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Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-?-1,4-glucanase from glycoside hydrolase family 12.

ABSTRACT: Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 M ammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space group P3(2)21. The unit-cell parameters were a = b = 103.24, c = 48.96 Å.

SUBMITTER: Segato F

PROVIDER: S-EPMC3936441 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-β-1,4-glucanase from glycoside hydrolase family 12.

Segato Fernando F Berto Gabriela L GL Ares de Araújo Evandro E Muniz João Renato JR Polikarpov Igor I

Acta crystallographica. Section F, Structural biology communications 20140128 Pt 2

Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such as Aspergillus terreus are effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). The A. terreus GH12 endoglucanase was cloned and overexpressed in A. nidulans, purified and crystallized. A single crystal was obtained from a solution consist ...[more]

PMID: 24637772

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Project description:As classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identified Arcticibacterium luteifluviistationis carboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase. AlCMCase originated from an Arctic marine bacterium, Arcticibacterium luteifluviistationis SM1504T It shows low identity (<35%) with other GH10 xylanases. The gene encoding AlCMCase was overexpressed in Escherichia coli Biochemical characterization showed that recombinant AlCMCase is a cold-adapted and salt-tolerant enzyme. AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity of AlCMCase is negligible. Correspondingly, AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides. AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity as AlCMCase. These results suggest that AlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters of AlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCE Cellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase, AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover, AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.

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

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-?-1,4-glucanase from glycoside hydrolase family 12.

Publications

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Aspergillus terreus endo-β-1,4-glucanase from glycoside hydrolase family 12.

Similar Datasets

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