Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae.

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

Rapid MALDI biotyper-based identification and cluster analysis of Streptococcus iniae.

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either "no reliable identification" or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.