Dataset Information

Characterization of an endo-processive-type xyloglucanase having a ?-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

ABSTRACT: We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to ?-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

SUBMITTER: Ichinose H

PROVIDER: S-EPMC3485957 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Characterization of an endo-processive-type xyloglucanase having a β-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

Ichinose Hitomi H Araki Yuko Y Michikawa Mari M Harazono Koichi K Yaoi Katsuro K Karita Shuichi S Kaneko Satoshi S

Applied and environmental microbiology 20120831 22

We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. ...[more]

PMID: 22941084

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Project description:Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, for example, by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is a valuable source for CAZyme and CBM discovery and characterization due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (X181), and a glycoside hydrolase family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase (M. A. Attia, C. E. Nelson, W. A. Offen, N. Jain, et al., Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6). In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest products of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. IMPORTANCE This study reveals carbohydrate-binding module family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the carbohydrate-active enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

Dataset Information

Characterization of an endo-processive-type xyloglucanase having a ?-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

Publications

Characterization of an endo-processive-type xyloglucanase having a β-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

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