microbiology, Vol 144, 457-465, Copyright © 1998 by Society for General Microbiology

ARTICLES

Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile 'Anaerocellum thermophilum' with separate glycosyl hydrolase family 9 and 48 catalytic domains

V Zverlov, S Mahr, K Riedel and K Bronnenmeier
Institute of Molecular Genetics, Russian Academy of Science, Moscow, Russia.

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum'. Full- length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and beta-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 degrees C (CM- cellulase) and 85 degrees C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of 'A. thermophilum' by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from 'A. thermophilum'. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

This article has been cited by other articles:

				V. V. Zverlov, M. Klupp, J. Krauss, and W. H. Schwarz Mutations in the Scaffoldin Gene, cipA, of Clostridium thermocellum with Impaired Cellulosome Formation and Cellulose Hydrolysis: Insertions of a New Transposable Element, IS1447, and Implications for Cellulase Synergism on Crystalline Cellulose J. Bacteriol., June 15, 2008; 190(12): 4321 - 4327. [Abstract] [Full Text] [PDF]

				F. Mingardon, A. Chanal, C. Tardif, E. A. Bayer, and H.-P. Fierobe Exploration of New Geometries in Cellulosome-Like Chimeras Appl. Envir. Microbiol., November 15, 2007; 73(22): 7138 - 7149. [Abstract] [Full Text] [PDF]

				M. B. Howard, N. A. Ekborg, L. E. Taylor II, R. M. Weiner, and S. W. Hutcheson Chitinase B of "Microbulbifer degradans" 2-40 Contains Two Catalytic Domains with Different Chitinolytic Activities J. Bacteriol., March 1, 2004; 186(5): 1297 - 1303. [Abstract] [Full Text] [PDF]

				K.-P. Fuchs, V. V. Zverlov, G. A. Velikodvorskaya, F. Lottspeich, and W. H. Schwarz Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-{beta}-1,3-glucanase bound to the outer cell surface Microbiology, April 1, 2003; 149(4): 1021 - 1031. [Abstract] [Full Text] [PDF]

				T. Tanaka, S. Fujiwara, S. Nishikori, T. Fukui, M. Takagi, and T. Imanaka A Unique Chitinase with Dual Active Sites and Triple Substrate Binding Sites from the Hyperthermophilic Archaeon Pyrococcus kodakaraensis KOD1 Appl. Envir. Microbiol., December 1, 1999; 65(12): 5338 - 5344. [Abstract] [Full Text]

				I. K. O. Cann, S. Kocherginskaya, M. R. King, B. A. White, and R. I. Mackie Molecular Cloning, Sequencing, and Expression of a Novel Multidomain Mannanase Gene from Thermoanaerobacterium polysaccharolyticum J. Bacteriol., March 1, 1999; 181(5): 1643 - 1651. [Abstract] [Full Text]