Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana
- Rutgers Univ., New Brunswick, NJ (United States)
Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA is optimally active at pH 6.0 at 95 C, while CelB has a broader optimal pH range (pH 6.0 to 6.6) at 106 C. Both enzymes are characterized by a high level of activity with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the V{sub max} values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the K{sub m} values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The K{sub i} values for CelA are 0.44 M for glucose and 1.5 mM for cellobiose; the K{sub i} values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106 C and 26 min at 110 C. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.
- Sponsoring Organization:
- National Renewable Energy Lab., Golden, CO (United States); USDOE, Washington, DC (United States)
- OSTI ID:
- 302303
- Journal Information:
- Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 12 Vol. 64; ISSN AEMIDF; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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