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Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition.

ABSTRACT: The aim of this study is to determine the key laccase-encoding gene in the life cycle of Morchella importuna SCYDJ1-A1, and to characterize the biochemical properties of the laccase. Two laccase-like multicopper oxidase (LMCO) genes were identified in the genome of M. importuna SCYDJ1-A1 as putative laccase-encoding genes. The two genes, belonging to Auxiliary Activity family 1 subfamily 3, were named as MiLacA and MiLacB. Phylogenetic analysis of deduced amino acid sequences showed that MiLacA is closest to a LMCO of M. importuna 22J1, while MiLacB had low similarity with known Morchella LMCOs. Real-time quantitative PCR results showed that MiLacA was expressed at much higher levels than MiLacB throughout the entire course of artificial cultivation. MiLacA was overexpressed in Pichia pastoris as a recombinant protein. Biochemical characterization of the purified enzyme showed that MiLacA simultaneously possessed laccase and polyphenol-oxidase activities. MiLacA could be strongly inhibited by Fe2+, which is unusual. The optimum pH was four and optimum temperature was 60 °C. The enzyme retained over 74% of the laccase activity after 16-h incubation at 60 °C, which means that its thermostability is at the forefront among the currently known laccases. Our findings may help to elucidate how the laccase of M. importuna is involved in decaying lignin in plant litter, and could also provide a candidate thermostable laccase for potential industrial application.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC6456629 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition.

Zhang Qiang Q Miao Renyun R Liu Tianhai T Huang Zhongqian Z Peng Weihong W Gan Bingcheng B Zhang Xiaoping X Tan Hao H

3 Biotech 20190409 5

The aim of this study is to determine the key laccase-encoding gene in the life cycle of Morchella importuna SCYDJ1-A1, and to characterize the biochemical properties of the laccase. Two laccase-like multicopper oxidase (LMCO) genes were identified in the genome of M. importuna SCYDJ1-A1 as putative laccase-encoding genes. The two genes, belonging to Auxiliary Activity family 1 subfamily 3, were named as MiLacA and MiLacB. Phylogenetic analysis of deduced amino acid sequences showe ...[more]

PMID: 30997308

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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.

Project description:BackgroundMany filamentous fungal genomes contain complex groups of multicopper oxidase (MCO) coding genes that makes them a good source for new laccases with potential biotechnological interest. A bioinformatics analysis of the Aspergillus niger ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed.ResultsA bioinformatic analysis of the A. niger ATCC 1015 genome revealed the presence of 13 MCO genes belonging to three different subfamilies on the basis of their phylogenetic relationships: ascomycete laccases, fungal pigment MCOs and fungal ferroxidases. According to in silico amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (mcoA, mcoB, mcoC, mcoD, mcoE, mcoF, mcoG, mcoI, mcoJ and mcoM) were placed under the control of the glaA promoter and overexpressed in A. niger N593. Enzyme activity plate assays with several common laccase substrates showed that all genes are actually expressed and code for active MCOs. Interestingly, expressed enzymes show different substrate specificities. In addition, optimization of fungal pigment MCOs extracellular production was investigated. The performance of the widely used glucoamylase signal sequence (ssGlaA) in McoA secretion was studied. Results obtained suggest that ssGlaA do not yield higher levels of secreted McoA when compared to its native secretion signal. Also, McoB synthesis was investigated using different nitrogen sources in minimal medium liquid cultures. Higher yields of extracellular McoB were achieved with (NH4)2 tartrate.ConclusionsAspergillus niger is a good source of new laccases. The different substrate specificity observed in plate assays makes them interesting to be purified and biochemically compared. The homologous signal sequence of McoA has been shown to be a good choice for its extracellular overexpression. From the nitrogen sources tested (NH4)2 tartrate has been found to be the most appropriate for McoB production in A. niger.

Dataset Information

Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition.

Publications

Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable <i>Morchella importuna</i> provides insights into plant-litter decomposition.

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