Food-grade expression of multicopper oxidase with improved capability in degrading biogenic amines

Abstract

Biogenic amines (BAs) are potential amine hazards that are detected in fermented foods and alcoholic beverages. Excessive intake of BAs may lead to allergic symptoms such as difficulty in breathing, nausea, and vomiting. Degradation of BAs by multicopper oxidase (MCO) is a promising method as it has little effect on the fermentation process, food nutrition, and flavor. However, the application of MCO in food industry was restricted due to its poor catalytic properties and low productivity. In this work, food-grade expression of the Bacillus amyloliquefaciens MCO (MCOB) and its three mutants were successfully constructed in Lactococcus lactis NZ3900. The expression level of MCOB in L. lactis NZ3900 was dramatically enhanced by optimizing the cultivation conditions, and the highest expression level reached 4488.1 U/L. This was the highest expression level of food-graded MCO reported so far, to our knowledge. Interestingly, the optimal reaction pH of MCOB expressed in L. lactis NZ3900 switched to 4.5, it would be more suitable for degrading BAs in food as the pH value of most fermented foods was found to be 4.5. Moreover, MCOB expressed in L. lactis NZ3900 was quite stable (with more than 80% residual activity) in the pH range of 4.0-5.5, the catalytic rate constant (k cat ) and specific activity of MCOB LS were all dramatically increased compared with that of MCOB expressed in Escherichia coli. Using histamine as the substrate, the degradation of BAs within 24 h by MCOB expressed in L. lactis NZ3900 was 69.7% higher than that expressed in E. coli. The results demonstrated the potential applications of MCOB in food industry for reduction of biogenic amines.

Functional genomic analysis of the isolated potential probiotic Lactobacillus delbrueckii subsp. indicus TY-11 and its comparison with other Lactobacillus delbrueckii strains

“…More importantly, the TY-11 genome carried a multicopper oxidase domain-containing protein (LPJCKDFM_00777). Multicopper oxidase (MCO)-mediated BAS degradation is an auspicious method as the fermentation process, food nutrition, and flavor are unaffected ( 42 ). This genome also contained nitroreductase (LPJCKDFM_00111), a flavoenzyme that catalyzes the reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds in a NAD ( P )H-dependent manner.…”

TEM Experiment and Complete Genome Sequencing with Comparative Genomic Analysis to Uncover Potential Probiotic Features of IsolatedLactobacillus delbrueckiisubsp.indicusTY-11

“…Another aspect, the TY-11 genome carried multicopper oxidase domain-containing protein (LPJCKDFM_00777). Multicopper oxidase (MCO) mediated BAS degradation is an auspicious method as the fermentation process, food nutrition, and flavour are unaffected [44]. This genome also contains nitroreductase (LPJCKDFM_00111), a flavoenzyme that catalyzes the reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds in a NAD (P)H-dependent manner.…”

“…These involve important enzymes, such as D-lactic acid Another aspect, the TY-11 genome carried multicopper oxidase domain-containing protein (LPJCKDFM_00777). Multicopper oxidase (MCO) mediated BAS degradation is an auspicious method as the fermentation process, food nutrition, and flavour are unaffected [44].…”

Biochemical Characterization of Three Heterologous Lactic Acid Bacteria Laccases from Pediococcus, Lactobacillus, and Lactococcus Genus and Their Potential to Degrade Biogenic Amines Using ABTS and Epicatechin as Mediators

“…Additionally, Xu and Fang (2019) reported that L. fermentum recombinant laccase can degrade histamine and tyramine by 51.6% and 40.9%, respectively, as well as other BAs such as tryptamine, PEA, putrescine, cadaverine, and spermidine [16]. Ni et al (2022) demonstrated that a salt-tolerant, mutant recombinant laccase from B. amyloliquefaciens effectively degraded histamine, tyramine, and putrescine by 25%, 20%, and 7%, respectively, in a 50 mM sodium phosphate buffer (pH 4.5) [54]. Recently, Wang and colleagues (2022) reported the ability of L. sakei recombinant laccase to degrade histamine and tyramine in the absence of mediators at various ethanol concentrations, with degradation rates of approximately 20% and 25%, respectively, when exposed to 20% ethanol, and in food matrices such as surimi and grape juice [20].…”