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

Abstract

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 Km and Vmax 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.

Selection of a versatile Lactobacillus plantarum for wine production and identification and preliminary characterisation of a novel histamine‐degrading enzyme

“…Only Spanish research team, that is Callejón and co‐authors, attempted to purify such enzyme from L. plantarum via Sulfopropyl Sepharose FF and Macro‐Prep Q chromatographic steps, but failed in obtaining a single band on the SDS‐PAGE gel (Callejón et al , ). They still made great progress in this field since a probable BA‐degrading enzyme candidate, a laccase was acquired, the gene of which was cloned and heterologously overexpressing in E. coli , and recombinantly purified laccase was proved effective in oxidising BA, especially tyramine (Callejón et al , ).…”

“…Members in this team attempted to purify the enzyme by different chromatographic techniques, but no single band was obtained on the SDS‐PAGE gel. Later, the gene of the laccase was cloned and heterologously overexpressed in E. coli (Callejón et al , ). The recombinant laccase protein was purified, with a molecular weight of 62.5 kDa, and showed the capability to oxidise biogenic amines, especially tyramine.…”

Climate Changes and Food Quality: The Potential of Microbial Activities as Mitigating Strategies in the Wine Sector

“…Enzymes isolated and purified from L. plantarum and P. acidilactici strains, and identified as multicopper oxidases, were able to degrade histamine, tyramine, and putrescine [99]. Such a finding opens a new perspective on the opportunity of employing purified microbial enzymes to deal with the problem of high BA concentrations in wine [103].…”

Enzymes for Wine Fermentation: Current and Perspective Applications

“…Responsible enzymes have been isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and have been identified as intracellular laccase-like multicopper oxidases. When the L. plantarum J16 laccase was overexpressed in Escherichia coli, it oxidized some BA, mainly tyramine [108].…”