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Heterologous Expression and Biochemical Characterization of a Novel Lytic Polysaccharide Monooxygenase from Chitinilyticum aquatile CSC-1.

ABSTRACT: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of recalcitrant polysaccharides. There are limited reports on LPMOs capable of concurrently catalyzing the oxidative cleavage of both cellulose and chitin. In this study, we identified and cloned a novel LPMO from the newly isolated bacterium Chitinilyticum aquatile CSC-1, designated as CaLPMO10. When using 2, 6-dimethylphenol (2, 6-DMP) as the substrate, CaLPMO10 exhibited optimal activity at 50 °C and pH 8, demonstrating good temperature stability at 30 °C. Even after a 6 h incubation at pH 8 and 30 °C, CaLPMO10 retained approximately 83.03 ± 1.25% residual enzyme activity. Most metal ions were found to enhance the enzyme activity of CaLPMO10, with ascorbic acid identified as the optimal reducing agent. Mass spectrometry analysis indicated that CaLPMO10 displayed oxidative activity towards both chitin and cellulose, identifying it as a C1/C4-oxidized LPMO. CaLPMO10 shows promise as a key enzyme for the efficient utilization of biomass resources in future applications.

SUBMITTER: Shao X

PROVIDER: S-EPMC11278713 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Heterologous Expression and Biochemical Characterization of a Novel Lytic Polysaccharide Monooxygenase from Chitinilyticum aquatile CSC-1.

Shao Xuezhi X Fang Hongliang H Li Tao T Yang Liyan L Yang Dengfeng D Pan Lixia L

Microorganisms 20240708 7

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of recalcitrant polysaccharides. There are limited reports on LPMOs capable of concurrently catalyzing the oxidative cleavage of both cellulose and chitin. In this study, we identified and cloned a novel LPMO from the newly isolated bacterium Chitinilyticum aquatile CSC-1, designated as CaLPMO10. When using 2, 6-dimethylphenol (2, 6-DMP) as the substrate, CaLPMO10 ex ...[more]

PMID: 39065150

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Project description:BackgroundCyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca.ResultsTwo variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L-1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.ConclusionsWe have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.

Dataset Information

Heterologous Expression and Biochemical Characterization of a Novel Lytic Polysaccharide Monooxygenase from Chitinilyticum aquatile CSC-1.

Publications

Heterologous Expression and Biochemical Characterization of a Novel Lytic Polysaccharide Monooxygenase from <i>Chitinilyticum aquatile</i> CSC-1.

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