Mamoru Wakayama

Isolation and characterization of salt-tolerant glutaminases from marine Micrococcus luteus K-3

Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The Km values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases.

Nitrite oxidation by heterotrophic bacteria under various nutritional and aerobic conditions

Abstract The nitrite transforming activities of heterotrophic bacteria from culture collections and isolates from activated sludge were studied under various nutritional and aerobic conditions. Among the 48 organisms tested, 17 strains, many of which are reported as denitrification negatives, consumed 1–5 mM of nitrite and accumulated corresponding amounts of nitrate. Twelve strains, many of which are denitrification-positive, consumed nitrite with the accumulation of less nitrate, while more than 1 mM nitrite was consumed but little nitrate was accumulated by 14 strains, many of which are Enterobacteriaceae or lactic acid bacteria. None of the organisms formed significant nitrate in the medium without nitrite, though a considerable amount of ammonia was also accumulated by most strains. Although the growth and nitrate accumulation of Bacillus badius I-73 was affected by the concentrations of sodium nitrite and peptone and by the culture volume, the amount of nitrate accumulated was always proportional to that of the nitrite consumed. Intact cells of B. badius I-73 produced almost the same amount of nitrate as the decrease in nitrite. On the other hand, in B. subtilis I-41, a denitrification-positive isolate, the ratio of the amount of nitrate accumulated to that of nitrite consumed varied from 0–100% depending on the culture conditions.

Cloning, expression, and nucleotide sequence of the N-acyl-D-aspartate amidohydrolase gene from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Abstract The gene (termed daa) encoding N-acyl- d -aspartate ( d -Asp) amidohydrolase ( d -AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. coli) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of d -AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified d -AAase from both Alcaligenes A-6 and cloned E. coli, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of d -AAase with that of d -aminoacylase from Alcaligenes A-6 showed high overall homology (56%). d -AAase from Alcaligenes A-6 showed 25∼29% homology with Bacillus stearothermophilus, porcine, and human l -aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield.

Role of Conserved Histidine Residues in D -Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a k cat/K m 6.3×104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant K m, but greatly decreased k cat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The k cat/K m of H250N mutant was reduced by a factor of 2.5×104-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the K m and k cat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.

Change in Nitrite Conversion Direction from Oxidation to Reduction in Heterotrophic Bacteria Depending on the Aeration Conditions

Abstract For investigation of the effects of aeration on nitrite- and nitrate-transforming activities of various heterotrophic bacteria, a series of coefficients of the oxygen absorption rate ( K d , 8–99 × 10 −7 mol/ml·min·atm) in 500-ml shaking flasks were determined by varying plug types and culture volumes. Bacillus badius I-73, which neither shows denitrification activity nor utilize nitrate as a nitrogen source, consumed nitrite and accumulated nitrate at all K d values at which experiments were conducted. In B. subtilis I-41, which dose show denitrification activity, the manner of nitrite and nitrate conversion was influenced by the culture time and K d , and the direction of conversion was changed from reduction to oxidation, as the K d of the culture increased. Pseudomonas pavonaceae , another denitrification-positive strain, metabolized both nitrite and nitrate to more reduced compounds at low K d , and the direction of conversion changed from reduction to oxidation at K d =20 × 10 −7 mol/ml·min·atm. Such switching behavior was also observed when P. pavonaceae was cultured continuously during variation of the aeration conditions with supply of pure oxygen. Many other denitrification-positive strains behaved similarly to P. pavonaceae , and showed their own critical K d , the point at which the direction of nitrite metabolism changed. The results of intact-cell reaction experiments indicate that this switching might be caused by inhibition and repression of nitrite-reducing activity, and by stimulation of nitrite-oxidizing activity by oxygen.

Comparative biochemistry of bacterial N-acyl-d-amino acid amidohydrolase

Abstract N-acyl- d -amino acid amidohydrolases can be classified into three types based on substrate specificity. d -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- d -aspartate amidohydrolase ( d -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- d -glutamate amidohydrolase ( d -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- d -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with l -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- d -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].

Isolation, enzyme production and characterization of d-aspartate oxidase from Fusarium sacchari var. elongatum Y-105

Abstract A microorganism that produces d -aspartate-oxidizing enzyme by induction was isolated from soil, and identified as Fusarium sacchari var. elongatum Y-105. The enzyme catalyzed the oxidative deamination of d -aspartate ( d -Asp) and produced oxaloacetate, ammonia, and hydrogen peroxide, stoichiometrically. The enzyme is designated “ d -Asp oxidase” (EC 1.4.3.1). In addition to d -Asp, the enzyme oxidized d -glutamate ( d -Glu) and N- methyl - d - aspartate (NMDA). N- Acetyl - d - Asp and other d - or l -amino acids, however, were inert as substrates. The optimum pH and temperature were 7.5 and 40°C, respectively. The enzyme was stable at pH 9.0 and temperature of 50°C, respectively. The enzyme activity was not inhibited by sodium benzoate which is a specific inhibitor of d -amino acid oxidase from mammals. The enzyme activity was also not affected by carboxylates such as meso- or d -tartarate, citrate, and fumarate which inhibit d -Asp oxidase from rabbits.

Molecular cloning and determination of the nucleotide sequence of a gene encoding salt-tolerant glutaminase from Micrococcus luteus K-3

Abstract The gene encoding the salt-tolerant glutaminase I from marine Micrococcus luteus K-3 ( M. luteus K-3) was cloned in Escherichia coli ( E. coli ) JM109. Clones were screened by hybridization with degenerate oligonucleotide probes designed using the known N-terminal amino acid sequence of glutaminase I from M. luteus K-3. A 2.4-kb Hin cII fragment from a 8-kb primary cloned DNA fragment was subcloned and sequenced. This fragment had an open reading frame of 1,368 nucleotides encoding 456 amino acids. The molecular weight of the deduced amino acid sequence of glutaminase I was determined to be 48,247. Comparison of the amino acid sequence of glutaminase I with those of kidney, brain, liver, and Caenorhabditis elegans glutaminases revealed high degrees of homology in several local regions.

Purification and characterization of l-aminoacylase from Pseudomonas maltophila B1

Abstract The constitutive l -aminoacylase, which is used for optical resolution of dl -α-aminosuberic acid ( dl -Asu), has been purified and characterized from Pseudomonas maltophila B1. The crude enzyme showed a specific activity of 0.062 units/mg for N -acetyl(Ac)- l -Asu. This value is very high compared with those from Aspergillus melleus , porcine kidney, and Bacillus stearothermophilus . Molecular masses of 108 kDa for the native enzyme and 50 kDa for the subunit were determined, indicating a dimer. The enzyme activity was optimal at pH 8.0 and at 55°C. The enzyme hydrolyzed N -acyl derivatives of various neutral l -amino acids and acidic l -amino acids, l -glutamate and l -Asu. The enzyme also had dipeptidase activity. The K m values for N -Ac- l -alanine and N -Ac- dl -Asu were determined at 2.32 and 12.7 mM, respectively. The apoenzyme was activated using Zn 2+ , Ca 2+ , and Co 2+ . Glyoxylate, dl -lactate, phenylboronic acid (PBA), butaneboronic acid (BBA), diethylpyrocar-bonate (DEP), and phenylglyoxal (PGO) inhibited enzyme activity.

Production of d-amino acid oxidase from Aspergillus sojae

d-Amino acid oxidase activities for d-glutamate (d-Glu), d-aspartate (d-Asp) and d-alanine (d-Ala) were found in cell-free extract of Aspergillus sojae (A. sojae). The enzyme activities for these three substrates increased over 30-fold by the addition of 0.25% d-Ala to the culture medium. Glycerol was an effective carbon source for increasing the enzyme activities. d-Ala, d-serine (d-Ser), and d-tryptophan (d-Trp) were better inducers than other d-amino acids. d-Glu and d-Asp were oxidized at rates of 70 and 6%, respectively, relative to the rate of oxidation of d-Ala which was taken as 100%. A. sojae d-amino acid oxidase showed no inhibition by sodium benzoate or dicarboxylates and had a molecular weight of 129,000, which differed substantially from those of d-amino acid oxidases of porcine and rabbit kidney.