Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry.

Monica Abbondi,Elisa Binda,Elena Rosini,Loredano Pollegioni,Giorgia Letizia Marcone

doi:10.3389/fmicb.2019.02786

Monica Abbondi, Elisa Binda + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2019.02786

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Abstract

Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O2-dependent oxidative deamination of D-enantiomer of amino acids generating α-keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37°C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O2-scavenger thus further hampering food deterioration.

Highlights

The presence of bacterial pathogens in food may be responsible for spoilage and foodborne disease incidence
We selected D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis for this application since it can be overexpressed in huge amounts in E. coli cells (Romano et al, 2009); it shows a strong interaction with the FAD cofactor (Pollegioni et al, 2007), a high kinetic efficiency, a broad substrate preference for D-amino acids, and absence of inhibition by the L-enantiomer
We tested the antibacterial activity of DAAO by diffusion agar test and liquid growth kinetics: in both cases, we demonstrated that active wild-type DAAO and its variant mDAAO are able to inhibit bacterial growth producing an inhibition halo on cultures grown on agar medium and to reduce by half the growth in liquid culture

Summary

Introduction

The presence of bacterial pathogens in food may be responsible for spoilage and foodborne disease incidence. Preservative agents are added to ensure food safety and prevent spoilage Both chemical and biological preservatives are used, for a review, see Brul and Coote (1999). Pathogenic bacteria, like Listeria monocytogenes cannot be fully eliminated in food products by chemical preservatives, which do not delay the DAAO as Food Preservative growth of spoilage microorganisms (Tajkarimi et al, 2010). On this side, the antibacterial effects of hydrogen peroxide have been extensively investigated due to its possible involvement in a number of important biological events in which bacterial cells are either killed or their growth inhibited. Hydrogen peroxide has a potential to be used in a variety of ways in the food industry as antimicrobial agent in water and dairy products (Juven and Pierson, 1996)

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