碩士 / 國立臺灣海洋大學 / 食品科學系 / 102 / The aim of this study is to observe the oligosaccharide product of algal alkaline-extracted xylan hydrolyzed by xylanase XylIH1 which was produced from xylI gene cloned to endotoxin free E. coli CleanColi BL21 (CCBL21). Activity of xylooligosaccharide from Ulva sp. (UXOS3k) as anticoagulant, antioxidant, disaccharidases inhibitor and Angiotensin converting enzyme (ACE) inhibitor, prebiotic, and antivirus. The xylI forward primer encoded SpeI restriction site and the reverse primer encoded SacI restriction site, and using the gene of Pseudomonas vesicularis MA103 xylI gene as a template for PCR amplification. The xylI gene ligated into pGEM-T vector called recombinant vector I, and then electroporation into the competent cells of E. coli DH5a. The recombinant vector I which carried the cloned xylI gene was further ligated into the pET39b vector and transformed into the E. coli CCBL21. After Selection and purification of the suspected colonies, and using sequence alignment was used to identify the cloned DNA sequence which completely identical to the xylI gene, was named E. coli CCBL21-xylIH1. The crude intracellular enzyme of recombinant strain E. coli CCBL21-xylIH1 showed 3.73 U/mg xylanase specific activity when it was induced by 0.2 mM IPTG at 26oC for 6 hr. SDS-PAGE result showed this enzyme had 63 kDa molecular weight compared to protein ladder as standard. Substrate specificity of xylanase XylI was tested using six alkaline-extracted-polysaccharides (AEPS) and the enzyme xylanase XylI only reacted with alkaline-extracted-polysaccharide from Ulva sp. (UAEPS). The 2.5% (w/v) UAEPS was hydrolyzed by xylanase XylI at pH 6, 45oC for 12 hr, TLC results showed that the main xylooligosaccharide (XOS) were estimated at DP 4 and DP 5. The yield of XOS from Ulva sp. (UXOS3k) obtained from UAEPS after ultrafiltration (< 3 kDa) was 2.78% (g/100 g dry seaweed). FT-IR analysis showed that UXOS3k correspond to the sulfate ester group in wave numbers 1,260 cm-1 (S=O) and 846 cm-1 (C-O-S) stretching vibration. The sulfated content of UXOS3k was 0.52% (w/w). UXOS3k had 23% DPPH radical scavenging capacity and 7.23 ug/mL Trolox of reducing ability. In the ferrous ion chelating ability, UXOS3k and 50 ug/mL EDTA had relatively equal chelating ability (90.44%). The prebiotic effect potentials of UXOS3k was tested against Lb. acidophilus and Lb. plantarum. The results showed UXOS3k as a carbon source had significant difference with control, the bacterial counts of Lb. acidophilus and Lb. plantarum were 8.06 and 8.10 log CFU/mL after 24 hour-incubation, respectively. In antivirus activity against japanese encephalitis virus Beijing 1 strain and dengue virus type 2 experiments, the results show the inhibition activity at prevention assay and had significant difference with control (p＜0.05). The antivirus activity indicated UXOS3k can inhibit the infection of virus to host cell receptor. In disaccharidase test, the UXOS3k had higher inhibition effect on maltase activity (87.26), but showed lower inhibition activity on lactase and sucrase (< 10%). This study provides the technique for cloning xylanase XylI to strain E. coli CCBL21-xylIH1 as well as the technique to produce UXOS3k from UAEPS and its utilization as antivirus and prebiotic.
| Identifer | oai:union.ndltd.org:TW/102NTOU5253066 |
| Date | January 2014 |
| Creators | Wang, Yi-Shian, 王議賢 |
| Contributors | Pan, Chorng-Liang, 潘崇良 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 127 |
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