|摘要: ||一、Aeromonas hydrophila JP101幾丁酶92幾丁結合部位之研究|
Chi 92為A. hydrophilaJP101在幾丁質誘導下所產生之外泌型幾丁酶，其ORF為2,598 bp，可以產生由865個胺基酸組成之蛋白質。Chi 92是一種具有多重功能部位的幾丁酶，分別由family 18的催化功能部位，未知的A功能部位及三個幾丁結合部位 (Chi92-N, ChBDCI及ChBDCII)所組成。利用Chi 92之C端刪除突變株進行吸附親和力及催化活性之測定可以證實Chi 92之功能部位，ChBDCI及ChBDCII負責不溶性幾丁質之吸附，並可影響Chi 92對此類幾丁質之水解能力。除此之外，將重覆幾丁結合部位ChBDCICII刪除的突變株 (Chi 92△CICII) 對不溶性幾丁質之親和力及催化活性皆比刪除單一幾丁結合部位之突變株 (Chi 92△CII) 明顯降低。以GST融合蛋白進行吸附實驗時，含有單一ChBD之GST融合蛋白對於膠狀幾丁質有最大結合活性，粉狀幾丁質次之，而對於纖維素也有微弱之親和力。相對於C末端的兩個ChBDs，Chi N-92功能部位與S. marcecens ChiA之Chi N功能部位具有相似性，據推測此區域具有結合幾丁質之能力。由於只剩ChiN-92功能部位及催化功能部位之Chi 92突變株仍保有對不溶性幾丁質之水解及結合能力，因此，Chi N-92功能部位應該是除了ChBDCI及ChBDCII 之外的第三個幾丁結合部位。
為了探討ChBDCI及ChBDCII之間的協同作用，我們利用pET系統構築單一及雙重ChBD。經E. coli表現後，再利用Ni2+螯合親和性管柱層析法進行蛋白質之純化，並分別測定雙重ChBD及單一ChBD之吸附等溫線。ChBDCICII對於膠狀及粉狀幾丁質之分配係數分別為ChBDCII的69倍及4倍，此結果指出，ChBDCI及ChBDCII之間存在著互助合作關係，雙重ChBD之結合機制可以利用雙步驟模式來解釋。為了研究芳香族胺基酸是否在ChBDCICII與幾丁質之交互作用上扮演重要角色，將4個高度保留芳香族胺基酸個別突變為glycine產生W773G, W792G, Y796G, W797G的突變株。突變株 (W773G, W792G, Y796G及W797G) 對於膠狀及粉狀幾丁質之親和力均遠低於野生株。上述數據指出，這四個芳香族胺基酸對於幾丁質之結合相當重要。
Vibrio vulnificus是一種會引發人類致命性感染的伺機性的革蘭氏陰性菌，本研究發展出一種利用聚合酉每連鎖反應快速檢測此種病原菌的方法。以vllY基因為目標序列，設計可以擴增452 bp之DNA片段的專一性引子對（vP1及vP2）。在專一性檢測方面，自臨床檢體所分離的16株V. vulnificus皆可產生正反應。除此外，非Vibrio vulnificus之Vibrio屬及非Vibrio 屬之菌株皆無法呈現正反應。此聚合酉每連鎖反應系統可以至檢測100 pg的染色體DNA，若配合 1 mM EDTA-0.5% Triton溶液之單一步驟萃取法進行模板DNA的萃取，其檢測靈敏度可達到102 CFU。
1.The Studies of Chitin Binding Domains From A. hydrophila JP101 Chitinase 92
The Chi92 is the chitinase secreted by A. hydrophila JP101 when induced with chitin and it is encoded by an ORF of 2,598 bp coding for 865 amino acids. The mature form of Chi92 is a modular enzyme comprised of a family 18 catalytic domain, an unknown function domain (A domain) and triple chitin binding domains (Chi92-N, ChBDCI and ChBDCII). The C-terminal repeated chitin binding domains, ChBDCI and ChBDCII, were grouped into family V of cellulose binding domain on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminal deleted Chi92 mutants lacking ChBDs demonstrated that the ChBDs is responsible for its adhesion to powdered and colloidal chitins. In addition, the deleted mutants without double ChBDs (Chi92ΔCICII) exhibited lower affinities and catalytic activities toward powdered and colloidal chitin than those without single ChBD (Chi92ΔCII). Further adsorption experiments with GST fusion proteins (GST-CI and GST-CICII) demonstrate that single ChBD (ChBDCI) could promote efficient chitin and cellulose binding. In contrast to C-terminal two ChBDs, the Chi92-N domain is similar to the ChiN of Serratia marcecens ChiA that has been proposed to participate in chitin binding. A truncated derivative of Chi92 that only contains catalytic and Chi92-N still exhibited insoluble chitin binding and hydrolytic activity. Thus, it would appear that the Chi92 contains ChiN-92 as the third ChBD in addition to two ChBD domains (ChBDCI and ChBDCII).
In order to understand the cooperative effect between ChBDCI and ChBDCII, we have constructed single and double ChBD by pET system. After expression in E. coli, the proteins were purified from cell extract by Ni2+ chelating column chromatography. Adsoption isotherm for the double ChBD and single ChBD were determined on colloidal and powdered chitin. The partition coefficients of the isotherm showed 69-fold and 4-fold increase for ChBDCICII the as compared with ChBDCII toward colloidal and powdered chitin. The results indicated interplay between ChBDCI and ChBDCII. A two-step model is used to explain the binding behavior of the double ChBD. In order to investigate whether the aromatic amino acids play an important role in the interaction of ChBDCICII and chitin, the four aromatic amino acids were independently mutated to glycine or phenylanaline, to create W773G, W773F, W792G, Y797G and W797G. The mutant proteins (W773G, W792G, Y797G and W797G) exhibited much weaker affinity for colloidal and powdered chitin than wild type. These data indicate that all four aromatic amino acids are important for binding to chitin. .
2. Development of the polymerase chain reaction Method for Direct Detection of Vibrio vulnificus
Vibrio vulnificus is an ubiquitous pathogen that is capable of causing a fatal infection in human. This study developed a PCR-based assay for the rapid identification of V. vulnificus. Specific primers (vP1 and vP2), which derived from vlly gene coding for V. vulnificus hemolysin , were designed for the amplification of 452 bp fragment within vlly gene specific for V. vulnificus. All 16 strains of V. vulnificus isolated from clinical specimens could generate positive results. In addition, Vibrio other than V. vulnificus and non-vibrio isolates did not yield positive results. Study on the detection sensitivity for this PCR system showed that the developed method was able to detect 100 pg of total genomic DNA. The PCR amplification, coupled with single-step DNA extraction method using a 1mM EDTA-0.5% Triton solution provided sensitivity limit to 102 CFU of V. vulnificus.