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Journal of Bacteriology, March 2004, p. 1297-1303, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1297-1303.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Chitinase B of "Microbulbifer degradans" 2-40 Contains Two Catalytic Domains with Different Chitinolytic Activities

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742,¹ National Science Foundation, Division of Molecular and Cellular Biosciences, Arlington, Virginia 22230²

Received 25 September 2003/ Accepted 24 November 2003

Chitinase B of "Microbulbifer degradans" 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18_N) releases methylumbelliferone from 4'-methylumbelliferyl-N,N'-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18_C) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18_C releases methylumbelliferone from 4'-methylumbelliferyl-N,N',N"-triacetylchitotriose 2.7-fold faster than GH18_N and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37°C and from pH 7.2 to 8.0 and was not affected by Mg²⁺, Mn²⁺, Ca²⁺, K⁺, EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni²⁺, Sr²⁺, and Cu²⁺, while Hg²⁺ completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.

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