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Journal of Bacteriology, December 2002, p. 6859-6865, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6859-6865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of Two Noncellulosomal Subunits, ArfA and BgaA, from Clostridium cellulovorans That Cooperate with the Cellulosome in Plant Cell Wall Degradation

Akihiko Kosugi,1 Koichiro Murashima,2 and Roy H. Doi1*

Section of Molecular and Cellular Biology, University of California Davis, Davis, California 95616,1 Health Care Company Unit, Meiji Seika Kaisha, Ltd., Sakado-shi, Saitama 350-0289, Japan2

Received 22 July 2002/ Accepted 11 September 2002

Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the {alpha}-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with {alpha}-L-arabinofuranosidase or {alpha}-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to {alpha}-L-arabinofuranosidase and ß-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl {alpha}-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl {alpha}-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl {alpha}-L-arabinopyranoside but also p-nitrophenyl ß-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl {alpha}-L-arabinopyranoside and p-nitrophenyl ß-D-galactopyranoside were compared, the Km values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for {alpha}-L-arabinopyranose residues than for ß-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 ß-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with ß-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

* Corresponding author. Mailing address: Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Ave., Davis, CA 95616-8535. Phone: (530) 752-3191. Fax: (530) 752-3085. E-mail: rhdoi{at}ucdavis.edu.

Journal of Bacteriology, December 2002, p. 6859-6865, Vol. 184, No. 24
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.24.6859-6865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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