Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus: Fusion with the β-1,4-glycanase (cex) from the cellulolytic bacterium cellulomonas fimi yields a robust, catalytically active product

Authors: Duncan, Gilian¹; Tarling, Chris²; Bingle, Wade¹; Nomellini, John¹; Yamage, Mat¹; Dorocicz, Irene¹; Withers, Stephen²; Smit, John³

Source: Applied Biochemistry and Biotechnology, Volume 127, Number 2, November 2005 , pp. 95-110(16)

Publisher: Humana Press

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Abstract:

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the β-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-β-d-cellobioside with a K _M similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the K _cat value was significantly reduced (0.08 s⁻¹ for Cex/RsaA vs 15.8 s⁻¹ for Cex)^cat, likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.

Keywords: Caulobacter surface (S)-layer protein RsaA; type I secretion; cellulomonas β-1,4-glycanase; cellulase; Cex; cellobiohydrolase; crosslinked enzyme crystals; particulate enzymes; immobilized biocatalysts

Document Type: Research article

DOI: http://dx.doi.org/10.1385/ABAB:127:2:095

Affiliations: 1: Department of Microbiology and Immunology, University of British Columbia, V6T 1Z3, Vancouver, British Columbia, Canada, 2: Department of Chemistry, University of British Columbia, V6T 1Z3, Vancouver, British Columbia, Canada, 3: Department of Microbiology and Immunology, University of British Columbia, V6T 1Z3, Vancouver, British Columbia, Canada, Email: jsmit@interchange.ubc.ca

Publication date: 2005-11-01

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus: Fusion with the β-1,4-glycanase (cex) from the cellulolytic bacterium cellulomonas fimi yields a robust, catalytically active product

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