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Applied and Environmental Microbiology, December 1998, p. 4774-4781, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB from Thermotoga neapolitanadagger

Jin-Duck Bok,Dagger Dinesh A. Yernool, and Douglas E. Eveleigh*

Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08901

Received 11 May 1998/Accepted 10 September 1998

Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) 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 (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km 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 Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki 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.

* Corresponding author. Mailing address: Department of Biochemistry and Microbiology, Cook College, Rutgers University, 76 Lipman Drive, New Brunswick, NJ 08901-8525. Phone: (732) 932-9763, ext. 328. Fax: (732) 932-8965. E-mail: Eveleigh{at}aesop.rutgers.edu.

dagger Paper no. D-01111-01-98 of the New Jersey Agricultural Experiment Station.

Dagger Present address: Department of Animal Science & Technology, College of Agriculture & Life Sciences, Seoul National University, Suweon 441-744, and Animal Resources Research Center, Kon-Kuk University, Seoul 133-701, Korea.

Applied and Environmental Microbiology, December 1998, p. 4774-4781, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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