Secretion and cell surface display of chitosanase from Bacillus subtilis DB104 in Bacillus licheniformis and Bacillus subtilis

Abstract

地衣芽孢桿菌(Bacillus licheniformis)和枯草桿菌(Bacillus subtilis)為革蘭氏陽性（gram-positive）、兼性厭氧（facultative anaerobe）之產孢桿菌。廣泛應用於工業生產蛋白酶，並經FDA（美國食品暨藥物管理局）認可為GRAS（generally recognized as safe）級之宿主。由於地衣芽孢桿菌及枯草桿菌具有分泌蛋白質之能力，使其成為工業上具發展潛力之蛋白質分泌宿主。幾丁聚醣為幾丁質經由乙醯化而獲得之產物。使用幾丁聚醣酶來分解幾丁聚醣，可產生具有多種生理活性之幾丁寡醣。幾丁寡醣之生理活性有抑制細菌和真菌生長、具抗腫瘤活性以及在高等植物中誘導抗菌素產生。 在先前研究過程中，本實驗室已建立地衣芽孢桿菌及枯草桿菌之持續表現系統。於本研究中，我們建立了地衣芽孢桿菌和枯草桿菌之分泌及細胞表層展示系統來表現枯草桿菌之幾丁聚醣酶基因。 實驗中使用了3個不同表現元件，包括ECSveg、SECS-3、SEC-6-lacO，於地衣芽孢桿菌及枯草桿菌中進行測試，結果顯示此3個表現元件均可被地衣芽孢桿菌及枯草桿菌之RNA polymerase所辨識，而表現元件SECS-6-lacO的表現效率高於ECSveg及SECS-3。本論文建立了分泌表現載體pSL-CSPCSN及pSL-YSPCSN，經轉形於地衣芽孢桿菌和枯草桿菌中後，可順利表現幾丁聚醣酶。在活性方面，地衣芽孢桿菌和枯草桿菌中帶有幾丁聚醣酶訊息胜肽之表現載體pSL-CSPCSN所表現之重組幾丁聚醣酶酵素活性高於帶有鹼性彈性蛋白酶訊息胜肽之載體pSL-YSPCSN。實驗中亦建立了地衣芽孢桿菌誘導表現系統，其含有可表現lacI基因之載體pBL1，於此誘導表現系統中可誘導表現含SECS-6-lacO表現元件之載體。本實驗建構了細胞表層展示載體pSLB-CSPCSN及pVEGB-CSPCSN，pSLB-CSPCSN上帶有SECS-6-lacO表現元件，可順利轉形於地衣芽孢桿菌誘導表現系統B. licheniformis (pBL1)及枯草桿菌誘導表現系統B. subtilis (pBL1)中，經誘導可表現細胞表層展示蛋白質融合之重組幾丁聚醣酶；而pVEGB-CSPCSN載體則無法順利轉形成功。在地衣芽孢桿菌及枯草桿菌誘導表現系統中，可藉由蛋白質電泳及西方墨點法來偵測融合有細胞表層展示蛋白質之重組幾丁聚醣酶的表現。以1 mM及0.5 mM之IPTG來比較不同誘導濃度對其表現量之影響，結果並無明顯之差異。而細胞表層蛋白表現後所位於細胞之部位可藉由蛋白質電泳及西方墨點法來偵測，結果發現經表現後，細胞表層蛋白可鍵結於細胞壁上，經由全菌體活性測試後可發現其具有酵素活性。

Bacillus licheniformis and Bacillus subtilis are gram-positive, facultative anaerobic endospore-forming bacteria. They have been used for industrial production of several proteases and are classified as a GRAS (generally recognized as safe) microorganisms by FDA. Some properties of B. licheniformis and B. subtilis such as the capability to secret protein have led these strains to be attractive hosts for secretory production of heterologous proteins. Chitosan is obtained by the deacetylation of chitin. Enzymatic hydrolysis of chitosan by using chitosanase could be used for preparation of chitosan oligosaccharides which have received growing attention because they perform a variety of biological activities, such as inhibiting the growth of bacteria and fungi, exerting antitumor activity, acting as immunopotential effectors, and inducing the phytoalexin product in higher plants. A constitutive expression system for B. licheniformis and B. subtilis has been established in our laboratory. In this study, we have developed secretory and cell surface display system for expression of chitosanase gene from B. subtilis in B. licheniformis and B. subtilis. Three expression control sequences including ECSveg, SECS-3, SECS-6-lacO were used as expression signals for B. licheniformis and B. subtilis. The results showed that all three expression signals could be recognized by B. licheniformis and B. subtilis RNA polymerase. The expression efficiency of SECS-6-lacO was higher than that of ECSveg and SECS-3. The secretory expression vectors including pSL-CSPCSN and pSL-YSPCSN were constructed and transformed into B. licheniformis and B. subtilis and could be expressed recombinant chitosanase. The activity of recombinant chitosanase expressed by pSL-CSPCSN is higher than that expressed by pSL-YSPCSN in B. licheniformis and B. subtilis. An inducible expression system based on SECS-6-lacO and lacI-containing plasmid pBL1 was established for B. subtilis in previous research and for B. licheniformis in this study. The cell surface display expression plasmids including pSLB-CSPCSN and pVEGB-CSPCSN were constructed and plasmid pSLB-CSPCSN, but not pVEGB-CSPCSN, could be transformed into B. licheniformis (pBL1) and B. subtilis (pBL1). Expression of cell wall binding protein fused chitosanase in B. licheniformis (pBL1) and B. subtilis (pBL1) was detected by SDS-PAGE and western blot. The expression level of different induction concentration by 1 mM and 0.5 mM IPTG is similar. The location of cell wall binding protein fused chitosanase in the cell fractions was detected by SDS-PAGE and western blot. The result showed that the cell wall binding protein fused chitosanase could be displayed on the cell wall, and the enzyme activity can be detected by whole cell activity test.