Mitsuaki Moriguchi

Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry

Abstract Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, l -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of glutaminase, the enzymatic properties, applications of glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of glutaminase. Finally, to facilitate the future applications of glutaminase in food fermentations, the mechanisms of action of salt-tolerant glutaminase are briefly discussed.

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.

Isolation of a Thermophilic Poly-L-Lactide Degrading Bacterium from Compost and Its Enzymatic Characterization

Thermophilic poly-L-lactide-degrading bacteria were isolated from a garbage fermentor. One of the isolates, strain PL21, was identified as Bacillus smithii based on its physiological properties, sugar assimilation pattern, and partial 16S rDNA sequence. The degradation activity of poly-L-lactide exibited by the culture fluid was parallel to the esterase activity, and the purified enzyme was active against various fatty acid esters and poly-L-lactide, at 60 degrees C and pH 5.

Production of d-amino acids by N-acyl-d-amino acid amidohydrolase and its structure and function

Abstract d -Amino acids have been widely used as synthetic materials for various compounds such as pharmaceuticals and agrochemicals. The manufacture of d -amino acids by fermentation is difficult, and enzymatic methods are mainly employed. At present, the optical resolution method using N-acyl- d -amino acid amidohydrolase is the most useful and convenient. In this review, the application of N-acyl- d -amino acid amidohydrolase to the production of d -amino acids and recent progress in the study of structure–function relationships from the standpoint of improving this enzyme for industrial application are discussed.

Crystalline lysine decarboxylase

Lysine decarboxylase (L-lysine carboxy-lyase, E.C. 4.1.1.18) was discovered and partially purified from Bacterium cadaveris and Escherichia coli by Gale and his coworker (Gale and Epps, 1944; Gale, 1945). Despite the wide-spread use of this enzyme for the manometric determination of L-lysine, it has not been enzymologically well-characterized. This communication describes the purification, crystallization and some of the properties of lysine decarboxylase from Bacterium cadaveris.

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.

Purification and properties of d-aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4

Abstract d -Aminoacylase has been purified 144-fold to electrophoretic homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl and affinity column chromatographies, and Sephadex G-100 gel filtration from the crude extracts of Alcaligenes denitrificans subsp. xylosoxydans MI-4. The enzyme was composed of a single polypeptide of about 51,000. The enzyme catalyzed hydrolysis of N -acyl-derivatives of neutral d -amino acids. Optimal pH and temperature were 7.8 and 50°C. The apparent K m and the V max for N- acetyl- d -phenylalanine were 14.1 mM and 1331 units/mg protein, respectively. The activity of the enzyme was inhibited by N- acetyl- d -valine ( K i =2.15 mM) and N- acetyl- d -alloisoleucine ( K i =1.47 mM), but not by its products ( i.e. , amino acids and acetate). The enzyme also had dipeptidase activity. Activation by metal ions was not observed.