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Poster Presentation 6-18
New Glycosyl Hydrolases from Acidothermus cellulolyticus
Shi-You Ding, William S. Adney, Stephen R. Decker, John O. Baker, Ed Jennings, Todd B. Vinzant, and Michael E. Himmel
National Bioenergy Center Biotechnology for Fuels and Chemicals Division National Renewable Energy Laboratory Golden, CO 80401
Acidothermus cellulolyticus was isolated from 55-60oC acidic water and mud samples collected in Yellowstone National Park in the early 1980s (Mohagheghi et al. Int. J. System. Bacteriol. 1986). Biochemical studies have shown that its purified enzymes are highly thermotolerant with maximal activities at temperatures of 75-83oC. One of them, endoglucanase Cel5A, has been cloned and expressed in E. coli and other hosts. A lambda clone isolated from a genomic library of A. cellulolyticus which was grown on biomass was chosen for sequencing by activity on Avicel plates. A 9-kb BamHI fragment from this clone was subcloned into pDR540 to generate a plasmid, which was first sequenced by a primer walking method and then subcloned into ipUC19 using restriction enzymes PstI and EcoRI. An inverse PCR technique was applied to continue sequencing the genomic DNA and the primer walking method was used to sequence the large PCR products. Sequence analysis revealed four additional ORFs downstream of the Cel5A gene. These genes all indicate glycosyl hydrolases with multidomain structure. Domains from GH families 5, 6, 12, 48, and 74 have been identified through sequence homology. The biochemical and kinetic properties of these enzymes will be discussed.
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