Display of β-glucanase on the cell surface of Lactobacillus using collagen-binding protein
Date Issued
2010
Date
2010
Author(s)
Huang, Shu-Jung
Abstract
Application of enzymes as feed additives is common in the livestock industry, especially in poultry, to eliminate the antinutritional factors present in the diets of chickens. However, enzyme supplementation substantially increases the cost of feed and is used for only short-term solution in enhancing digestion of cereals. Since lactobacilli possess the mucosal surface-colonizing property and have the potential to express proteins at specific sites, an alternative and less expensive strategy might be designed to develop lactobacilli with the capacity to digest plant structural carbohydrates by inducing of heterologous genes encoding polysaccharide-degrading enzyme.
Thus, the aim of this study was to display the β-glucanase (Glu) from Fibrobacter succinogenes on the cell surface of Lactobacillus reuteri Pg4 via collagen-binding proteins (Cnb), an adhesin in L. reuteri and capable to bind collagen type I. The Glu was fused to the C-terminus of Cnb as a recombinant Cnb-Glu-His6 fusion protein. The analysis of β-glucanase activity revealed that the enzyme was successfully displayed on the cell surface of the Lactobacillus cell. In addition, the specific activity of the displayed Glu on the cell surface of Lactobacillus cells was much improved compared with the free form. Localization of the Cnb-Glu-His6 fusion protein on the cell surface was also confirmed by immunofluorescence microscopy and flow cytometric analysis. Finally, the results of probiotic characterization indicated that the properties of recombinant strain, such as acid tolerance, bile-salt tolerance, and adhesion capability, were similar to that of the parental strain L. reuteri Pg4.
In summary, I constructed a Lactobacillus strain that displayed β-glucanase, anchoring on the cell wall in its active form. The probiotic characteristics were not affected by the transgenic construction.
Subjects
cell surface display
Lactobacillus
β-glucanase
collagen-binding proteins
Type
thesis
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