title: Bg10: A Novel Metagenomics Alcohol-Tolerant and Glucose-Stimulated GH1 ß-Glucosidase Suitable for Lactose-Free Milk Preparation.
abstract: New ß-glucosidases with product (glucose) or ethanol tolerances are greatly desired to make industrial processes more marketable and efficient. Therefore, this report describes the in silico/vitro characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a Vmax of 10.81 ± 0.43 μM min-1, Kcat of 175.1± 6.91 min-1, and Km of 0.49 ± 0.12 mM at a neutral pH and 37°C when pNP-ß-D-glucopyranoside was used as the substrate, and the enzyme retained greater than 80% activity within the respective pH and temperature ranges of 6.5 to 8.0 and 35 to 40°C. The enzyme was stimulated by its product, glucose; consequently, the Bg10 activity against 50 and 100 mM of glucose were increased by 36.8% and 22%, respectively, while half of the activity was retained at 350 mM. Moreover, the Bg10 was able to hydrolyse 55% (milk sample) and 100% (purified sugar) of the lactose at low (6°C) and optimum (37°C) temperatures, respectively, suggesting the possibility of further optimization of the reaction for lactose-free dairy production. In addition, the enzyme was able to fully hydrolyse 40 mM of cellobiose at one hour and was tolerant to ethanol up to concentrations of 500 mM (86% of activity), while a 1 M concentration still resulted in 41% residual activity, which could be interesting for biofuel production.
authors.affiliation: Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal. Via de Acesso Prof. Paulo Donato Castellane S/N, km 5, CEP, Jaboticabal, São Paulo State, Brazil.|Molecular Biology Laboratory; Institute for Research in Bioenergy (IPBEN), UNESP-Jaboticabal, SP, Brazil.|Agricultural Microbiology postgraduate program of UNESP, Jaboticabal, São Paulo State, Brazil.
authors.forename: Elisângela Soares
authors.initials: ES
authors.lastname: Gomes-Pepe
authors.affiliation: Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal. Via de Acesso Prof. Paulo Donato Castellane S/N, km 5, CEP, Jaboticabal, São Paulo State, Brazil.|Molecular Biology Laboratory; Institute for Research in Bioenergy (IPBEN), UNESP-Jaboticabal, SP, Brazil.
authors.forename: Elwi Guillermo
authors.initials: EG
authors.lastname: Machado Sierra
authors.affiliation: Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal. Via de Acesso Prof. Paulo Donato Castellane S/N, km 5, CEP, Jaboticabal, São Paulo State, Brazil.|Molecular Biology Laboratory; Institute for Research in Bioenergy (IPBEN), UNESP-Jaboticabal, SP, Brazil.
authors.forename: Mariana Rangel
authors.initials: MR
authors.lastname: Pereira
authors.affiliation: Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal. Via de Acesso Prof. Paulo Donato Castellane S/N, km 5, CEP, Jaboticabal, São Paulo State, Brazil.|Molecular Biology Laboratory; Institute for Research in Bioenergy (IPBEN), UNESP-Jaboticabal, SP, Brazil.
authors.forename: Tereza Cristina Luque
authors.initials: TC
authors.lastname: Castellane
authors.affiliation: Department of Technology, São Paulo State University (Unesp), School of Agricultural and Veterinarian Sciences, Jaboticabal. Via de Acesso Prof. Paulo Donato Castellane S/N, km 5, CEP, Jaboticabal, São Paulo State, Brazil.|Molecular Biology Laboratory; Institute for Research in Bioenergy (IPBEN), UNESP-Jaboticabal, SP, Brazil.
authors.forename: Eliana Gertrudes de Macedo
authors.identifier: http://orcid.org/0000-0002-1119-7748
authors.initials: EG
authors.lastname: Lemos
mesh_terms: D000818:Animals; D002475:Cellobiose / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry*; D005947:Glucose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000737:chemistry*; D056186:Metagenomics; D008892:Milk / Q000378:metabolism*; D010802:Phylogeny; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000145:classification / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D002475:Cellobiose; D000431:Ethanol; D001617:beta-Glucosidase; D005947:Glucose; D007785:Lactose