|Abstract: ||海洋天然資源給予我們很大的能力讓我們利用，使人類更健康，過更好的生活。我們從海洋可獲得一些具活性的有益化合物，也給予這些化合物功能的改進。本研究檢驗由海藻來的醣類以作為具淺力的抗病毒製劑。為了強化它們的活性，海藻多醣被萃取出來，並降解成生物可利用性之低聚合度的醣類。將海藻多醣降解的工具是由海洋細菌來源的酵素。其中用來做本研究探討的細菌(Pseudomonas vesiculiaris MA103)對於洋菜酶合成最好的條件也同時被探討。然而，本論文一開始是基於利用商用標準品來建立最適當之利用海洋醣類的條件。 瓊脂(agarose)所製備出的粗產物混合物所含之新洋菜寡糖(NAOS, neoagaro-oligosaccharides)或洋菜寡糖(AOS, agaro-oligosaccharides)主要是藉由兩種高效能液相層析系統(HPLC):分子篩(SEC)和氨基管柱(NH2-HPLC)及串聯一台揮發光散射偵測器(ELSD)進行監測。兩種HPLC系統清楚的解析出個別的醣類產物；然而SEC管柱可以製備出醣類產物在毫克的範圍，相對於NH2-HPLC管柱只允許製備出產物在微克的範圍。 為了要精準的測量洋菜酶水解活性，本研究也利用SEC和ELSD串聯系統發展出一個新穎的方法；此方法又與傳統評估洋菜酶活性所使用之還原醣含量的方法(RSC)進行比較。結果確認出SEC-ELSD方法的精準性，然而RSC方法顯示出當酵素用量少時對洋菜酶活性有低估的情況，而酵素用量多時會導致洋菜酶活性高估情況。 其次，利用海洋菌(P. vesiculiaris MA103)的MA103海藻酶與海洋菌(Aeromonas salmonicida MAEF108)的MAEF108海藻酶作用在兩種海藻(Gracilaria sp. 和 Monostroma nitidum)，獲得到低聚合度之含硫酸多醣(low-DP sulfated saccharides)。低聚合度之含硫酸醣類的產出與洋菜酶活性的測量於前段所述之操作方法相同。由海藻產出之聚合度為6，24，30的含硫醣類藉由1H核磁共振光譜及ESIMS質譜確認其結構，分子量及純度；此部分探討顯示出前段所使用的商用標準品(瓊酯及β-洋菜酶)所建立的方法，可以成功的應用在從洋菜多醣經過特定活性之洋菜酶作用後所製備出的低聚合度含硫醣。 接著，製備出的低聚合度含硫醣用於測試對抗日本腦炎病毒(Japanese encephalitis virus ；JEV)的活性探討，並與未降解的海藻多醣作比較。在體外探討是使用MTT方法，由兩種藻類所製備出低聚合度含硫醣對於抗病毒活性都比未降解的洋菜多醣要低；然而ELISA實驗結果顯示出低聚合度含硫醣會和未降解的多醣結合在JEV的外鞘膜蛋白的強度相當。結果也顯示出低聚合度含硫醣比未降解的多醣對於JEV所感染的C3H/HeN小鼠相對於控制組有較高之存活率的效應(65-100% vs. 0-33%)。在體內抗病毒活性方面，藉由測量在小鼠的血清中的醣含量證實低聚合度含硫醣比未降解的多醣似乎較好吸收，此結果指出低聚合度含硫醣是進一步發展出具有前瞻性候選之抗病毒製劑。 海洋菌P. vesiculiaris MA103，培養於含有不同碳源的基質(modified marine broth; MMB)當中，用於海藻酶MA103的合成。不同的碳源包括半乳糖、聚合度為4-20的NAOS、瓊酯和洋菜。當細菌在含Gal-N8的基質中進行90秒培養，會合成主要蛋白質分子範圍在約85-145 kDa，然而當培養在含N16-20、瓊酯和洋菜的基質中，起初會合成之主要蛋白質分子範圍在約25 kDa和29 kDa，然後是45 kDa和85 kDa。有趣的是，從不同碳源MMB所培養之海洋菌產出MA103洋菜酶具有相似的水解能力；最終，所有的產出的洋菜酶總類會將洋菜降解成相當含量的N4，以及小部分的N6、N2及單體。本探討顯示在基質中的碳源可能對於P. vesiculiaris MA103之洋菜酶合成有影響，然而也觀測到他們作用在洋菜的水解能力的獨特差異性。|
Ocean natural resources give us huge capabilities to utilize them for our healthier and better life. From the ocean we gain compounds with beneficial activity, and also additional instruments for their improvement. In this study saccharides from marine algae were examined as potential antiviral agents. For enhancement of their activity, the extracted algae-polysaccharides were digested to low-degree-polymerization (low-DP) saccharides; these are better available by life form. Tools for the polysaccharide digestions were enzymes, from marine bacteria. The best conditions for enzyme synthesis by one of the bacteria using in this study, Pseudomonas vesiculiaris MA103, were also studied. However, at the beginning, methods, basing on commercial standards, for optimal utilization of marine resources were established. Neoagaro-oligosaccharides (NAOS) or agaro-oligosaccharides (AOS) crude product mixtures from agarose were prepared by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) or NH2-column chromatography (NH2-HPLC) and monitored by an evaporative light-scattering detector (ELSD). Both HPLC systems clearly resolved individual saccharide products. However, SEC column enable the production of the saccharide products in milligram scale, while NH2-HPLC column allowed preparation of the products only in microgram scale. For the precise measurement of agarase hydrolysis activity a novel method, also utilizing SEC coupled with ELSD, was developed. The SEC-ELSD method was then compared with reducing sugar content methods, using routinely for assessment of the agarase activity. Results confirmed the high precision of SEC-ELSD method, whereas, RSC methods showed the agarase activity was underestimated when small amount of enzyme was used, and high amount of enzyme resulting in overestimation of agarase activity. Subsequently, low-DP sulfated saccharides from two algae, Gracilaria sp. and Monostroma nitidum were obtained by their polysaccharides (PS) digestion using MA103-agarases from P. vesiculiaris MA103 and MAEF108-agarases from Aeromonas salmonicida MAEF108. The production of the low-DP sulfated saccharides as well as activity of the agarases was performed as in preceding sections. The sulfated saccharides from algae with DP 6, 24 and 30 were inspected by 1H NMR spectroscopy and ESIMS spectrometry confirming structure, molecular mass, and purity. This part of study showed the methods established on commercial available standards (agarose and β-agarase) were successfully applied for preparation of low-DP sulfated saccharides from algal PS by digestions of agarases with defined activity. Then, the low-DP sulfated saccharides were tested for anti-Japanese encephalitis virus (anti-JEV) activity. Their activities were compared with undigested algal-PS. During in vitro studies performed by MTT assay, low-DP sulfated saccharides from both algae have slightly lower antiviral activities than their undigested-PS. However, the ELISA experiment results showed the low-DP sulfated saccharides bind to the JEV envelope protein at least strongly as undigested-PS. Results also showed, the low-DP sulfated saccharides have a distinctly higher positive effect on survivability in JEV infected C3H/HeN mice in comparison to undigested-PS (65-100% vs. 0-33% higher than control). The in vivo antiviral activity seems to be connected with better absorption of low-DP sulfated saccharides than undigested-PS, what was proved by measurement of sugar content in mice blood plasma. Our results point out that the low-DP sulfated saccharides are promising candidates for further development as antiviral agents. P. vesiculiaris MA103 was utilized for synthesis of MA103-agarases, when cultivating in medium (modified marine broth; MMB) with different carbon sources. Galactose, NAOS with DP from 4 to 20, agarose and agar were utilized as the carbon sources. Bacteria culturing in media with Gal-N8 at 90 sec mainly synthesized enzymes with MW 85-145 kDa, whereas these cultivating in media with N16-20, agarose and agar, initially produced enzymes with MW 25 and 29 kDa, then enzymes with MW 45 and 85 kDa. Interestingly, MA103-agarases from bacteria cultivating in various MMBs with different carbon sources had similar cleavage ability. Finally, all of them digested agarose to significant amount of N4, as well as smaller portion of N6, N2 and monomers. This study showed that the carbon source in media might have influence on synthesis of agarases by P. vesiculiaris MA103; however any distinct difference of their cleavage abilities on agarose were observed.