This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, Y.-H. P. Articles by Lynd, L. R. Search for Related Content PubMed PubMed Citation Articles by Zhang, Y.-H. P. Articles by Lynd, L. R. Agricola Articles by Zhang, Y.-H. P. Articles by Lynd, L. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, March 2004, p. 1563-1569, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1563-1569.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Yi-Heng Percival Zhang¹ and Lee R. Lynd^1,2*

Thayer School of Engineering,¹ Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755²

Received 2 September 2003/ Accepted 2 December 2003

Rates of phosphorolytic cleavage of ß-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by 20-fold for both cellobiose and cellopentaose over a 10-fold range of ß-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of ß-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V_max values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K_m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

---

* Corresponding author. Mailing address: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755. Phone: (603) 646-2277. Fax: (603) 646-2231. E-mail: Lee.R.Lynd{at}dartmouth.edu.

---

Applied and Environmental Microbiology, March 2004, p. 1563-1569, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1563-1569.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Stevenson, D. M., Weimer, P. J. (2005). Expression of 17 Genes in Clostridium thermocellum ATCC 27405 during Fermentation of Cellulose or Cellobiose in Continuous Culture. Appl. Environ. Microbiol. 71: 4672-4678 [Abstract] [Full Text]  
• Zhang, Y.-H. P., Lynd, L. R. (2005). Cellulose utilization by Clostridium thermocellum: Bioenergetics and hydrolysis product assimilation. Proc. Natl. Acad. Sci. USA 102: 7321-7325 [Abstract] [Full Text]  
• Demain, A. L., Newcomb, M., Wu, J. H. D. (2005). Cellulase, Clostridia, and Ethanol. Microbiol. Mol. Biol. Rev. 69: 124-154 [Abstract] [Full Text]  
• Zhang, Y.-H. P., Lynd, L. R. (2005). Regulation of Cellulase Synthesis in Batch and Continuous Cultures of Clostridium thermocellum. J. Bacteriol. 187: 99-106 [Abstract] [Full Text]