Biochemical Reduction of 3-Oxoalkanoic Esters by a Bottom-fermentation Yeast,Saccharomyces cerevisiae IFO 0565

The scope and limitation of a bottom-fermentation yeast (Saccharomyces cerevisiae IFO 0565) toward the reduction of 3-oxoalkanoic esters were examined. The substrate specificity of this microorganism for various kinds of 3-oxoalkanoic esters was studied. This microorganism was distinct from converntional bakers’ yeast in terms of its selectivity in the reduction and its high expression of a hydrolytic enzyme. 3-Oxoalkanoic ester with an aromatic substituent, a halogen substituted 3-oxoalkanoic ester, an aliphatic longer-chain 3-oxoalkanoic ester and its α,α-difluoro analog were also accepted by this microorganism. The products are useful intermediates in the synthesis of physiologically active compounds.     

Differences in Protein Structure and Similarities in Catalytic Function of Two l-Stereoselective Carbonyl Reductases from Bakers’ Yeast

We purified and studied two l-stereoselective carbonyl reductases from bakers’ yeast (Saccharomyces cerevisiae). One catalyzed exclusively the enantioselective reduction of carbonyl compounds such as β-keto esters and the other acted on α-acetoxy ketones and β-keto esters. The enzymes had identical molecular weights and catalyzed the l-stereoselective reduction of various carbonyl compounds with similar substrate specificity, but they were different proteins coded by different genes.     

Studies on Transfer of Antiseptics to Microbes and their Toxic Effect

The transfer of alkyl esters of sorbic, benzoic and salicylic acid from the medium to bakers’ yeast cell was investigated, and both the quantity of the ester dissolved in the lipid phase of the cell and the quantity adsorbed on the solid phase were determined.

The dissolved quantity of these esters was very great in comparison with the adsorbed quantity. At the ester concentration which gives a definite inhibiting effect on the yeast growth, the adsorbed quantity remained constant, being independent of the kind of ester, while the dissolved quantity greatly varied according to the kind of ester. From this fact it was concluded that the toxic effect of these esters, as well as esters of p-hydroxybenzoic acid (cf. Part III), is exclusively limited by the adsorbed quantity.     

Evaluation of the Suppressive Effect on Bitter Taste of Gluconate

Improved conditions for the production and characterization of 1-arylpropane-1, 2-diols and related compounds were developed. Experimental conditions providing highly enhanced activity of pyruvate decarboxylase in bakers’ yeast in the presence of pyruvate, thiamine pyrophosphate, and magnesium(II) salt were applied to the preparation of (R)-1-hydroxy-1-phenyl-2-propanone from benzaldehyde. Subsequent reduction with bakers’ yeast efficiently afforded 1-phenyl-1, 2-propanediol (35%). The composition of its stereoisomers was precisely determined, and the major (1R, 2S)-isomer (89% of the total mixture) could be isolated by recrystallizing the corresponding benzoate. The analytical method for identifying the stereoisomeric composition was also effective for the determination of 5-phenyl-4-pentene-2, 3-diol, the biotransformation product from cinnamaldehyde, the vinylogous substrate of benzaldehyde. Furthermore, the structural characterization of 1-(2-furyl)propane-1, 2-diol, which was obtained from furfural (28%) by the action of brewers’ yeast Saccharomyces cerevisiae (carlsbergensis), is described. The major (1S, 2S)-isomer could be isolated by recrystallizing the crude product.     

Lysis of Yeast Cell Wall by Enzymes from Streptomycetes

This paper deals with yeast cell-wall lytic enzymes formed by Streptomyces with regard to the connection with the cell-wall structure.

In the first place, 29 organisms of β-glucanase-producing Streptomycetes were selected among 777 strains belonging to genus Streptomyces by means of a cylinder-plate method employing the yeast glucan as a substrate. As for these organisms, the depolymerizing activity against the yeast glucan was considered to be mainly due to β-1,3-glucanase activity. Against the heat-treated cell of bakers’ yeast, the crude enzymes merely showed poor lytic activities, however, in the combined employment with some protease preparations, especially with an alkaline protease from St. satsumaensis nov. sp., a remarkable increase of the lytic activities was demonstrated. On the other hand, the intact cell wall of bakers’ yeast, or both the heat-treated and the intact cells of Sacch. cerevisiae 18.29 strain were dissolved very easily by a sole action of β-glucanase or of protease, respectively. In consequence, it seemed that the lysis occurred with different mechanisms in response to differences of substrates. On this subject, the results of investigations and discussions were described in special measure. In addition, the possibility, that some other enzymes than β-glucanase or protease might concern to the lysis of the cell wall, was also investigated and discussed.     

Purification and characterization of a novel keto ester reductase from the green alga, <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>: comparison of enzymological properties with other microbial keto ester reductases

A novel keto ester reductase (Chlorella sorokiniana keto ester reductase, CSKER) from Chlorella sorokiniana SAG 211-8k cells was purified. The CSKER had a monomeric structure based on gel filtration chromatography (37 kDa) and SDS–polyacrylamide gel electrophoresis (34 kDa). The purified CSKER showed a high reducing activity with β-keto esters, in particular, ethyl 4-chloro-3-oxobutanoate and ethyl 2-chloro-3-oxobutanoate. However, the purified enzyme did not show any reducing activity with α-keto esters and 2-chlorobenzoylformamide (aromatic α-keto amide). The CSKER catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate, ethyl 3-oxobutanoate, and methyl 3-oxobutanoate to the corresponding (R)-, (S)-, and (S)-hydroxy ester, respectively, with high enantioselectivity (>99% e.e.), respectively. Furthermore, the reduction of ethyl 2-methyl-3-oxobutanoate by CSKER exclusively yielded the corresponding syn-(2R, 3S)-hydroxy ester. The purified CSKER was inactive with NADH, used instead of NADPH. None of the keto ester-reducing enzymes already isolated from other microorganisms was identical to the CSKER. These results suggested that CSKER is a novel keto ester reductase that has not yet been reported.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

It was confirmed that washed yeast cells produced isoamyl alcohol and isobutanol from either pyruvic acid or α-ketoisovaleric acid. At the same time α-ketoisocaproic acid, a presumed intermediate to isoamyl alcohol, was found.

These results seem to support the presumptive scheme that pyruvic acid converts to α-ketoisocaproic acid via acetolactic acid and α-keto,isovaleric acid, from which isoamyl alcohol and isobutanol are formed.     

Permeability and Selective Toxicity of Nitrofurane Compounds

The bacterial growth is inhibited by nitrofurane compounds, although the yeast growth is hardly affected. In relation to the selective toxicity of nitrofuranes for bacteria, the interaction between microbes (Escherichia coli, Staphylococcus aureus and bakers– yeast) and nitrofurane compounds (5-nitro-2-furfural semicarbazone and 5-nitro-2-furylacryl amide) was examined.

Apparently, in the bacterial suspension containing energy substrate, nitrofuranes are continuously reduced to corresponding aminofuranes, respectively. The velocity of the bacterial reduction at the growth inhibiting condition was evaluated as great as above 30 per cent of the limit of supplying velocity of coenzymes in the cell, the reduction velocity of such value is enough to inhibit the bacterial growth, because the electron transfer in the cell metabolism is disordered.

On the other hand, in the yeast suspension, the reduction velocity was negligibly small. The difference of the reduction ability between bacteria and yeast was seemingly owing to the fact that the permeability of the nitrofuranes differs by the kind of microbe so that it was concluded that the antimicrobial effect of nitrofuranes is limited by the permeability for the microbe cell.     

Pyridoxamine-alpha-keto acid transamination activity of aspartate apoaminotransferases

Pyridoxamine-α-keto acid transamination activities of homogeneous aspartate apoaminotransferases from various organisms were determined. Aspartate apoaminotransferases from pig heart cytosol and bakers' yeast utilized both oxalacetate and α-keto-glutarate as amino acceptors, while those from pig heart mitochondria and bacteria (Escherichiacoli B and Pseudomonas striata) showed reactivity only toward oxalacetate. Specific activities of bacterial aspartate apoaminotransferases were very high compared to those of the yeast and animal apoenzymes. Phosphate and various anions, including sulfate, raised the pyridoxamine-α-keto acid transamination activity of all the aspartate apoaminotransferases examined. However, a high concentration of phosphate inhibited the reaction.     

Reduction by a model of NAD(P)H: XI. Stereochemistry of a lactate dehydrogenase-model reaction

Stereochemistry of the biomimetic reduction of α-keto esters with NAD(P)H-model compounds has been investigated. The model compound with the R-configuration reduces the α-keto esters to the (R)-α-hydroxy esters, whereas (S)-α-hydroxy esters are afforded by the reduction with the S-configurational model compounds. It has been concluded that pro-R and -S hydrogens of the model compounds with R- and S-configuration, respectively, contribute predominantly to the reduction.     

Stereoselective Reduction of Carbonyl Compounds with Actinomycete: Purification and Characterization of Three α-Keto Ester Reductases from Streptomyces avermitilis

We achieved the purification of three α-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for α-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the α- and β-keto esters, but also toward α-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.     

A-nor steroids. 1. The stereochemistry of A 2-carbomethoxy-A-nor-cholestan-3-one obtained via the Dieckmann condensation

The Dieckmann condensation of dimethyl 3, 4-$\text{seco}$-5α-cholestan-3, 4-dioate ($\text{1}$), using sodium methoxide in benzene under reflux for two hours, is shown to give 2α-carbomethoxy-A-nor-5α-cholestan-3-one ($\text{2}$). Confirmation of the stereochemistry of the β-keto ester $\text{2}$ was obtained through its sodium borohydride reduction product, 2α-carbomethoxy-A-nor-5α-cholestan-3β-ol ($\text{4}$).     

Mechanism of Antimicrobial Effect of Quinone Compounds

It has been observed that, in the culture medium, the toxicity of p-benzoquinone on bakers’ yeast decreases with time, and the decreasing process was examined from the view point of chemical reaction of quinone with the component of the medium.

As a result, it was shown that quinone concentration decreases by its 1,4-addition reaction with amino radicals of the component of the medium, and it was concluded that the inhibiting effect of quinone on the yeast growth is determined by the velocity of death of the yeast by the toxicity of quinone and that of inactivation of the toxicity by the addition reaction.     

Synthesis of novel 13α-18-nor-16-carboxamido steroids via a palladium-catalyzed aminocarbonylation reaction

13α-18-nor-16-Carboxamido steroids were synthesized via a palladium-catalyzed aminocarbonylation reaction of the corresponding iodoalkenes. The starting material was an unnatural 13α-16-keto steroid, obtained by a Wagner–Meerwein rearrangement of a 16α,17α-epoxide in the presence of [BMIM][BF₄]. The 13α-16-keto steroid was converted to a mixture of 16-iodo-16-ene and 16-iodo-15-ene derivatives in two steps by Barton’s methodology. Aminocarbonylation of the steroidal alkenyl iodides was carried out using different primary and secondary amines as nucleophiles. The products, 16-carboxamido-16-ene and 16-carboxamido-15-ene derivatives, were obtained in good yields and were characterized by ¹H and ¹³C NMR, IR and MS.The reduction of the above two unsaturated carboxamides resulted in the same product, 17α-methyl-16α-carboxamido-androstane.     

Amine Transaminase

An enzyme “amine transaminase”, which catalyzed transamination between amines and α-keto acids, was found to occur in certain fermentative bacteria, such as Escherichia coli and Aerobacter aerogenes. Using a partially purified enzyme preparation obtained from cell extract of E. coli, some properties of the enzyme were investigated. α-Ketoglutaric acid appeared to be the most efficient amino acceptor and substitution of α-ketoglutaric acid by other α-keto acid resulted in much lower activity. Putrescine, cadaverine and hexamethylenediamine were found to be active as amino donors, but the other monoamines, diamines and polyamines were inert. Treatment of the enzyme with acid ammonium sulfate resolved the enzyme into apo- and coenzyme. The apoenzyme was well reactivated by pyridoxal phosphate as well as pyridoxamine phosphate. Physiological role of the amine transaminase was suggested in relation to the metabolism of amines in bacterial cells.     

Stereoselective Reduction of α- and β-Keto Esters with Aerobic Thermophiles,Bacillus Strains

The first example of stereoselective reduction with aerobic thermophiles is reported. Various α- and β-keto esters were reduced stereoselectively to the corresponding alcohols by the aerobic thermophiles, Bacillus strains. In particular, the reduction of ethyl 3-methyl-2-oxobutanoate with B. stearothermophilus DSM 297 gave the corresponding (R)-alcohol with high yield in excellent enan-tioselectively (> 99% e.e.). The conversions of keto esters to the corresponding hydroxy esters with Bacillus strains were increased by introduction of glycerol in the reaction mixture as an additive.     

Engineering of NADPH-dependent aldo-keto reductase from <Emphasis Type="Italic">Penicillium citrinum</Emphasis> by directed evolution to improve thermostability and enantioselectivity

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Efficient production of phenylpropionic acids by an amino-group-transformation biocatalytic cascade

Phenylpropionic acids are commonly used in the synthesis of pharmaceuticals, cosmetics, and fine chemicals. However, the synthesis of phenylpropionic acids faces the challenges of high cost of substrates and a limited range of products. Here, we present an artificially designed amino-group-transformation biocatalytic process, which uses simple phenols, pyruvate, and ammonia to synthesize diverse phenylpropionic acids. This biocatalytic cascade comprises an amino-group-introduction module and three amino-group-transformation modules, and operates in a modular assembly manner. Escherichia coli catalysts coexpressing enzymes from different modules achieve whole-cell simultaneous one-pot transformations of phenols into the corresponding phenylpropionic acids including (S)-α-amino acids, α-keto acids, (R)-α-amino acids, and (R)-β-amino acids. With cofactor recycling, protein engineering, and transformation optimization, four (S)-α-amino acids, four α-keto acids, four (R)-α-amino acids, and four (R)-β-amino acids are synthesized with good conversion (68–99%) and high enantioselectivities (>98%). Therefore, the amino-group-transformation concept provides a universal and efficient tool for synthesizing diverse products.     

Preparation of Diphosphopyridine Nucleotide from Bakers’ Yeast

A method for the preparatoin of diphosphopyridine nucleotide (DPN) from bakers’ yeast is described. This method consists of partial purification of crude extract of yeast by charcoal chromatography according to Pontis and coworkers, ion-exchange chromatography on Dowex-l acetate, and precipitation of DPN as the free acid with ethanol. 0.71~.1.1 g of DPN with a purity of 85~90% was obtained from 5 kg of fresh bakers’ yeast by this method.