Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition
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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.
KeywordsMorchella importuna SCYDJ1-A1 AA1_3 family Laccase-like multicopper oxidase Transcript level Overexpression Biochemical characterization
This research was supported by the Sichuan Science and Technology Program (Applied Fundamental Research Project, 2018JY0637), Innovative Improvement Projects of Sichuan Province (2016ZYPZ-028, 2016LWJJ-007), and the Special Fund for Agro-scientific Research in the Public Interest (201503137).
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Conflict of interest
The authors declare no conflict of interest.
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