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研究生:卡伯特
論文名稱:低聚合度洋菜醣的分離,鑑定和應用以及洋菜多醣降解的機制探討
論文名稱(外文):Studies on the Isolation, Identification and Applications of Low-Degree-Polymerization Algal Saccharides and Mechanism of Agaro-Polysaccharide Degradations.
指導教授:潘崇良 博士吳彰哲 博士柯源悌 博士
指導教授(外文):Chorng-Liang Pan, Ph.D.Chang-Jer Wu, Ph.D.Yuan-Tih Ko, Ph.D.
學位類別:博士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:192
中文關鍵詞:低聚合度洋菜醣海藻多醣海洋菌P. vesiculiaris MA103
外文關鍵詞:Low-Degree-Polymerization Algal SaccharidesAgaro-Polysaccharide DegradationJapanese Encephalitis VirusGracilaria sp.Monostroma nitidumPseudomonas vesicularis MA103Aeromonas salmonicida MAEF108
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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.

Contents

Contents……………………………………………………………………………
Table list……………………………………………………………………………
Figure list…………………………………………………………………………..
Appendix list……………………………………………………………………….
Preface……………………………………………………………………………...
Organization of the dissertation………………………………………………….
Chinese abstract…………………………………………………………………...
Abstract…………………………………………………………………………….

Chapter 1
Background………………………………………………………………………..
1.1. Seaweed and seaweed polysaccharide functional properties……..........
1.2. Marine polysaccharides………………………………………………...
1.2.1 Agarose……………………………………………………………..
1.2.2 Agar…………………………………………………………………
1.2.3 Carragenan……………………………………………….................
1.2.4 Heparinoids………………………………………………………....
1.3. Agarolytic bacteria - sources of agarases…………………………….…
1.4. Agarases……………………………………………………………..….
1.4.1 α-Agarase………………………………………………...................
1.4.2 β-Agarase…………………………………………………...............
1.4.3 Neoagarobiose hydrolasae………………………………………….
1.5. Systems used for the preparation and quantification of low-DP saccharides……………………………………………………………...
1.5.1 Polymer amino phase HPLC (NH2-HPLC)………………………...
1.5.2 HPSEC……………………………………………………………...
1.6 System used for the monitoring of low-DP saccharides separation………
1.6.1 ELSD………………………………………………………………..
1.7 Biological activities of saccharides derived from phyla Rodophyta and Chlorophyta………………………………………………………………
1.8 Japanese encephalitis virus………………………………………………

Chapter 2
Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by -agarase and HCl in liquid chromatography systems………………………………………………………….
Abstract…………………………………………………………………………
2.1 Introduction…………………………...…………………………………
2.2 Experimental……………………………………………………………..
2.2.1 Chemicals and reagents……………………………………………..
2.2.2 Preparation of NAOS products……………………………………..
2.2.3 Preparation of AOS products……………………………………….
2.2.4 Product detection by HPSEC-ELSD and NH2-HPLC-ELSD systems……………………………………………………………...
2.2.5 Purification and isolation of the NAOS and AOS product series by HPSEC and NH2-HPLC……………………………………………
2.2.6 1H nuclear magnetic resonance spectroscopy (1H NMR)…………..
2.2.7 Electrospray-ionization mass spectrometry (ESIMS)………………
2.2.8 Data analysis………………………………………………………..

2.3 Results……………………………………………………………………
2.3.1 Establishing the calibrations of NAOS and AOS in two HPLC systems……………………………………………………………...
2.3.2 Optimization and determining the DP of the NAOS products from β-agarase digestion…………………………………………………
2.3.3 Preparation of NAOS with defined DP……………………………..
2.3.4 Optimization and determining the DP of the AOS products from HCl hydrolysis……………………………………………………...
2.3.5 Preparation of AOS with defined DP……………………………….
2.3.6 Structure, molecular mass, and purity confirmation of AOS and NAOS……………………………………………………………….
2.4 Discussion………………………………………………………………..
2.5 Table….………………………………………………………………….
2.6 Figures……………………………………………………………………

Chapter 3
Evaluation of HPSEC-ELSD method for precise measurement of β-agarase activity……………………………………………………………………………...
Abstract…………………………………………………………………………
3.1 Introduction………………………………………………………………
3.2 Experimental……………………………………………………………..
3.2.1 Chemicals and reagents…………………………………………..
3.2.2 Preparation of NAOS product-mixtures……………………………
3.2.3 Miscellaneous reducing sugar content methods……………………
3.2.4 Product detection by HPSEC-RI and HPSEC-ELSD systems……..
3.2.5 Isolation of the NAOS product-mixture……………………………
3.2.6 Kinetic characterization of β-agarase……………………………….
3.2.7 Data analysis and evaluation for accuracy and precision…………..
3.3 Results……………………………………………………………………
3.3.1 Establishing the calibrations of the amount of NAOS by reducing sugar content methods and HPSEC-based RI and ELSD detection system methods……………………………………………………..
3.3.2 Detection limits of standards and optimization of chromatography solvent………………………………………………………………
3.3.3 Reducing sugar content measurements of NAOS product-mixture from β-agarase digestion……………………………………………
3.3.4 HPSEC-RI monitoring of NAOS product-mixture from β-agarase digestion…………………………………………………………….
3.3.5 HPSEC-ELSD monitoring of NAOS product-mixture from β-agarase digestion…………………………………………………
3.3.6 Comparison of the amount of released NAOS product-mixtures calculated from different assay methods with the true recovered amount of the isolated forms………………………………………..
3.4 Discussion………………………………………………………………..
3.5 Figures……………………………………………………………………

Chapter 4
Separation, quantification and identification of low-degree- polymerization Gracilaria- and Monostroma-saccharide products generated from their polysaccharide digestion by MA103-agarases from Pseudomonas vesiculiaris and MAEF108-agarases from Aeromonas salmonicida in size exclusion chromatography system…………………………………………………………..
Abstract…………………………………………………………………………
4.1 Introduction………………………………………………………………
4.2 Experimental……………………………………………………………..
4.2.1 Chemicals and reagents…………………………………………….
4.2.2 Preparation of MA103-crude agarase enzymes solution…………...
4.2.3 Preparation of MAEF108 crude-agarase enzymes solution………...
4.2.4 Concentration of crude enzyme solutions…………………………..
4.2.5 Agarase activity measurement……………………………………...
4.2.6 Extraction of algal polysaccharides………………………………......
4.2.7 Preparation of Gra- and Mon-crude product mixtures……………..
4.2.8 Product detection of Gra- and Mon- product series with DP 6,
DP 24 and DP 30……………………………..……………………..
4.2.9 Purification and isolation of the Gra- and Mon- product series by HPSEC……………………………………………………………...
4.2.10 1H NMR and ESIMS………………………………………………..
4.2.11 Sulfate content determination………………………………………
4.2.12 Polyphenols content determination…………………………………
4.2.13 Protein determination……………………………………………….
4.3 Results……………………………………………………………………
4.3.1 Determining the DP of the Gra-saccharide products from agarases digestion…………………………………………………………….
4.3.2 Preparation of Gra-saccharide products with defined DP………….
4.3.3 Determining the DP of the Mon-saccharide products from agarases digestion…………………………………………………………….
4.3.4 Preparation of Mon-saccharide products with different DP………..

4.3.5 Structure, molecular mass, and purity confirmation of Gra-saccharides and Mon-saccharides……………………………..
4.4 Discussion………………………………………………………………..
4.5 Table……………………………………………………………………...
4.6 Figures……………………………………………………………………

Chapter 5
Effective prevention of Japanese encephalitis virus infection by low-degree-polymerization sulfated saccharides from Gracilaria sp. and Monostroma nitidum……………………………………………………………….
Abstract…………………………………………………………………………
5.1 Introduction………………………………………………………………
5.2 Experimental……………………………………………………………..
5.2.1 Chemicals and reagents……………………………………………..
5.2.2 Preparation of low-DP saccharides…………………………………
5.2.3 Virus and animals…………………………………………………...
5.2.4 The antiviral activity by MTT assay………………………………..
5.2.5 The binding activity of saccharides to JEV detected by ELISA……
5.2.6 Transmission electron microscopy (TEM)…………………………
5.2.7 NO measurement…………………………………………………...
5.2.8 Measurement of iNOS expression by Western blotting…………….
5.2.9 In vivo antiviral assay.........................................................................
5.2.10 Statistical analysis…………………………………………………..
5.3 Results……………………………………………………………………
5.3.1 Preparation and characterization of low-degree-polymerization (DP) saccharides……………………………………………………
5.3.2 Anti-JEV activity of the sulfated saccharides from Gracilaria sp. and M. nitidum………………………………………………………
5.3.3 Direct action of sulfated saccharides on JEV virions………………
5.3.4 NO production ability via iNOS induction in RAW264.7 macrophage cells of sulfated compounds…………………………..
5.3.5 In vivo anti-JEV activity of sulfated saccharides…………………...
5.4 Discussion………………………………………………………………..
5.5 Tables…………………………………………………………………….
5.6 Figures……………………………………………………………………

Chapter 6
Examination of saccharides with different size as medium components for Pseudomonas vesiculiaris MA103 growth and agarases production…………...
Abstract…………………………………………………………………………
6.1 Introduction………………………………………………………………
6.2 Experimental……………………………………………………………..
6.2.1 Chemical and reagents……………………………………………...
6.2.2 Culture of Pseudomonas vesicularis MA103 bacteria strain……….
6.2.3 Preparation of MA103-agarases for monitoring of their pattern by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)……………
6.2.4 SDS-PAGE………………………………………………………….
6.2.5 Monitoring of MA103-agarases activity produced in MMB with different carbon source by NH2-HPLC-ELSD……………………..
6.2.6 Kinetic characterization of crude MA103-agarases…………...........
6.2.7 BLAST……………………………………………………………...
6.3 Results……………………………………………………………………
6.3.1 Culture of Pseudomonas vesicularis MA103 bacteria in MMB supplementing with different carbon source………………………..
6.3.2 Identification of end products of hydrolysis activity of MA103-agarases produced by bacterium P. vesiculiaris MA103 culturing in MMB with different carbon source……………………
6.3.3 BLAST analysis…………………………………………………….
6.4 Discussion………………………………………………………………..
6.5 Table……………………………………………………………………...
6.6 Figures……………………………………………………………………

References………………………………………………………………………….
Abbreviations and acronyms……………………………………………………...
Appendix…………………………………………………………………………...












Table list

Table 2.1 The isolated oligomer product yields from the HPSEC- and NH2-HPLC systems……………………………………………………
Table 4.1 The isolated saccharide product yields with DP6, DP24 and DP30 from HPSEC- system…………………………………………………...
Table 5.1 Analytical analysis of prepared fractions, influence on cell viability and their anti-JEV activities…………………………………………….
Table 5.2 Measurement of the significantly different values (P < 0.05) of the Gra-saccharides compounds and controls after in vivo antiviral test obtained by all pairwise multiple comparison procedures (Holm-Sidak method)…………………………………………………………………
Table 5.3 Measurement of the significantly different values (P value < 0.05) of the Mon-saccharides compounds and controls after in vivo antiviral test obtained by all pairwise multiple comparison procedures (Holm-Sidak method)…………………………………………..………
Table 6.1 BLAST analysis of nucleotide sequences of the genes from Pseudomonas vesicularis MA103 coding for agarases………………...








Figure list

Fig. 1.1 Scheme of the structure of agarose molecule and the two product types derived from HCl acid hydrolysis and agarases digestion……………...
Fig. 1.2 Chemical structure of the repeat unit of agar type molecules with the different types of sugar units and the different substituents……………
Fig. 1.3 Proposed structure of Rhamnan sulfate from Monostroma nitidum…….
Fig. 1.4 Scientific classification of Monostroma nitidum and Gracilaria sp……
Fig. 1.5 Scientific classification of agarolytic bacteria……………………….....
Fig. 1.6 Diagram of evaporative light scattering detector……………………….
Fig. 1.7 Schematic representations of flavivirus particles, their genome organization and the viral life cycle…………………………………….
Fig. 2.1 Calibration curves for calculating the DP of oligomer products separated in the HPSEC and NH2-HPLC systems……………………..
Fig. 2.2 Calibration curves for calculating the quantity of oligomer products separated in the HPSEC and NH2-HPLC systems…………….……….
Fig. 2.3 HPLC chromatograms of NAOS crude product mixtures generated by different units of agarase cleavage on agarose in SEC and NH2-HPLC systems………………………………………………………………….
Fig. 2.4 HPLC inspection of the isolated NAOS fractions…………………..….
Fig. 2.5 HPLC chromatograms of AOS crude product mixtures derived from HCl hydrolysis under different concentration in SEC and NH2-HPLC systems………………………………………………………………….
Fig. 2.6 HPLC inspection of the isolated AOS fractions………………..………
Fig. 2.7 1H NMR spectra of the isolated N4 and A4 fractions………..…………

Fig. 2.8 The ESIMS spectra of 12 isolated NAOS and AOS fractions from DP 2-12 and two commercial standards, N2 and N6……………………….
Fig. 3.1 Calibration curves of oligosaccharide standards by the reducing sugar content methods: Arsenomolybdate, Dinitrosalicilic acid, Ferricyanide as well as HPLC-based methods: HPSEC-RI, and HPSEC-ELSD for measuring the activity of b-agarase…………………………………….
Fig. 3.2 Comparison of product concentrations from the total isolated products with those calculated from reducing sugar content methods: Arsenomolybdate, Dinitrosalicilic acid, Ferricyanide, using three standards: Gal, N2 and N6……………………………………………...
Fig. 3.3 The absorbance intensities of the NAOS product-mixture generated by different unit of b-agarase cleavage on agarose using three reducing sugar methods…………………………………………………………..
Fig. 3.4 Correlation of b-agarase activity unit and peak area of NAOS product-mixture in HPSEC coupled with RI and ELSD methods……...
Fig. 3.5 (A) Comparison of product concentrations from the total isolated products with those calculated from five monitoring methods under various b-agarase activity unit of digestion on agarose, or five methods monitoring recovered total isolated product. (B) Percentage deviation of the product concentration obtained by the five monitoring methods to that obtained by the total isolated product or five methods monitoring recovered total isolated product, where the total isolated product was normalized to 0 deviation…………………………………
Fig. 3.6 Henri-Michaelis-Menten and Lineweaver-Burk plots for b-agarase kinetics measurement using agarose as substrate………………………

Fig. 4.1 HPSEC-ELSD chromatogram of Gra-crude product mixture generated by agarases cleavage on Gra-polysaccharides from Gracilaria sp……...
Fig. 4.2 HPSEC-ELSD inspection of the isolated Gra-fractions……………..…
Fig. 4.3 HPSEC-ELSD chromatograms of Mon-crude product mixtures generated by agarases cleavage on M. nitidum…………………………
Fig. 4.4 HPSEC-ELSD inspection of the isolated Monostroma-fractions……....
Fig. 4.5 1H NMR spectra of the isolated Gra-6 and Mon-6 fractions……..…….
Fig. 4.6 ESIMS spectra of isolated Gra-6 and Mon-6 fractions…….…….……
Fig. 4.7 Plausible structures of Gra-saccharides………………………………...
Fig. 4.8 Plausible structure of Mon-saccharides………………………..……….
Fig. 5.1 Influence of addition time of sulfated and non-sulfated compounds from: Gracilaria sp., M. nitidum as well as agarose and heparin on anti-JEV activity………………………………………………………..
Fig. 5.2 Blocking effect of samples from Gracilaria sp., M. nitidum as well as agarose and heparin on JEV attachment to the cell membrane…………
Fig. 5.3 Binding effect of JEV to compounds from Gracilaria sp., M. nitidum and agarose……………………………………………………………...
Fig. 5.4 TEM images of the JEV incubated alone and with Mon-6……..............
Fig. 5.5 Effect of compounds from Gracilaria sp. and M. nitidum on nitrite production in the RAW264.7 macrophages…………………………….
Fig. 5.6 Effect of compounds from Gracilaria sp. and M. nitidum on the expression of iNOS in RAW264.7 macrophages……………………….
Fig. 5.7 HPSEC-ELSD chromatograms of detected saccharides in mice plasma after 15th days of sample delivering by i.p. or oral route………………
Fig. 5.8 Mice survivability within 15 days of samples administrated by i.p. or oral route………………………………………………………………..
Fig. 5.9 In vivo anti-JEV activities of compounds from Gracilaria sp………….
Fig. 5.10 In vivo anti-JEV activities of compounds from M. nitidum……………
Fig. 5.11 HPSEC-ELSD chromatograms of detected saccharides in mice plasma after 15th day of feeding………………………………………………..
Fig. 6.1 Pseudomonas vesiculiaris growth profiles cultured in MMB contained different carbon source…………………………………………………
Fig. 6.2 Effect of different carbon sources on activities and cleavage abilities of MA103-agarases produced by Pseudomonas vesiculiaris……………...
Fig. 6.3 SDS-PAGE electrophoresis of crude agarases produced by Pseudomonas vesiculiaris, cultured in MMB contained different carbon source…………………………………………………………...
Fig. 6.4 Henri-Michaelis-Menten (A) and Lineweaver-Burk (B) plots for MA103-agarases kinetics measurement using agarose as substrate……













Appendix list

Appendix 1 Simplified scheme of neoagaro-oligosaccharide (NAOS) series production by HPSEC system………………………………………
Appendix 2 Simplified scheme of Gra-saccharide series production by HPSEC system………………………………………………………………
Appendix 3A Simplified scheme of Mon-saccharide series (digested by 1 U MA103-agarases and 1 U MAEF108-agarases) production by HPSEC system……………………………………………………...
Appendix 3B Simplified scheme of Mon-saccharide series (digested by 2 U MA103-agarases and 2 U MAEF108-agarases) production by HPSEC system……………………………………………………...
Appendix 4 Standard curve for the ester sulfate content…………………………
Appendix 5 Calibration curves for calculating the nitrite production……………
Appendix 6 The process scheme of in vivo anti-JEV activity of Gra- and Mon-saccharide samples……………………………………………
Appendix 7 Natural prestained SDS-PAGE protein standards……..……………











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