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Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp.
B V Thage1, F P Rattray, M W Laustsen
1Department of Dairy and Food Science, Dairy Technology, The Royal Veterinary and Agricultural University, Rolighedsvej, Frederiksberg C, Denmark. birgittevedel.thage@dk.chr-hansen.com
Aim:
Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115.
Methods And Results:
The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively.
Conclusions:
The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese.
Significance And Impact Of The Study:
The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.
