Backgroundβ-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant β-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications.ResultsThe β-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of β-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel β-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2–7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (Km value of 0.62 mM) and cellobiose (Km value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat for cellobiose was 67.7 s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290 mM. It was activated by glucose at concentrations lower that 200 mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600 mM glucose.ConclusionsThe article provides a useful novel β-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

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Isoorientin is a C-glycosylated derivative of luteolin and exhibits a number of biological properties. In this study, multiple strategies were adopted to improve isoorientin production from luteolin in Escherichia coli. Isoorientin production was improved substantially by adjusting induction strategies and controlling acetic acid accumulation, with maximum isoorientin production reaching 826 mg/L. Additionally, a novel UDP-glucose synthesis pathway was reconstructed in E. coli through cellobiose phosphorylase-catalyzed phosphorolysis of cellobiose for the production of glucose 1-phosphate, which serves as a precursor in UDP-glucose formation. The results from two mechanisms of UDP-glucose formation in E. coli, cellobiose phosphorolysis and sucrose phosphorolysis, were compared. Increasing the UDP-glucose supply resulted in maximal isoorientin production reaching 1371 mg/L. Finally, isoorientin (1059 mg) was obtained from 1 L of fermentation broth by simple purification steps with a yield of 81.5%. Therefore, this study provides an efficient method for isoorientin production and a novel UDP-glucose synthesis pathway.