Characterization of an extremely thermo-active archaeal β-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase
A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 β-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant β-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP β-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active β-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP β-D-mannopyranoside in comparison with 4NP β-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the β-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley β-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with β-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.
Keywordsβ-Glucosidase Extremozymes Heat-active Glucose-tolerant Synergism
Carola Schröder planned the study and drafted the manuscript with Garabed Antranikian. Characterization of β-glucosidase was carried out by Daniela Eixenberger. Concerted endoglucanase and β-glucosidase experiments were performed by Marcel Suleiman and Christian Schäfers performed the genome binning and drafted the respective paragraphs. All authors commented on previous versions of the manuscript and all authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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