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Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides L: -glutamine, which was hydrolyzed with the highest specific activity (100%), L: -asparagine (74%), D: -glutamine (75%), and D: -asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60 degrees C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0-10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70 degrees C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

Expression of the ansZ gene encoding a putative L-asparaginase II (BsAII) from Bacillus subtilis in Escherichia coli was examined. No expression was detected in E. coli transformed with a plasmid containing the full-length ansZ gene. Three N-terminal truncated enzymes (BsAIIT18M, BsAIIS40M, and BsAIID49M) were prepared based on comparison with the N-terminal sequences of other type II L-asparaginases. BsAIIT18M became easily inactivated during DEAE-Toyopearl column chromatography. The purified N-terminal-truncated enzymes BsAIIS40M and BsAIID49M had tetrameric subunit structures and V max values of 45.5 and 45.8 U/mg towards L-asparagine, respectively. Their K m values were 2.06 and 7.02 mM, respectively. The enzymes differed from asparaginase II from E. coli and Erwinia carotovora in substrate specificity and affinity for L-asparagine. BsAIIS40M and BsAIID49M retained over 80% of their original activities in the presence of 15% NaCl, and thus may find application in the food industry for products in which NaCl is used. This study also revealed that BsAII is rather different from the type I enzyme (BsAI) from B. subtilis in substrate specificity and salt-tolerance.

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