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Journal of Bacteriology, March 1999, p. 1643-1651, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Molecular Cloning, Sequencing, and Expression of a Novel Multidomain Mannanase Gene from Thermoanaerobacterium polysaccharolyticum

Isaac K. O. Cann,dagger Svetlana Kocherginskaya, Michael R. King, Bryan A. White, and Roderick I. Mackie*

Department of Animal Sciences, University of Illinois at Urbana-Champaign Urbana, IL 61801, USA

Received 21 August 1998/Accepted 16 December 1998

The manA gene of Thermoanaerobacterium polysaccharolyticum was cloned in Escherichia coli. The open reading frame of manA is composed of 3,291 bases and codes for a preprotein of 1,097 amino acids with an estimated molecular mass of 119,627 Da. The start codon is preceded by a strong putative ribosome binding site (TAAGGCGGTG) and a putative -35 (TTCGC) and -10 (TAAAAT) promoter sequence. The ManA of T. polysaccharolyticum is a modular protein. Sequence comparison and biochemical analyses demonstrate the presence of an N-terminal leader peptide, and three other domains in the following order: a putative mannanase-cellulase catalytic domain, cellulose binding domains 1 (CBD1) and CBD2, and a surface-layer-like protein region (SLH-1, SLH-2, and SLH-3). The CBD domains show no sequence homology to any cellulose binding domain yet reported, hence suggesting a novel CBD. The duplicated CBDs, which lack a disulfide bridge, exhibit 69% identity, and their deletion resulted in both failure to bind to cellulose and an apparent loss of carboxymethyl cellulase and mannanase activities. At the C-terminal region of the gene are three repeats of 59, 67, and 56 amino acids which are homologous to conserved sequences found in the S-layer-associated regions within the xylanases and cellulases of thermophilic members of the Bacillus-Clostridium cluster. The ManA of T. polysaccharolyticum, besides being an extremely active enzyme, is the only mannanase gene cloned which shows this domain structure.

* Corresponding author. Mailing address: University of Illinois at Urbana-Champaign, 458 Animal Sciences Laboratory, 1207 W. Gregory Dr., Urbana, IL 61801. Phone: (217) 244-2526. Fax: (217) 333-8809. E-mail: r-mackie{at}staff.uiuc.edu.

dagger Present address: Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874 Japan.

Journal of Bacteriology, March 1999, p. 1643-1651, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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