Characterization of a Novel Metagenome-Derived 6-Phospho-β-Glucosidase from Black Liquor Sediment

Abstract

The enzyme 6-phospho-␤-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenomederived gene sequence, encoding a novel 6-phospho-␤-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37°C and pH 7; Ca 2؉ , Mg 2؉ , and Mn 2؉ stabilized the activity of Pbgl25-217, whereas Ni 2؉ , Fe 2؉ , Zn 2؉ , Cu 2؉ , and Fe 3؉ inhibited its activity. The K m and V max of Pbgl25-217 were 4.8 mM and 1,987.0 U mg ؊1 , respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as "gatekeepers" in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-␤-glycosidases in GH1 and be useful for designing further directed evolution.

Potential Correlation between Dietary Fiber-Suppressed Microbial Conversion of Choline to Trimethylamine and Formation of Methylglyoxal

“…The gut bacterial genomes from red meat and low fiber diet-fed animals showed higher sequence identity with the gene encoding 6-phospho-β-glucosidase (EC3.2.1.86), which catalyzes the conversion of 6-phospho-β-D-glucosyl-(1,4)-Dglucose to D-glucose and D-glucose 6-phosphate; the latter can be metabolized by microbiota and used for glycolysis. 44 This was in agreement with the results of the KEGG analysis.…”

“…These carbohydrate enzymes in CAZy families assigned to the fiber-rich diet groups revealed considerable levels of similarity with each other, which may be because the animals were fed with the same soluble fiber sources. The gut bacterial genomes from red meat and low fiber diet-fed animals showed higher sequence identity with the gene encoding 6-phospho-β-glucosidase (EC3.2.1.86), which catalyzes the conversion of 6-phospho-β- d -glucosyl-(1,4)- d -glucose to d -glucose and d -glucose 6-phosphate; the latter can be metabolized by microbiota and used for glycolysis . This was in agreement with the results of the KEGG analysis.…”

Transcriptomic analysis of stress response to novel antimicrobial coatings in a clinical MRSA strain

“…These enzymes hydrolyse β-glycosides in various carbohydrates. β-D-glucosidase hydrolyses cellulose with the help of the phosphotransferase system by converting cellobiose to glucose [30]. Differential expression of these genes is presented below in Table 1.…”

Beta-Glucosidase: Key Enzyme in Determining Efficiency of Cellulase and Biomass Hydrolysis

“…After the advancement of third generation sequencing, the discovery of complete genes from metagenomic assemblies is still limited [34]. Congregated mining of the genetic information of β-glucosidase was explored across different environments including hydrothermal spring [35], wetlands [36], sludge [37], pulp wastewater [38], gut and rumen [39][40][41], anaerobic digesters [42] and compost [43].…”