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Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars

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Abstract

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.

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Acknowledgements

Author (YHPZ) thanks the Biological Systems Engineering Department at Virginia Polytechnic Institute and State University. This work was also supported partially by grants DE-FG02-02ER15350 (to LRL and YHPZ) from the Department of Energy and 60NANB1D0064 (to LRL) from the National Institute of Standards and Technology.

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Authors and Affiliations

  1. Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, 210-A Seitz Hall (0303), Blacksburg, VA, 24060, USA

    Y. -H. P. Zhang

  2. Thayer School of Engineering, 8000 Cummings Hall, Dartmouth College, Hanover, NH, 03755, USA

    L. R. Lynd

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  1. Y. -H. P. Zhang
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  2. L. R. Lynd
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Correspondence to Y. -H. P. Zhang.

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Zhang, Y.H.P., Lynd, L.R. Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars. Appl Microbiol Biotechnol 70, 123–129 (2006). https://doi.org/10.1007/s00253-005-0278-1

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  • DOI: https://doi.org/10.1007/s00253-005-0278-1

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