|Abstract: ||本研究目的在於探討 P. vesicularis MA103 經可溶性澱粉誘導後所產之澱粉誘導粗酵素液 (SI-Enzyme solution)，分離純化後分別探討其生化特性和水解產物組成；另外嘗試以無限制酶剪切 (RF cloning) 方式將 MA103 澱粉酶基因 (Amy-00047) 轉殖至大腸桿菌中表現。MA103 澱粉誘導酵素液經過離心、超過濾和膠體過濾層析法無法得到單一純化後蛋白質，因而使用澱粉親和力純化法暨膠體過濾層析法，成功得到單一純化後酵素 Pv-AM-I，純化倍率為 7.94，其分子質量約為 37 kDa。MA103 澱粉誘導粗酵素液與純化後澱粉酶 Pv-AM-I 之最適反應溫度及 pH 皆為 50oC, pH 7.0，而且 Ca2+ 皆可以使二者之相對活性提昇 (124%, 103%)，但 Sn2+ 會使澱粉誘導粗酵素液及澱粉酶 Pv-AM-I 完全失去澱粉酶活性。在基質特異性部分Pv-AM-I 及澱粉誘導粗酵素液對龍鬚菜多醣與海菜多醣都具有降解能力，而 Pv-AM-I 亦具有降解馬尾藻多醣之能力。水解澱粉產物分析方面，MA103 澱粉誘導酵素液與純化後澱粉酶 Pv-AM-I 水解 0.2% 之可溶性澱粉產物，經高效能液相層析法 (HPLC) 以及薄層層析法 (TLC) 分析比對標準品後，推測其主要產物為麥芽糖。另一方面探討澱粉酶基因轉殖技術部分，嘗試從 MA103 genomic DNA定序報告中挑選一個澱粉酶序列，命名為 Amy-00047 且長度為 1071 bp，依序列設計專一性引子，以 RF cloning 進行基因轉殖至大腸桿菌表現，實驗過程中，使用二種不同之 DNA 聚合酶 (Taq, Pfu polymerase) 經過三次聚合酶連鎖反應 (polymerase chain reaction, PCR) 成功擴增後，利用 DpnI 剪切處理後以熱休克之方式轉型至宿主細胞 E. coli DH5 以及 BL21 皆發現具有抗生素抗性之轉殖株生長，但使用不具基因校正能力之 Taq polymerase 組別經 PCR 擴增後，於轉殖株中並未發現有與 Amy-00047基因大小相似之產物出現；反觀使用具校正能力之 Pfu polymerase 之組別於轉殖株中可以成功擴增出與欲轉殖之基因大小相似約1000 bp 之產物產生，此結果仍待進一步轉殖株之重組質體基因定序確認。|
The aim of this study is using soluble starch to induce Pseudomonas vesicularis MA103 for amylase production, after enzyme purification the biochemical properties and hydroltsate of starch induced enzyme solution (SI-Enzyme solution) and purified amylase Pv-AM-I were both investigated; MA103 amylase sequence Amy-00047 was cloned to E. coli. MA103 starch induced enzyme solution was centrifuged, ultrafiltrated and chromatogramed by gel permeation. The purification methods above were not able to acquire single protein band. On the other hand, using starch affinity method and gel permeation chromatography successfully obtained single protein band by SDS-PAGE, named as Pv-AM-I. The purification fold was 7.94 and the molecular weight was approximately 37 kDa. The optimal reaction pH and temperature of SI-Enzyme solution and purified amylase Pv-AM-I were pH 7.0 and 50oC. Ca2+ was able to raise the relative activity of both SI-Enzyme solution and Pv-AM-I (103-124%), on the other side, Sn2+ was with the ability to completely inhibit the enzyme activity of both enzymes. On substrate specificity , SI-Enzyme solution and Pv-AM-I can both hydrolyze Garcilaria sp. and Monostroma sp. polysaccharide, in addition, purified amylase Pv-AM-I was capable to hydrolyze Sargassum sp. polysaccharide. Analyzing the hydrolysate of SI-Enzyme solution and Pv-AM-I by HPLC and TLC, compared to the standards, discovered that the main hydrolysate were both assumed to be maltose. Further, research on amylase cloning. Amy-00047, 1071 base pairs, was one of the amylase gene sequence from the sequencing report of MA103 genomic DNA. According to Amy-00047 gene sequence a pair of primer was designed, and the primer pair was used in 3 times of PCR (RF cloning) to acquire recombinant plasmid DNA. During the 3 times PCR process, two different kinds of DNA polymerase (Taq, Pfu) were used. After 3 times of PCR, the Amy-00047 inserted plasmid DNA was transformed into E. coli DH5 and BL21 by heat shock. The plasmid accepted E. coli colonies were capable to grow on ampicilin added LA plate, but there were no colony PCR products with the colonies which plasmids were amplified by Taq polymerase. In contrast, the colonies with Pfu polymerase amplified plasmids was discovered with approximately 1000 bp product. The result still needs further recombinant plasmid DNA sequencing of to confirm the inserted gene sequence.