Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation

• Published in: Applied microbiology and biotechnology Vol. 100; no. 7; pp. 3113 - 3124
• Main Authors: Callejón, S, Sendra, R, Ferrer, S, Pardo, I
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.04.2016; Springer; Springer Nature B.V
• Subjects: Amines; Amino Acid Sequence; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Benzothiazoles - chemistry; Benzothiazoles - metabolism; Beverages; Biocatalysis; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalytic Domain; Cell division; Cloning; Cloning, Molecular; Copper; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; fermented foods; Fungi; Gene Expression; gene overexpression; genes; Genetic aspects; Genetic engineering; Gram-positive bacteria; Histamine; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; laccase; Laccase - chemistry; Laccase - genetics; Laccase - metabolism; lactic acid bacteria; Lactobacillus plantarum; Lactobacillus plantarum - chemistry; Lactobacillus plantarum - enzymology; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular Weight; Natural products; Observations; Oxidases; Oxidation; Physiological aspects; Plasmids; Proteins; Pyrogallol - analogs & derivatives; Pyrogallol - chemistry; Pyrogallol - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; spectral analysis; Studies; Substrate Specificity; Sulfonic Acids - chemistry; Sulfonic Acids - metabolism; toxic substances; tyramine; Tyramine - chemistry; Tyramine - metabolism; Spain; Tyramine; Histamine; Putrescine; Biogenic amines; Laccase; Lactobacillus plantarum
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-7158-0

In our search for degrading activities of biogenic amines (BAs) in lactic acid bacteria, a protein annotated as laccase enzyme was identified in Lactobacillus plantarum J16 (CECT 8944). In this study, the gene of this new laccase was cloned and heterologously overexpressed in Escherichia coli. The recombinant laccase protein was purified and characterized biochemically. The purified laccase showed characteristic spectroscopic properties of blue multicopper oxidases. The enzyme has a molecular weight of ∼62.5 kDa and activity toward typical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). The pH optima on ABTS and 2,6-DMP were 3.5 and 7.0, respectively. Kinetic constants K ₘ and V ₘₐₓ were of 0.21 mM and 0.54 U/mg for ABTS and 1.67 mM and 0.095 U/mg for 2,6-DMP, respectively. The highest oxidizing activity toward 2,6-DMP was obtained at 60 °C. However, after a preincubation step at 85 °C for 10 min, no residual activity was detected. It has been demonstrated that recombinant L. plantarum laccase oxidizes biogenic amines, mainly tyramine, and thus presents new biotechnological potential for the enzyme in eliminating toxic compounds present in fermented food and beverages.