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.
Highlights
The construction of protein structure models based on homology and the identification of conserved catalytic residues combined with heterologous expression and a kinetic study of Bg10 revealed that this enzyme is an alcohol-tolerant and glucose-stimulated ß-glucosidase with high lactase activity that has a great deal of potential as a biotechnological product in the dairy industry [22]
The 23-kb-length cosmid DNA insert B5p37metaSE was submitted to sequence comparison with GenBank sequences by use of the BLAST tool provided by the National Center for Biotechnology Information (NCBI: http://www.ncbi.nlm.nih.gov), and the results demonstrated that only 38% of this sequence was similar to other sequences deposited in the NCBI database but shared 78% identity with Kitasatospora setae KM-6054 (AP010968.1 NCBI code), an Actinobacteria from Streptomycetaceae family
The gene annotation for B5p37metaSE showed that the metagenomic DNA fragment harboured one sequence for an aromatic polyketide antibiotic cluster and sequences related to carbohydrate degradation: one ß-glucosidase (ORF10, corresponding to Bg10 enzyme, target of this study), one chitinase (ORF11), and one laccase (ORF12)
Summary
The construction of protein structure models based on homology (template 1GNX, with 70% identity) and the identification of conserved catalytic residues combined with heterologous expression and a kinetic study of Bg10 revealed that this enzyme is an alcohol-tolerant and glucose-stimulated ß-glucosidase with high lactase activity that has a great deal of potential as a biotechnological product in the dairy industry [22]. The principal residues for the pocket cavity within the active site of Bg10 (Fig 5D) were predicted on the basis of sequence alignment with previously identified ß-glucosidases, which were studied of their kinetic, mutational, and catalytic properties [3,60].
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