可分解蘭花病毒之放線菌蛋白酶的生產與應用

Production and Application of an Orchid Virus Degrading Actinomycete Secreting Proteases

陳宏榮(Hong-Rong Chen)

指導教授 ： 張清安

朝陽科技大學/理工學院/生物技術與化學工程研究所碩士班/碩士(2009年)

摘要

蘭花產業為我國觀賞花卉最具產值者，其中蝴蝶蘭更被農委會選為四大外銷旗艦作物之一。近年來我國蝴蝶蘭外銷之品目逐漸轉以種苗為主，因此病毒之感染與否即成為界定種苗品質優劣標準之一。蘭花病毒中，齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV) 與蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 是感染蘭花之近五十九種病毒中分佈最普遍影響最嚴重的二種。在田間此二病毒並不藉由媒介昆蟲傳播，而是以污染栽培環境與工作器具，再經由傷口入侵而造成感染。本研究乃針對過去本實驗室所篩選具有分解蘭花病毒鞘蛋白能力的放線菌菌株 (CA5) 之培養流程及實際應用方式進行研究，期能提升 CA5 所分泌鞘蛋白分解酵素之生產效率，並能掌握 CA5 鞘蛋白分解酵素之有效應用方式，以降低此二種病毒在生產健康蘭花種苗的過程中之危害。
試驗中首先針對培養之三角瓶結構進行研究，比較一般與二種具溝槽或檔板設計之三角瓶對培養 CA5 菌數量及所生產酵素活性之影響，結果發現以具有溝槽之三角瓶由於可以在震盪培養時產生較高的溶氧量，因而可在較短的時間內獲得較高之菌量與酵素相對活性 (relative activity unit, RU)。此外本研究特別針對大量培養前之接種源之培養方式進行探討，確定將菌種先於大豆粉液態培養基於 32 ℃ 下培養至第 5 天可作為最佳之接種源。將其以 1 % (v/v) 比例接種於相同培養基中，於30 ℃ 與150 rpm震盪下培養 6 天所獲得之濾液，即可達到4149 RU/ml之最高分解蘭花病毒鞘蛋白之相對活性，顯著高於過去以菌絲孢子懸浮液做為接種源，於一般三角瓶內培養14天所獲之3029 RU/ml相對活性。此一培養模式之確立估計可使 CA5 酵素之總相對活性比過去所建立之培養程序提升 4 倍，而時程可以縮短 3 天。
利用CA5 培養濾液再經 60 % 飽和硫酸銨濃縮沉澱之酵素材料 (CA5P) 進行實際應用方式與效果之評估。測試結果發現以CA5酵素處理確定污染ORSV或CymMV病毒之蘭花種子超過一分鐘後，即無法再以 ELISA 或 RT-PCR 偵測到病毒存在。但以相同方式處理含有蘭花絨毛組織之種子時，則仍可以偵測到病毒訊號，顯示蘭花種子僅外表上污染病毒，因此可以藉由CA5鞘蛋白分解酵素加以清除。另以酵素材料處理污染 ORSV 或 CymMV 之固體表面試驗中，亦確定稀釋4 倍之酵素濃縮液處理30秒後，已明顯無法利用 ELISA 再偵測到病毒的殘留，且於接種試驗中亦確定已完全摧毀此二病毒之感染能力。若改以稀釋 8 及16 倍之酵素濃縮液但延長處理時間，發現延長時間助益不大，此證明 30秒內即可達到主要分解病毒之效果。對於蘭園經常使用之可能造成病毒污染與傳播的材料用具，以固體表面試驗之方法實際以 CA5 酵素材料處理 30 秒，不論材質如刀片、魔繩、塑膠夾、塑膠名牌、手套及蘭花組培苗，甚至指甲，其表面汙染之二種蘭花病毒也都能被分解，且利用 ELISA 已偵測不到有病毒訊號。
本研究亦測試CA5 培養濾液與酵素濃縮液之保存方式，將其冷凍保存於 - 20 ℃ 下，每隔 30 天取出回溫，取樣測試其分解蘭花病毒鞘蛋白之活性變化，歷經150 天發現 CA5 培養濾液經反覆冰凍和解凍，仍可維持原保存前之 83 % 之相對活性 (RU)。相較於保存在 4 ℃ 或 25 ℃ 之兩種方式，分別僅剩餘 53 % 及 6 % 之相對活性，可見 CA5 酵素活性之保存應以冷凍方式較佳。
總之，針對 CA5 菌株之培養與病毒鞘蛋白分解酵素之生產，本研究已經建立一個較過去培養方式更為有效與高產之培養模式。並且證實應用所生產之酵素濃縮液可以在 30 秒的處理時間下，可以將污染於各種蘭園中常見之工具材料表面上之蘭花病毒，清除到無法以接種方式偵測到病毒感染力及以ELISA無法偵測到病毒訊號之程度。另外也證實可以應用此酵素濃縮液在一分鐘內完全清除污染於蘭花種子表面之 ORSV 與 CymMV。

關鍵字

放線菌 ； 蛋白分解酵素 ； 蘭花

並列摘要

Among the various ornamentals produced in Taiwan, orchids are the one with the highest annual output value. Phalaenopsis orchids, especially, are selected by the Council of Agriculture as one of the four major representative agricultural products in Taiwan. In recent years, tissue culture plantlets have become the major exportation item for Taiwan’s Phalaenopsis industry. Therefore, the cleanness of the plantlets free from virus infection has become one of the major concerns for quality assessment of the exporting Phalaenopsis plantlets. At present, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) are recognized as the most widely spread and economically important viral agents to orchid industry among the 30 known viruses infecting orchids. These two viruses are not disseminated by any known insect vectors, but they can be transmitted easily through mechanical wounds on the host plants by tools contaminated with virus particles. In the previous studies, our laboratory has successfully screened and obtained a pure culture of an isolate of actinomycetes, namely CA5, which can secrete a protease-like substance in its culture filtrate that can degrade coat proteins of ORSV and CymMV and abolish their infectivities. This research attempts to establish a standard cultivation protocol that can produce higher yield of CA5 protease and also to test the feasible application scheme for this novel protease to sanitize the tools and growing environment of orchids.
In the first part of this research, we tested the cultivation effects on the total cell output and the relative activity unit (RU) of CA5 protease activity by conventional flasks comparing with two special designed flasks that could stimulate higher oxygen input into the culture media. Of the three types of flasks tested, the baffled shake flask (BSF) showed the highest cell output and RU value within a shorter period of cultivation. Using BSF as cultivation flasks, we further found that a best CA5 inoculum for subsequent large scaled production could be prepared by culturing CA5 in soybean meal liquid media (SMLM) at 32 C, 150 rpm, for 5 days. With this standardized inoculum, we were able to establish an optimum standard cultivation protocol for CA5 and its secreted protease by starting with the addition of 2 ml ( 1 % (v/v) ) inoculum into a BSF containing 200 ml of SMLM (pH 8.0) and cultured at 30 C, 150 rpm, for 6 days. The greatest cell output and RU of CA5 protease activity could be obtained from this 6 days culture filtrate. With this new protocol, about 5.2 times increase of the RU value of CA5 protease activity was harvested from the culture filtrate comparing to that obtained from our previous protocol. To test the application scheme of CA5 protease, we generally prepared the materials, subsequently named as CA5P, by concentrating the CA5 culture filtrate with 60 % of ammonium sulfate and followed by dialyzing CA5P overnight. Treating orchid seeds contaminated by ORSV or CymMV with 8x H2O-diluted CA5P for 1 min, it was found that virus signals could not be detected from the treated seeds by ELISA or RT-PCR. In another treatment with virus-contaminated orchid seeds and fiber tissues, virus signals could still be detected by RT-PCR after treating similarly with CA5P. This result indicated that ORSV and CymMV were only contaminated on the surface of orchid seeds instead of residing inside the cells while the fiber tissues were actually infected by viruses which were not accessible by CA5P. In another experiment, 4x H2O-diluted CA5P was used to treat 30 seconds on the materials commonly used in orchid nurseries including razor blade, scissors, plastic clip, plastic labels, plastic gloves, orchid plantlets and nail pieces coating previously with ORSV and CymMV particles and the result confirmed that no virus signals could be detected by ELISA. In the last part of our study, we tested the effect of storage temperature on the maintenance of the relative activities of CA5 protease. Three different storage temperature (-20, 4, and 25 C) were tested to store the CA5P for 180 days and every one month the storage materials were allowed to revert to room temperature and part of samples were taken to check their RU of protease activity. The result showed that storing CA5P at -20 C was the best treatment that could preserve at least 83% of RU value comparing with the original one before storage. Instead, preserving at 4 and 25 C could only maintain 53 and 6 % of the original RU value, respectively.
Altogether, we have succeeded in establishing an optimum cultivation protocol for the production of CA5 protease. It takes only 11 days to reach a production peak of CA5 protease comparing to our old protocol taking at least 14 days to reach its maximum. Furthermore, we are confident that CA5P could be practically applied in routine orchid cultivation for sanitizing the growing environment to free from possible viral contamination. In addition, CA5P can also be used to disinfect orchid seeds to free from possible ORSV and CymMV contamination before seeding in the cultivation medium.

並列關鍵字

Actinomycete ； Protease ； Orchid

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