利用膠原吸附蛋白在乳酸菌細胞表面展現聚葡萄糖酶

Abstract

於飼料中添加β-聚葡萄糖酶，可以消除非澱粉多醣的抗營養作用，提高單胃動物的飼料效率。然而，酵素的添加不僅會增加飼料成本，且僅能短暫提高穀物的消化率。乳酸菌為安全且能定殖於腸黏膜，並可在宿主體內發揮許多生理功能，如改善腸胃道菌相、提高免疫能力、減少疾病發生等。微生物表面展現系統（cell surface display）目前已廣泛應用於活疫苗（live vaccines）、生物感測器（biosensor）、生物催化反應（biocatalytic reaction）等領域。 因此，本論文即利用 Lactobacillus reuteri 細胞表面上的膠原吸附蛋白（collagen-binding protein；Cnb）做為攜帶蛋白，將瘤胃細菌 Fibrobacter succinogenes 之 β-聚葡萄糖酶（Glu）接合於Cnb的C端，再由L. reuteri Pg4表達產生Cnb-Glu-His6融合蛋白，並評估L. reuteri Pg4轉形株之β-聚葡萄糖酶活性。經酵素擴散法及聚丙烯醯胺膠體電泳活性染色後發現，β-聚葡萄糖酶可固定於基因重組乳酸菌表面，且比活性顯著高於表達游離型β-聚葡萄糖酶之轉形株。透過間接免疫螢光染色及流式細胞儀分析，證實Cnb-Glu-His6融合蛋白可成功表達並錨定在乳酸菌表面。在益生菌的特性測試中，結果顯示L. reuteri Pg4轉形株與非轉形株具有相似的益生特性。 綜上所述，本研究成功利用乳酸菌細胞表面蛋白質Cnb，將瘤胃微生物來源的β-聚葡萄糖酶固定於L. reuteri Pg4表面，且基因重組乳酸菌的益生菌特性不受其細胞表面展現的重組蛋白所影響。

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.