Abstract
Glycoside hydrolases (GHs) are enzymes found in all living kingdoms that are involved in multiple physiological functions. Due to their multiple enzymatic activities, GHs are broadly applied in bioethanol, food, and paper industry. In order to increase the productivity of these industrial processes, a constant search for novel and efficient enzymes has been proved to be necessary. In this context, metagenomics is a powerful approach to achieve this demand. In the current study, we describe the discovery and characterization of a novel member of GH16 family derived from the sugarcane soil metagenome. The enzyme, named SCLam, has 286 amino acid residues and displays sequence homology and activity properties that resemble known laminarases. SCLam is active against barley beta-glucan, laminarin, and lichenan (72, 33, and 10 U mg−1, respectively). The optimal reaction conditions were identified as 40 °C and pH 6.5. The low-resolution structure was determined using the small-angle X-ray scattering technique, revealing that SCLam is a monomer in solution with a radius of gyration equal to 19.6 Å. To the best of our knowledge, SCLam is the first nonspecific (1,3/1,3:1,4)-β-d-glucan endohydrolase (EC 3.2.1.6) recovered by metagenomic approach to be characterized.
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The authors would like to acknowledge FAPESP for financial support to FMS (08/58037-9) and TMA (2010/11469-1); CNPq for financial support to FMS (442333/2014-5 and 310186/2014-5), MVL (158882/2014-8), and MON (478900/2012-0); and SAXS2 (LNLS/CNPEM) and CTBE/CNPEM for technical support.
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Figure S1
SDS–PAGE analysis of SCLam purification. A) Affinity chromatography. Lane MW – molecular weight marker (labelled in kDa). Lane 1 – soluble fraction. Lane 2 – Flow-through. Lane 3 -Wash. Lanes 4 to 8 – Elutions. B) Size-exclusion chromatography. Lane MW - molecular weight marker (labelled in kDa). Lanes 1 to 3 - elution fractions from size-exclusion chromatography, in 20 mM sodium phosphate pH 7.4 containing 50 mM NaCl buffer. (JPEG 268 kb)
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Alvarez, T.M., Liberato, M.V., Cairo, J.P.L.F. et al. A Novel Member of GH16 Family Derived from Sugarcane Soil Metagenome. Appl Biochem Biotechnol 177, 304–317 (2015). https://doi.org/10.1007/s12010-015-1743-7
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DOI: https://doi.org/10.1007/s12010-015-1743-7