Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated beta-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome

Author(s)

Alves, Luana de Fatima ^[1] ; Meleiro, Luana Parras ^[2] ; Silva, Roberto N. ^[1] ; Westmann, Caua Antunes ^[3] ; Guazzaroni, Maria-Eugenia ^[4]

Affiliation

^[1] Univ Sao Paulo, Dept Biochem & Immunol, Fac Med Ribeirao Preto, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Dept Chem, Fac Filosofia Ciencias & Letras Ribeirao Preto, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Dept Cellular & Mol Biol, Fac Med Ribeirao Preto, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, Sao Paulo - Brazil

• Document type: Journal article
• Source: FRONTIERS IN MICROBIOLOGY; v. 9, OCT 29 2018.
• Web of Science Citations: 2

Abstract

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel beta-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel beta-glucosidase - named Lfa2 - displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50 degrees C, and showed hydrolytic activity toward several pNP synthetic substrates containing beta-glucose, beta-galactose, beta-xylose, beta-fucose, and alpha-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, beta-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that 13-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing. (AU)

• FAPESP's process: 16/05472-6 - Synthetic biology approaches to engineer/mine novel regulatory elements in bacteria
• Grantee: Cauã Antunes Westmann
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries
• Grantee: María Eugenia Guazzaroni
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 16/06323-4 - New tools for improving metagenomic cellulase screenings
• Grantee: Luana de Fátima Alves
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/17582-0 - Thermostability modulation of a ß-glucosidase stimulated by glucose and xylose using site directed glycosylation
• Grantee: Luana Parras Meleiro Garcia
• Support Opportunities: Scholarships in Brazil - Post-Doctoral