| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 107 === Celiac disease (CD) is a chronic autoimmune enteropathy with 1% prevalence in the western countries. CD is mainly triggered by the ingestion of gliadin, one of the components of wheat gluten, in the intestinal digestive tract. Upon ingestion, gluten is partially degraded by pepsin into gliadin peptide, which are resistant to further proteolysis due to their high proline and glutamine contents. These proteolytic-resistant oligopeptides are believed to trigger the autoimmune response, which causes many of the symptoms in celiac patients, including diarrhea, steatorrhea, nutrient malabsorption and development retardation due to atrophy of the villi. Until now, the only treatment is following gluten-free diet strictly for life. Gluten is ubiquitous in processed foods, so it is extremely difficult to avoid eating gluten completely. Recently, a novel concept for treating celiac disease is to hydrolyze gliadin peptides with proteases, preventing the gliadin peptide from eliciting the immune response. An oral enzyme therapeutic (OET) for celiac disease would have the following traits: (1) Activity of gliadin hydrolysis that removes the toxic peptides before entering the duodenum. (2) Resistance to the acidic environment (pH 2-4) of the stomach. (3) Resistance to pepsin. Therefore, screening conditions were designed to obtain a bacterium, BYGA01, with the potential to break down the gliadin peptide form Byblis liniflora. The next generation of genome analysis was performed on BYGA01. Combined with protein mass spectrometric data, four candidate proteases (BYGA_0323, BYGA_1527, BYGA_1903, and BYGA_5507) were identified. In the study, the corresponding gene sequences were individually constructed on the plasmid pETDuet-1 and expressed in Escherichia coli BL21(DE3) to test their activities. After preliminary analysis, BYGA_1903 had the highest degradation activity against gliadin. To define the most suitable conditions for secretion of BYGA_1903 into the medium, various culture conditions such as IPTG concentration, duration of induction, culture temperature, and culture medium were tested. The test showed that the optimal production of BYGA_1903 requires RNA protection and rare tRNA supplementation, so E. coli BL21Star-pRARE(DE3) was used to express BYGA_1903 in large amount with optimal conditions. To obtain more purified proteases, the extracellular fluid was concentrated and purified by gel filtration for subsequent characterization. BYGA_1903 has the best activity to degrade gliadin at pH 6, while still retaining 50% activity at pH 3.5. At 40℃, BYGA_1903 has the best activity to degrade gliadin. At temperature higher than 50℃, the enzyme loses its activity. In addition, BYGA_1903 retains the ability to degrade gliadin in the presence of pepsin. Based on the above characterization, BYGA_1903 has potential as an oral enzyme.
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