A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After ...A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.展开更多
Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme condition...Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme conditions, such as high temperature, salinity or extreme pH, is required for practical industrial applications. Here we focus on bacterial laccases from the phylum Actinobacteria, notably the order Actinomycetales. Currently, less than 10 enzymes have been properly characterized, all belonging to genus Streptomyces, but it is noteworthy that all of them have exhibited industrially important properties. Furthermore, studies with enzymes from this phylum revealed a novel molecular structure of laccases, providing the basis for a distinct family, the two-domain laccases. The relevant traits of actinomycetes laccases emphasize the need for more studies involving the isolation of this bacterial group from lignin-rich environmental samples, detection of their laccase activity and thereafter, characterization of the proteins and related genes. The nonhomogeneous responses of actinomycetes laccases to traditional inhibitors, substrates or metal ions have challenged the currently accepted “laccase concept”. Finally, considering that distinguishing laccase activity in vitro from other ligninolytic enzymes becomes a difficult task due to overlaps in catalytical properties of the enzymes, we proposed a simple flow chart to help experimental assays.展开更多
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 difculty in breathing,nausea,and vomiting.Degr...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 difculty in breathing,nausea,and vomiting.Degradation of BAs by multicopper oxidase(MCO)is a promising method as it has little efect on the fermentation process,food nutrition,and favor.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(kcat)and specifc activity of MCOBLS 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.展开更多
文摘A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.
文摘Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme conditions, such as high temperature, salinity or extreme pH, is required for practical industrial applications. Here we focus on bacterial laccases from the phylum Actinobacteria, notably the order Actinomycetales. Currently, less than 10 enzymes have been properly characterized, all belonging to genus Streptomyces, but it is noteworthy that all of them have exhibited industrially important properties. Furthermore, studies with enzymes from this phylum revealed a novel molecular structure of laccases, providing the basis for a distinct family, the two-domain laccases. The relevant traits of actinomycetes laccases emphasize the need for more studies involving the isolation of this bacterial group from lignin-rich environmental samples, detection of their laccase activity and thereafter, characterization of the proteins and related genes. The nonhomogeneous responses of actinomycetes laccases to traditional inhibitors, substrates or metal ions have challenged the currently accepted “laccase concept”. Finally, considering that distinguishing laccase activity in vitro from other ligninolytic enzymes becomes a difficult task due to overlaps in catalytical properties of the enzymes, we proposed a simple flow chart to help experimental assays.
基金This work was supported by the National Key Research and Development Program of China(2017YFC1600405)National Natural Science Foundation of China(31771955).
文摘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 difculty in breathing,nausea,and vomiting.Degradation of BAs by multicopper oxidase(MCO)is a promising method as it has little efect on the fermentation process,food nutrition,and favor.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(kcat)and specifc activity of MCOBLS 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.
