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Identification and characterization of a halotolerant, cold-active marine endo-?-1,4-glucanase by using functional metagenomics of seaweed-associated microbiota.

ABSTRACT: A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-?-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.

SUBMITTER: Martin M

PROVIDER: S-EPMC4135742 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Identification and characterization of a halotolerant, cold-active marine endo-β-1,4-glucanase by using functional metagenomics of seaweed-associated microbiota.

Martin Marjolaine M Biver Sophie S Steels Sébastien S Barbeyron Tristan T Jam Murielle M Portetelle Daniel D Michel Gurvan G Vandenbol Micheline M

Applied and environmental microbiology 20140606 16

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was p ...[more]

PMID: 24907332

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Project description:Endo-β-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted β-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-β-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-β-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-β-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley β-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley β-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-β-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Dataset Information

Identification and characterization of a halotolerant, cold-active marine endo-?-1,4-glucanase by using functional metagenomics of seaweed-associated microbiota.

Publications

Identification and characterization of a halotolerant, cold-active marine endo-β-1,4-glucanase by using functional metagenomics of seaweed-associated microbiota.

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