Ultra-high-molecular-weight poly-gamma-glutamic acid (gamma-PGA) is a most promising biodegradable polymer that is produced by Bacillus subtilis (chungkookjang). Attractive properties of gamma-PGA are that it is water soluble, anionic, biodegradable, and edible. Development of gamma-PGA has pursued in terms of cosmetics/skin care, bone care, nanoparticle for drug delivery system, hydrogel, and so on. Very recently, our research has shown that gamma-PGA can be used as an immune-stimulating agent, especially at high molecular weight. This review presents the synthesis and production of high-molecular-weight gamma-PGA and its various applications in industrial fields.
D-Amino acid aminotransferases (D-AATs) fromGeobacillus toebii SK1 and Geobacillus sp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly active Bacilli enzymes, with respective turnover rates of 47 and 55 s ؊1 at 50°C. The Geobacillus enzymes were unique and shared limited sequence identities of below 45% with D-AATs from mesophilic and thermophilic Bacillus spp., except for a hypothetical protein with a 72% identity from the G. kaustophilus genome. Structural alignments showed that most key residues were conserved in the Geobacillus enzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence in Bacillus D-AATs was 144-EYcY-147 in the Geobacillus D-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward ␣-ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from the Geobacillus D-AATs had a / interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.
Bacillus velezensis strain KMU01 showing γ-glutamyltransferase activity as a probiotic candidate was isolated from kimchi. However, the genetic information on strain KMU01 was not clear. Therefore, the current investigation was undertaken to prove the probiotic traits of B. velezensis strain KMU01 through genomic analysis. Genomic analysis revealed that strain KMU01 did not encode enterotoxin genes and acquired antibiotic resistance genes. Strain KMU01 genome possessed survivability traits under extreme conditions such as in the presence of gastric acid, as well as several probiotic traits such as intestinal epithelium adhesion and the production of thiamine and essential amino acids. Potential genes for human health enhancement such as those for γ-glutamyltransferase, nattokinase, and bacteriocin production were also identified in the genome. As a starter candidate for food fermentation, the genome of KMU01 encoded for protease, amylase, and lipase genes. The complete genomic sequence of KMU01 will contribute to our understanding of the genetic basis of probiotic properties and allow for the assessment of the effectiveness of this strain as a starter or probiotic for use in the food industry.
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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