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.

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.

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.

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.

High catalytic activity of alanine racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate.

We examined the effect of the pyridoxal 5'-phosphate (PLP) cofactor on the activity and stability of the psychrophilic alanine racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40 degrees C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40 degrees C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic alanine racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60 degrees C.

Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

Effect of ethyl alcohol on growth and intracellular alanine racemase of psychrotrophs.

The psychrotrophic alanine racemase from Pseudomonas fluorescens, a typical psychrotroph, is less resistant to organic solvents than the enzymes from thermophilic and mesophilic bacteria (Okubo et al., J. Home Econ. Jpn., 46: 1135-1140, 1995). To further elucidate this difference, we examined the effect of ethyl alcohol on the growth and intracellular alanine racemase activity of three typical psychrotrophs-P. fluorescens, Bacillus psychrosaccharolyticus and B. psychrophilus-in comparison with two mesophiles, Escherichia coli and B. subtilis. Although all the bacteria grew to the early stationary phase when cultivated at 22 degrees C for 36 h in the absence of ethyl alcohol, the growth of the psychrotrophs was more effectively suppressed by the addition of 3 and 5% ethyl alcohol to the medium than that of the mesophiles. The intracellular alanine racemase activity of the psychrotrophs was also more markedly reduced in the presence of ethyl alcohol than that of the mesophiles. When bacterial cells of each strain grown at 22 degrees C for 36 h in the absence of alcohol were suspended in 0-5 % ethyl alcohol solution and incubated at 30 degrees C for 1 h, both the survival ratio and intracellular alanine racemase activity of the psychrotrophs were lower than those of the mesophiles. Thus, ethyl alcohol effectively reduced both the growth of the psychrotrophs and their intracellular alanine racemase activity. Low concentrations of various other alcohols also repressed the growth of the psychrotrophs at 10 degrees C.

A new sterility test for cow's milk using alanine racemase gene as the index.

Oligonucleotide primers designed from consensus sequences of alanine racemase genes were used for a sterility test of cow's milk by polymerase chain reaction (PCR). Commercial cow's milk in two 250 ml packages was separately centrifuged at 5000 x g for 10 min, bacterial cells in each precipitate were cultivated at 30 and 55 degrees C for 5 h in Luria-Bertani medium, and the cells from each culture were mixed and used for the PCR after being treated with 0.1 N NaOH at 60 degrees C for 10 min. When we performed the PCR using DNAs from various bacteria and eukaryotes as the templates, a unique PCR product of about 390 bp was amplified only from the bacteria. The sensitivity of the PCR method was such that an initial inoculum of 1 CFU of Bacillus stearothermophilus, Escherichia coli, and Pseudomonas fluorescens per 250 ml of cow's milk could be detected. When we analyzed 14 types of commercial cow's milk, all samples which were positive by the standard sterility test at 30 or 55 degrees C were also found positive by the PCR method.

Difference between Escherichia coli O157:H7 and non-pathogenic E. coli: survival and growth in seasonings.

We examined the survival and growth of Escherichia coli O157:H7 cells incubated with several seasonings, in comparison with those of non-pathogenic E. coli. The cells were incubated at 25 degrees C for 24 h with several concentrations of NaCl, sucrose, soy sauce, worcester sauce and tomato ketchup, and their survival ratios were determined. The E. coli O157:H7 strains showed relatively higher survival ratios in 0.5-1.0 M sucrose, 25% soy sauce and 12.5-50% worcester sauce than the non-pathogenic strains, but slightly lower survival ratios in 0.5-2.0 M NaCl. A noteworthy difference between E. coli O157:H7 and the non-pathogenic strains was that incubation in the presence of 12.5% soy sauce allowed the growth of E. coli O157:H7 strains but reduced the viable cell numbers of non-pathogenic E. coli strains.

Trehalose 6-phosphate production with energy coupling fermentation by yeast cells.

We tried a method for the production of trehalose 6-phosphate (T6P) with energy-coupling fermentation by baker's yeast. T6P was produced in a reaction mixture containing glucose, 5'-UMP, MgSO4, inorganic phosphate, and dried cells of baker's yeast as the enzyme preparation, T6P was isolated from the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic methods. The reaction conditions suitable for T6P production were investigated. The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose, at various pHs and concentrations of substrates was examined. Accumulation of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM 5'-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100 mg/ml dried cells of baker's yeast shaken at 37 degrees C for 6 h. The yield of T6P as a percentage of glucose was 11% (mol/mol) under these reaction conditions.

Structural study of an exocellular polysaccharide of Bacillus circulans.

Bacillus circulans, a soil bacterium, produced an exocellular polysaccharide of high viscosity. On the basis of the results of methylation analysis, mild acid hydrolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a basic repeating unit composed of beta-L-rhamnopyranose, alpha-D-mannopyranose, alpha-D-galactopyranose, and alpha-D-glucopyranuronic acid with the following structure. [symbol: see text]

An alanine racemase gene as a new index for detecting Escherichia coli in foods.

A gene of alanine racemase, a typical prokaryotic enzyme, was evaluated as a new index for detecting Escherichia coli in foods. An alanine racemase gene fragment containing a non-conserved sequence of the gene was amplified from genomic DNA of E. coli by a polymerase chain reaction, and then labeled with digoxigenin as a probe for detecting E. coli. Food samples and bacteria were each treated as at 25 degrees C for 10 min in 0.1N NaOH containing 0.5% SDS, before being directly spotted on to nylon membranes for DNA hybridization. The probe was specific for E. coli; all 48 strains of E. coli examined, including such pathogenic strains as E. coli O157:H7, showed positive signals, whereas all 59 strains of non-E. coli species, except for one strain (Shigella sonnei), did not show a signal. Various foods inoculated with E. coli K-12 showed positive signals whereas no uninoculated foods showed any signal. Quantification of E. coli cells in the death phase by the hybridization method showed good correlation with that by the plate culture method. The alanine racemase gene could prove useful as an index for detecting E. coli in foods.

Lipid composition of commercial bakers' yeasts having different freeze-tolerance in frozen dough.

The lipid composition of some commercial bakers' yeasts having different freeze-sensitivity in frozen dough was investigated to clarify the correlation between their lipid composition and freeze-tolerance. The total lipid content including neutral lipid, free fatty acid, sterol, and phospholipid ranged between 23.0 to 32.2 mg/100 mg protein of the yeasts tested. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine were the main phospholipids found in all yeast strains, but no distinct difference in these components between freeze-tolerant and freeze-sensitive strains was observed. Palmitoleic (C16:1), oleic (C18:1), palmitic (16:0), and stearic (C18:0) acids were the major fatty acids present in total lipid and phospholipid, and unsaturation indices of fatty acid in these lipid components were almost equal by the strains. The molar ratios of sterol to phospholipid of freeze-sensitive strains were higher than those of freeze-tolerant strains. The difference in the sterol-phospholipid ratio that influences the fluidity of plasma membranes in yeast cells was supposed to reflect the difference in freeze-sensitivity of bakers' yeast.

Trehalase activity and trehalose content in a freeze-tolerant yeast, Torulaspora delbrueckii, and its freeze-sensitive mutant.

Two types of trehalase activities different in the optimum reaction pH (4.3 and 6.7) were found in a freeze-tolerant yeast, Torulaspora delbrueckii D2-4, and three types of the activities at pH 4.3, 5.7, and 6.7 were found in its freeze-sensitive mutant (60B3). The pH 4.3 activities in the cell extracts of the two strains were on the same level, but the pH 6.7 activity in the strain D2-4 was about half of that in the strain 60B3. The enzyme with the pH 4.3 activity and the other two enzymes were analogous to acid and neutral trehalases, respectively, in the sensitivity to metal ions and in response to phosphorylation and dephosphorylation. The trehalose content in the strain D2-4 was three times higher than that in the strain 60B3. The change of trehalose content during growth of the strain 60B3 correlated only to the change of the pH 5.7 activity.

Induction of Freeze-sensitive Mutants from a Freeze-tolerant Yeast, Torulaspora delbrueckii.

Freeze-sensitive strains of yeast were induced from a freeze-tolerant yeast Torulaspora delbrueckii by incubation with ethyl-methane sulfonate as a mutagen. A maximum ratio of mutation was attained by the incubation at 30°C for 75min. One-hundred and fifty strains of freeze-sensitive yeast were selected by plating-culture for the first screening. The freeze-tolerance ratio of each strain was examined based on the fermentative activity before and after freezing in liquid medium and dough. Strain 60B3 showed the highest freeze-sensitivity in a pre-fermented frozen dough (pre-fermented at 30°C for 2h, and frozen at -20°C for 7 days) among eight strains finally selected.

A new amino acid racemase with threonine alpha-epimerase activity from Pseudomonas putida: purification and characterization.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L- to D-allo-threonine and also from D- to L-allo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the alpha position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.

Thermolabile alanine racemase from a psychotroph, Pseudomonas fluorescens: purification and properties.

A psychotrophic bacterium that produces a thermolabile alanine racemase was isolated from raw milk, and identified as Pseudomonas fluorescens TM5-2. The enzyme was purified to homogeneity from the cell extract, and characterized to be compared with enzymes from mesophiles (Bacillus subtilis and Salmonella typhimurium) and a thermophile (Bacillus stearothermophilus). The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (38,000). The enzyme contains two mol of pyridoxal 5'-phosphate per mol as a coenzyme. The amino acid composition was different from those of other alanine racemases in content of valine. The amino acid sequence of the amino terminal region (from 1Met to 25Gly) had 21-33% homology with those of other alanine racemases. Kinetic parameters of the enzyme were similar to those of other alanine racemases. The enzyme is extremely labile over 30 degrees C, and shows the high catalytic activity even at 0 degrees C; it is thermolabile and psychotrophic.

Urokinase quality: analysis of different preparations.

Five commercially available German Urokinase (UK) preparations were examined for purity, fibrinolytic activity, and molecular weight composition. With the exception of one preparation (B) all samples showed comparable fibrinolytic activity (91-107% of the activity declared). Preparations A, D, and E contained almost exclusively high-molecular-weight UK (HMW-UK), i.e., 82-86%. However, preparation C (from kidney tissue cultures) contained 95% of low-molecular-weight-UK (LMW-UK), and sample B consisted of nearly equal amounts of both HMW-UK and LMW-UK (47:53). Purity criteria (coagulative and lysozyme activity, endotoxin content, protease activity, HBs antigen) were fulfilled by preparation E, and, with certain restrictions, also by preparations A and C.