possess mutarotase activity only after induction of cells in the presence of galactose. The mutarotase activity appears 1 h after transfer to a galactose-containing medium and rises in synchrony with the utilization of galactose. Cycloheximide blocks the induction completely at a concentration of 100 μg/ml. InSaccharomyces fragilis the mutarotase is constitutive but its activity is strikingly increased after growth on galactose. The yeast mutarotase resembles in some respects analogous enzymes from other cells (pH dependence, substrate specificity, heat lability). Its affinity ford-galactose is substantially greater than ford-glucose. There may exist a coupling between mutarotase activity and the anomer-specific galactokinase.     

Oxynitrilases for asymmetric C-C bond formation

Oxynitrilases for the preparation of (R)- or (S)-cyanohydrins are now readily available. The research efforts of a number of groups have established these enzymes as catalysts with significant potential for application to asymmetric synthesis. Advances made in molecular cloning and genetics have delivered information on the oxynitrilase mechanism of action and sufficient quantities of enzyme to satisfy industrial requirements.