A bacterium with high poly-gamma-glutamate (PGA) productivity was isolated from the traditional Korean seasoning, Chung-Kook-Jang. This bacterium could be classified as a Bacillus subtilis, but sporulation in culture was infrequent in the absence of Mn2+. It was judged to be a variety of B. subtilis and designated B. subtilis (chungkookjang). L-Glutamate significantly induced PGA production, and highly elongated PGAs were synthesized. The volumetric yield reached 13.5 mg ml(-1) in the presence of 2% L-glutamate. The D-glutamate content was over 50% in every PGA produced under the conditions used. During PGA production, glutamate racemase activity was found in the cells, suggesting that the enzyme is involved in the D-glutamate supply. Molecular sizes of PGAs were changed by the salt concentration in the medium; PGAs with comparatively low molecular masses were produced in culture media containing high concentrations of NaCl. B. subtilis (chungkookjang) harbors no plasmid and is the first B. subtilis strain reported with both naturally high PGA productivity and high genetic competence.
A thermophilic, spore-forming rod isolated from hay compost in Korea was subjected to a taxonomic study. The micro-organism, designated strain SK-1(T), was identified as being aerobic, Gram-positive, motile and rod-shaped. Growth of the isolate was observed at 45-70 degrees C (optimum 60 degrees C) and pH 6.0-9.0 (optimum pH 7.5). The G+C content of the genomic DNA was 43.9 mol%. Chemotaxonomic characteristics of the isolate included the presence of mesodiaminopimelic acid in the cell wall and iso-C15:0 and iso-C17:0 as the major cellular fatty acids. The predominant isoprenoid quinone was MK-7. The chemotaxonomic characteristics of strain SK-1(T) were the same as those of the genus Geobacillus. Phylogenetic analysis based on 16S rDNA sequences showed that strain SK-1(T) is most closely related to Geobacillus thermoglucosidasius. However, the phenotypic properties of strain SK-1(T) were clearly different from those of G. thermoglucosidasius. The level of DNA-DNA relatedness between strain SK-1(T) and the type strain of G. thermoglucosidasius was 27%. On the basis of the phenotypic traits and molecular systematic data, strain SK-1(T) represents a novel species within the genus Geobacillus, for which the name Geobacillus toebii sp. nov. is proposed. The type strain is strain SK-1(T) (= KCTC 0306BP(T) - DSM 14590(T)).
D-Tagatose was continuously produced using thermostable L-arabinose isomerase immobilized in alginate with D-galactose solution in a packed-bed bioreactor. Bead size, L/D (length/diameter) of reactor, dilution rate, total loaded enzyme amount, and substrate concentration were found to be optimal at 0.8 mm, 520/7 mm, 0.375 h(-1), 5.65 units, and 300 g/L, respectively. Under these conditions, the bioreactor produced about 145 g/L tagatose with an average productivity of 54 g tagatose/L x h and an average conversion yield of 48% (w/w). Operational stability of the immobilized enzyme was demonstrated, with a tagatose production half-life of 24 days.
A new thermostable d-methionine amidase was found in a cell-free extract of Brevibacillus borstelensis BCS-1. After five steps of purification, the specific activity increased approximately 207-fold and the purity was more than 98%. The molecular weight of the enzyme was estimated to be 199 kDa by gel permeation chromatography and 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the thermostable d-methionine amidase was a homo-hexamer consisting of a single subunit. The purified enzyme was stable up to 65 • C within a broad pH range from 6.5 to 10.0, and its maximum activity was measured at pH 9.5 and 70 • C. The enzyme activity increased about five-fold with the addition of Co 2+ , yet was strongly inhibited by Hg 2+ , 2-mercaptoethanol, dithiothreitol, and ethylenediaminetetracetic acid.The thermostable d-methionine amidase exhibited a high amidase activity and d-stereospecificity toward d-amino acid amides and esters, yet did not hydrolyze d-peptides. The catalytic efficiencies (k cat /K m , mM −1 s −1 ) of the enzyme for d-methioninamide and d-alaninamide were 3086 and 21.5, respectively, and the enantiomeric excess (ee) and enantiomeric ratio of d-phenylalanine produced from dl-phenylalaninamide were 97.1 and 196%, respectively.
A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acidcontaining peptides, and NH 2 -terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co 2؉ and Mn 2؉ . The k cat /K m for D-alaninamide was measured as 544.4 ؎ 5.5 mM ؊1 min ؊1 at 50°C with 1 mM Co 2؉ .D-Amino acids occur in bacterial cell wall peptidoglycan (28), mammalian cells (11), higher plants (25), and active peptides (5,13,14,24) and are important materials for various pharmaceuticals, herbicides, and food additives (1). Unlike L-amino acids, almost all D-amino acids are obtained by using enzymatic methods; otherwise it is difficult to obtain a high state of optical purity and productivity (1,22). Peptides incorporating D-amino acids exhibit stronger antimicrobial properties than peptides with L-isomers because D-isomers appear to be more stable against proteolytic digestion than L-isomers (12). These facts have already been verified by various studies on the fate of D-amino acids in peptides and proteins (21,23).Enzymatic biotransformations in which optically pure D-amino acids are produced from DL-amino acid racemic mixtures by D-amino acid-specific enzymes have been determined to be most feasible for the production of D-amino acids with a high optical purity and yield (1). To apply this system, many microbial D-amino acid-specific enzymes have already been screened and subjected to direct enzyme methods (22).Although the synthesis of bioactive peptides incorporating D-amino acids instead of their L-counterparts could lead to metabolically stable and long-acting products, this has been hampered because of the need to use expensive processes that suffer from low stereoselectivity, low temperature stability, and the production of undesired by-products due to the use of an undesirable biocatalyst (1). Accordingly, thermolabile enzymes have been considered inappropriate for the harsh reaction conditions required in industrial processes. However, D-amino acid-specific enzymes have recently attracted much attention in regard to the synthesis of useful bioactive D-peptides and enantioselective synthesis of D-amino acids from DL-amino acid racemic mixtures (16,18,19,22). Among these enzymes, Daminoacylase (9, 29), D-aminopeptidase (2), and D-amino acid a...
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