Contents lists available at ScienceDirect BBA - Proteins and Proteomics journal homepage: www.elsevier.com/locate/bbapap A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil) Danyelle Toyama a,1 , Mariana Abrahão Bueno de Morais b,1 , Felipe Cardoso Ramos b , Letícia Maria Zanphorlin b , Celisa Caldana Costa Tonoli c , Augusto Furio Balula a , Fernando Pellon de Miranda d , Vitor Medeiros Almeida e , Sandro Roberto Marana e , Roberto Ruller b , Mario Tyago Murakami b, ⁎ , Flavio Henrique-Silva a, ⁎⁎ a Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, SP, Brazil b Brazilian Bioethanol Science and Technology Laboratory, National Center for Research in Energy and Materials, Campinas, SP, Brazil c Brazilian Biosciences National Laboratory, National Center for Research in Energy and Materials, Campinas, SP, Brazil d Petróleo Brasileiro S.A. (Petrobras), Centro de Pesquisas e Desenvolvimento Leopoldo Américo Miguez de Mello, Rio de Janeiro, RJ, Brazil e Departamento de Bioquímica, Instituto de Química, SP, Brazil ARTICLE INFO Keywords: Amazon Metagenome Cellulose β-Glucosidase GH1-family 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 =6s -1 ·mM -1 ) than cellobiose (k cat /K m = 0.6 s -1 ·mM -1 ). AmBgl-LP showed maximum ac- tivity 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 under- standing of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their in- dustrial applications. 1. Introduction The Amazon comprises the largest hydrographic basin in the world. Despite its enormous biodiversity, the microorganisms present in their rivers and lakes remain unexplored. The great genetic and metabolic diversity of both cultivable and non-cultivable organisms present in this important region may result in the discovery of new enzymes of bio- technological interest, such as enzymes involved in the degradation of the plant cell walls. Cellulose is the most abundant component of plant cell wall [1], and the soils, rivers, and lakes of the Amazon have large amounts of organic matter derived from plants degraded by microorganisms. In this way, microorganisms present in this region must produce enzymes capable of degrading cellulose and also converting it into fermentable sugars (e.g., cellulases, cellobiosidases and β-glucosidases). Cellulases (EC 3.2.1.x) are enzymes synthesized by several organ- isms and are involved in cellulose degradation [2,3]. They are glycoside hydrolases (GH) and act by hydrolyzing β-1,4 glycosidic bonds in the cellulose chain. They are classified as endoglucanases (EC 3.2.1.4) and exoglucanases (EC 3.2.1.91) that act on cellulose to produce cellobiose https://doi.org/10.1016/j.bbapap.2018.02.001 Received 19 September 2017; Received in revised form 9 February 2018; Accepted 14 February 2018 ⁎ Correspondence to: M. T. Murakami, Brazilian Bioethanol Science and Technology Laboratory, National Center for Research in Energy and Materials, Giuseppe Maximo Scolfaro 10000, Campinas, SP, Brazil. ⁎⁎ Correspondence to: F. Henrique-Silva, Federal University of São Carlos, Department of Genetics and Evolution, Laboratory of Molecular Biology, Rodovia Washington Luiz, km 235, São Carlos, SP, Brazil. 1 Both authors have contributed equally. E-mail addresses: mario.murakami@ctbe.cnpem.br (M.T. Murakami), dfhs@ufscar.br (F. Henrique-Silva). BBA - Proteins and Proteomics 1866 (2018) 569–579 Available online 16 February 2018 1570-9639/ © 2018 Elsevier B.V. All rights reserved. T