A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil)

Danyelle Toyama; Mariana Abrahão Bueno de Morais; Felipe Cardoso Ramos; Letícia Maria Zanphorlin; Celisa Caldana Costa Tonoli; Augusto Furio Balula; Fernando Pellon de Miranda; Vitor Medeiros Almeida; Sandro Roberto Marana; Roberto Ruller; Mario Tyago Murakami; Flavio Henrique-Silva

doi:10.1016/j.bbapap.2018.02.001

A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil)

Biochim Biophys Acta Proteins Proteom. 2018 Apr;1866(4):569-579. doi: 10.1016/j.bbapap.2018.02.001. Epub 2018 Feb 16.

Affiliations

¹ Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, SP, Brazil.
² Brazilian Bioethanol Science and Technology Laboratory, National Center for Research in Energy and Materials, Campinas, SP, Brazil.
³ Brazilian Biosciences National Laboratory, National Center for Research in Energy and Materials, Campinas, SP, Brazil.
⁴ Petróleo Brasileiro S.A. (Petrobras), Centro de Pesquisas e Desenvolvimento Leopoldo Américo Miguez de Mello, Rio de Janeiro, RJ, Brazil.
⁵ Departamento de Bioquímica, Instituto de Química, SP, Brazil.
⁶ Brazilian Bioethanol Science and Technology Laboratory, National Center for Research in Energy and Materials, Campinas, SP, Brazil. Electronic address: mario.murakami@ctbe.cnpem.br.
⁷ Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, SP, Brazil. Electronic address: dfhs@ufscar.br.

PMID: 29454992
DOI: 10.1016/j.bbapap.2018.02.001

Abstract

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel β-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-β-d-glucopyranoside (pNPβG) and the natural substrate cellobiose, it showed higher specificity for pNPβG (k_cat/K_m = 6 s^-1·mM^-1) than cellobiose (k_cat/K_m = 0.6 s^-1·mM^-1). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPβG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a K_i of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.

Keywords: Amazon; Cellulose; GH1-family; Metagenome; β-Glucosidase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Brazil
Lakes / microbiology*
Metagenome*
Water Microbiology*
beta-Glucosidase / chemistry*
beta-Glucosidase / genetics
beta-Glucosidase / metabolism

Substances

beta-Glucosidase