在枯草桿菌及地衣芽孢桿菌宿主表現及純化重組幾丁聚醣&;#37238;及特性探討

Abstract

枯草桿菌（Bacillos subtilis）與地衣芽孢桿菌（Bacillus licheniformis）皆為革&;#63775;氏陽性（gram-positive）、兼性厭氧（facultative anaerobe）之產孢桿菌，並且都經FDA（Food and Drug Administration；美國食品暨藥物管&;#63972;局）認可為GRAS（generally recognized as safe）級之宿主，而且具有分&;#63848;蛋白質之能&;#63882;，因此，作為蛋白質分&;#63848;宿主於酵素工業之運用上是十分具有發展潛&;#63882;的。幾丁質（chitin）經由去乙醯化而獲得幾丁聚醣（chitosan），而幾丁聚醣經由幾丁聚醣&;#37238;分解後可產生具有多種生&;#63972;活性之幾丁寡醣（chitooligosaccharide）。幾丁寡醣之生&;#63972;活性有抑制細菌和真菌生長、增強免疫力、具抗腫瘤活性以及在高等植物中誘導抗菌素的產生。 本實驗室先前已建立枯草桿菌與地衣芽孢桿菌之持續表現系統，利用此系統來表現源自於枯草桿菌之重組幾丁聚醣&;#37238;。於本研究中，我們建立了重組幾丁聚醣&;#37238;的純化條件以及酵素特性的確立，並且利用隨機突變的方式來改造重組幾丁聚醣&;#37238;。實驗中以枯草桿菌與地衣芽孢桿菌作為表現重組幾丁聚醣&;#37238;之宿主，兩轉形菌株皆於12小時達到蛋白質最佳表現量，而且地衣芽孢桿菌產量明顯高於枯草桿菌。酵素之特性分析方面，枯草桿菌與地衣芽孢桿菌重組幾丁聚醣&;#37238;之最適作用溫度，分別為45℃與40℃；熱穩定性Tm值分別為44℃與46℃；最適作用pH值則分別是5.0與5.5，而兩轉形菌株皆具有大範圍pH值耐受性。酵素動力學分析結果，枯草桿菌重組幾丁聚醣&;#37238;的Vmax值為1.578 umole/sec/mg；Km值為3.1×102 mg/ml；kcat/Km值為14.6×10-2 ml/mg/sec。地衣芽孢桿菌重組幾丁聚醣&;#37238;的Vmax值為19.27 umole/sec/mg；Km值為25.6×102 mg/ml；kcat/Km值為21.6×10-2 ml/mg/sec。金屬離子Mg2+、Mn2+、Ca2可提升活性，其中又以Mn2+最為明顯，而Fe3+則明顯抑制酵素活性。 本實驗利用error-prone PCR對未含有訊息胜&;#32957;基因之幾丁聚醣&;#37238;基因進行突變，獲得一突變轉形菌株EP70，基因序列611由guanine突變為adenine（G611A），蛋白質序列194由aspartic acid突變為asparagine（D194N）。

Bacillus subtilis and Bacillus licheniformis are gram-positive, facultative anaerobic endospore-forming bacteria. They have been classified as GRAS (generally recognized as safe) microorganisms by FDA, and have the capability to secret protein. These strains were therefore considered potent hosts to express and secret heterologous proteins. Chitosan is a product obtained by the deacetylation of chitin. Chitosanase hydrolysis of chitosan could be used for preparation of chitosan oligosaccharides which have variety of biological activities, such as inhibiting the growth of bacteria and fungi, acting as immunopotential effectors, exerting antitumor activity, and inducing the phytoalexin product in higher plants. A constitutive secretory expression system for B. subtilis and B. licheniformis has been established in our laboratory, and express recombinant chitosanase gene originated from B. subtilis DB104. In this study, the recombinant chitosanase secreted by B. subtilis and B.licheniformis purified and analyzed the characteristics. Both transformants expressed highest recombinant chitosanase at 12 hours of culture, and the higher recombinant chitosanase activities secreted by B. licheniformis than B. subtilis. The recombinant chitosanase were purified and the optimal temperature of purified recombinant chitosanase of B. subtilis and B. licheniformis were 45℃ and 40℃ respectively, and the thermostabilities were Tm 44℃ and 46℃. The optimal pH were 5.0 and 5.5 for B. subtilis and B. licheniformis respectively, and the pH stability of recombinant chitosanase were both stable in pH 2.0 to 8.5. The kinetics of recombinant chitosanase by B. subtilis were Vmax of 1.578 umole/sec/mg;Km of 3.1×102 mg/ml;kcat/Km of 14.6×10-2 ml/mg/sec, and recombinant chitosanase secreted by B. licheniformis were Vmax of 19.27 umole/sec/mg;Km of 25.6×102 mg/ml;kcat/Km of 21.6×10-2 ml/mg/sec. The enzyme activities were increased by Mg2+, Mn2+, Ca2+, especially Mn2+.. And Fe3+ significantly inhibited the activity. In this study, mutated recombinant chitosanase gene obtained by error-prone PCR and got a mutant enzyme EP70. The guanine 611 of wild type chitosanase gene was replaced by adenine (G611A), and the amino acid aspartic acid 194 of wild type chitosanase was replaced by asparagine (D194N).