Characterization of a glucose-tolerant β-1,4-glucosidase BglC from Cytophaga hutchinsonii
September 24-28, 2017
rapidly and sufficiently through a unique mechanism without cellulosome and free cellulases. It was speculated to degrade cellulose with cell-bound cellulases. In this study, a putative GH3 β-glucosidase, BglC, was functionally expressed in Escherichia coli JM109, and the recombinant protein was purified and characterized. BglC was identified as a β-glucosidase with wide substrate specificity. It could not only degrade cellobiose and p-nitrophenyl β-D-glucopyranoside (pNPG) but also degrade cellodextrins such as cellotriose, cellotetrase and cellopentose. BglC had similar specific activity with both cellobiose and pNPG as the substrates. When cellobiose was used as the substrate, certain amount of cellotriose, cellotetrase and cellopentose could be produced besides glucose showing that BglC also had transglycosylation activity. Glucose could inhibite its transglycosylation activity rather than increase it. Study on the biochemical properties of BglC showed that its optimum reaction temperature was 420C and its optimum pH was pH6.5. Mg2 +, Co 2+, Mn2 +, Fe3 + could increase BglC’s activity, while Ni + and Cu2 + decreased its activity. Moreover, BglA was also found to be highly tolerant to glucose as it retained 60 % activity when the concentration of glucose was 100 times higher than that of the substrate (Fig. 1), showing potential application in the bioenergy industry. Point mutations including D303A, E513A, W443A, I336M, D122A, M268A, E188A/R, K224A, R185A/V and S271A led to disappearance of BglC activity indicating these residues were essential for the hydrolytic activity of the enzyme. Point mutations in S82A and I83A increased the hydrolysis activity of BglC by 20% and 30%, repectively. Mutations in I83A, I336S and I336L led to reduce of tolerance to glucose obviously indicating residues I83 and I336 were important not only for the hydrolytic activity of the enzyme but also for its tolerance to glucose.
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Xuemei Lu and Yuxiang Zhang, "Characterization of a glucose-tolerant β-1,4-glucosidase BglC from Cytophaga hutchinsonii" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/enzyme_xxiv/39