One-pot chemo-enzymatic enantiomerization of racemates

A new one-pot chemo-enzymatic procedure was developed for enantiomerization of racemates based on enzymatic enantiospecific oxidation of a substrate and chemical non-enantiospecific reduction of the product. The principle is shown as follows for the -proline production.

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-Proline and -pipecolate were produced from racemic proline and pipecolate by means of amino acid oxidase and sodium borohydride in high yield in this reaction system [J.W. Huh, K. Yokoigawa, N. Esaki, K. Soda, Biosci., Biotechnol., Biochem. 56 (1992) 2081]. - and -Lactate were -enantiomerized in a one-pot reaction system containing -lactate oxidase and sodium borohydride in the similar manner [S. Mukoyama, K. Yamanaka, T. Oikawa, K. Soda, Nippon Nogei Kagaku Kaishi 73 (1999) 62]. Pyruvate was also converted to an equimolar amount of lactate in the same system. α-Hydroxybutyrate can be produced from the - and -isomers, and α-ketobutyrate in the same manner though slowly. This method is applicable to production of other chiral compounds from the corresponding racemates.     

Leavening ability of baker's yeast exposed to hyperosmotic media

To develop a simple and rapid method for enhancing the leavening ability of baker's yeast, we examined the fermentation ability of baker's yeast exposed to hyperosmotic media. When baker's yeast cells were incubated at 25 degrees C for 1 h in a hyperosmotic medium containing 0.5% yeast extract, 0.5% peptone and 20% sucrose, the cells showed a higher fermentation ability in the subsequent fermentation test than those untreated. The increased ratios were from 40 to 60% depending on the strains used. Glucose and fructose showed a similar effect to that of sucrose, but sorbitol was less effective. A high correlation between the intracellular glycerol content and fermentation ability after the osmotic treatment suggested that glycerol accumulated during the hyperosmotic treatment was used in the subsequent fermentation as a substrate, lessened the lag time, and consequently enhanced the fermentation ability. Various baker's yeasts also showed a high leavening ability in dough after the hyperosmotic treatment.     

Properties of an extracellular polysaccharide produced by a strain of enterobacter isolated from pond water

A bacterium which was isolated from pond water and identified as Enterobacter cloacae produced a viscous extracellular polysaccharide when it was grown aerobically in a medium containing sucrose as a sole source of carbon. The maximum molecular weight of the polysaccharide was about 9.0 x 10(5). The polysaccharide was composed of fucose, galactose, glucose, and glucuronic acid in a molar ratio of 2:3:2:1, but the molecular weight and the molar ratio of the sugar component were different from those of the polysaccharide produced by the same species reported elsewhere.     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.     

Structure and function of psychrophilic alanine racemase

We describe the structure and function of psychrophilic alanine racemases from Bacillus psychrosaccharolyticus and Pseudomonas fluorescens. These enzymes showed high catalytic activities even at 0°C and were extremely labile at temperatures over 35°C. The enzymes were also found to be less resistant to organic solvents than alanine racemases from thermophilic and mesophilic bacteria, both in vivo and in vitro. Both enzymes have a dimeric structure and contain 2 mol of pyridoxal 5′-phosphate (PLP) per mol as a coenzyme. The enzyme from B. psychrosaccharolyticus was found to have a markedly large K_m value (5.0 μM) for PLP in comparison with other reported alanine racemases, and was stable at temperatures up to 50°C in the presence of excess amounts of PLP. The dissociation of PLP from the P. fluorescens enzyme may trigger the unfolding of the secondary structure. The enzyme from B. psychrosaccharolyticus has a distinguishing hydrophilic region around residue no. 150 in its deduced amino acid sequence, whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of this region in the three dimensional structure of this enzyme was predicted to be in a surface loop surrounding the active site. This hydrophilic region may interact with solvent, reduce the compactness of the active site, and destabilize the enzyme.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Improving the freeze tolerance of bakers' yeast by loading with trehalose

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.     

Catalase is the bacteria-derived detoxifying substance against paramecia-killing toxin in wheat grass powder infusion

Paramecium cells are usually cultured in a wheat grass powder infusion inoculated with Klebsiella pneumoniae. However, non-bacterized wheat grass powder infusion is toxic to paramecia, and bacteria-derived substance detoxifies the toxic substance. Here, the detoxifying substance from K. pneumoniae, which was found to be proteinaceous, was purified to homogeneity. The protein had an apparent molecular mass of about 200 kDa by gel filtration and 92 kDa by SDS-polyacrylamide gel electrophoresis. Although the amino acid sequence of the amino terminal region did not show a high sequence homology with any reported proteins, amino acid sequences of internal regions of the protein were nearly identical to catalase HPII from Escherichia coli. When the wheat grass powder infusion was treated at 25 degrees C for 1 h with commercially available catalase from bovine liver, the toxicity of the infusion against paramecia was completely abolished. The initial concentration of hydrogen peroxide in the wheat grass powder infusion was about 30 microM and was completely decomposed by the catalase treatment. Therefore, the toxic substance in the wheat grass powder infusion and the detoxifying substance from K. pneumoniae are considered as hydrogen peroxide and catalase, respectively.     

Subunit interaction of monomeric alanine racemases from four Shigella species in catalytic reaction

Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer). To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor. The enzymes were eluted on gel filtration as a monomer of about 39,000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine. An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer. The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60,000 and 76,000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively. D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium. The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes. The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands. These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction. Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.