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Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

ABSTRACT: Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development.

SUBMITTER: Ihssen J

PROVIDER: S-EPMC4464401 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

Ihssen Julian J Reiss Renate R Luchsinger Ronny R Thöny-Meyer Linda L Richter Michael M

Scientific reports 20150612

Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different e ...[more]

PMID: 26068013

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Project description:BackgroundFungal laccases are multicopper oxidases (MCOs) with high biotechnological potential due to their capability to oxidize a wide range of aromatic contaminants using oxygen from the air. Albeit the numerous laccase-like genes described in ascomycete fungi, ascomycete laccases have been less thoroughly studied than white-rot basidiomycetous laccases. A variety of MCO genes has recently been discovered in plant pathogenic ascomycete fungi, however little is known about the presence and function of laccases in these fungi or their potential use as biocatalysts. We aim here to identify the laccase-type oxidoreductases that might be involved in the decolorization of dyes by Leptosphaerulina sp. and to characterize them as potential biotechnological tools.ResultsA Leptosphaerulina fungal strain, isolated from lignocellulosic material in Colombia, produces laccase as the main ligninolytic oxidoreductase activity during decolorization of synthetic organic dyes. Four laccase-type MCO genes were partially amplified from the genomic DNA using degenerate primers based on laccase-specific signature sequences. The phylogenetic analysis showed the clustering of Lac1, Lac4 and Lac3 with ascomycete laccases, whereas Lac2 grouped with fungal ferroxidases (together with other hypothetical laccases). Lac3, the main laccase produced by Leptosphaerulina sp. in dye decolorizing and laccase-induced cultures (according to the shotgun analysis of both secretomes) was purified and characterized in this study. It is a sensu-stricto laccase able to decolorize synthetic organic dyes with high efficiency particularly in the presence of natural mediator compounds.ConclusionsThe searching for laccase-type MCOs in ascomycetous families where their presence is poorly known, might provide a source of biocatalysts with potential biotechnological interest and shed light on their role in the fungus. The information provided by the use of genomic and proteomic tools must be combined with the biochemical evaluation of the enzyme to prove its catalytic activity and applicability potential.

Project description:BackgroundLaccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O.ResultsThe structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, micro-eta 1:eta 1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (micro3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.ConclusionThis is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.

Project description:Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

Dataset Information

Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

Publications

Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli.

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