Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus: fusion with the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi yields a robust, catalytically active product.
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 beta-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-beta-D-cellobioside with a KM 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 kcat value was significantly reduced (0.08 s-1 for Cex/RsaA vs 15.8 s-1 for Cex), 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.
Duncan
Gillian
G
Departments of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
Tarling
Chris A
CA
Bingle
Wade H
WH
Nomellini
John F
JF
Yamage
Mat
M
Dorocicz
Irene R
IR
Withers
Stephen G
SG
Smit
John
J
D001426:Bacterial Proteins / Q000737:chemistry*; D016935:Caulobacter crescentus / Q000201:enzymology*; D040143:Cellulomonas / Q000201:enzymology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D006026:Glycoside Hydrolases; C020668:glycanase