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ABSTRACT: Dectin-1, a specific pattern recognition receptor for β-1,3/β-1,6-glucans, is expressed mainly on phagocytes. Human dectin-1 (hDectin-1) and mouse dectin-1 (mDectin-1) were separately expressed on HEK293 cell surfaces for examination of the binding abilities of a synthetic particulate β-glucan (pβG), a product extracted from Saccharomyces cerevisiae, in this study. The binding of zymosan-FITC to hDectin-1 and mDectin-1 was inhibited by pβG at similar concentrations for 50% inhibition of binding (IC₅₀). However, the kinetics of the time course and dose response to zymosan stimulation observed for U937 and J774A.1 differed. Superoxide anion production was increased in U937 but reduced in J774A.1 when cells were treated with pβG, zymosan, or laminarin, whereas ovalbumin endocytosis was enhanced in U937 and J774A.1 treated either with pβG, zymosan, laminarin, or barley-glucan. These results indicate that the binding affinity of pβG to hDectin-1 is similar to the binding affinity to mDectin-1, and that stimulation by pβG as well as various forms of β-1,3-glucans on U937 and J774A.1 resulted in upregulation of cell activity and ovalbumin endocytosis. Additionally, other coreceptors on U937 and J774A.1 may be involved in directing different responses to superoxide anion production in these two types of cells. These results will likely contribute to further investigations on identifying the biological forms of β-glucans capable of binding its specific receptor as the effective immunomodulators.
Biotechnology Progress 09/2010; 26(5):1391-9. · 2.34 Impact Factor
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ABSTRACT: The process of applying salt-stressed baker’s yeast during southern style Chinese steamed bread dough preparation was examined. Baker’s yeast was stressed in 7% salt solution then mixed into dough, which was then evaluated for dough fermentation producing gas, dough expansion, texture profile analysis (TPA), colour, specific volume, spread ratio and sensory analysis. The results of this study pointed out salt-stressed baker’s yeast produced significant amount of gas and dough expansion, particularly after 40 min of salt stressing. The texture of steamed bread was softer (463.08 g) than control (541.35 g) (P < 0.05), greater in specific volume (3.15 cm3 g−1) than control (2.89 cm3 g−1) (P < 0.05), had a lower spread ratio (1.45) than control (1.74) (P < 0.05) and a significantly improved sensory properties for taste (90.6) than control (81.6) (P < 0.05) were obtained.
International Journal of Food Science & Technology 11/2009; 44(12):2637 - 2643. · 1.26 Impact Factor
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ABSTRACT: Interleukin-18 (IL-18) can induce interferon-gamma (IFN-gamma) production and promote Th1 immunity, and hence, it modulates immune functions. In the present study, the in vitro and in vivo immunomodulatory activities of full length or mature chicken IL-18 expressed in a prokaryotic expression system (pCHIL18-F and pCHIL18-M, respectively) and chicken IL-18 expressed in a eukaryotic expression system (euCHIL18) were examined. Results showed that pCHIL18-F, pCHIL18-M and euCHIL18 significantly enhanced IFN-gamma mRNA expression in chicken splenocytes, which successfully increased IFN-gamma-induced nitric oxide (NO) synthesis by macrophages. Vaccination with cell-cultured Newcastle disease vaccine (NDTC) co-administrated with pCHIL18-F, pCHIL18-M or euCHIL18 resulted in significant increments of hemagglutination inhibition (HI) titers, cell proliferation of peripheral blood mononuclear cells (PBMC), and ratios of CD8(+) to CD4(+) in chickens compared with inoculation of PBS or NDTC alone. Thus, full length and mature chicken IL-18 expressed using a prokaryotic system and using a eukaryotic system showed equivalent in vitro and in vivo biological activities, and all forms effectively enhanced cell-mediated and humoral immunity, suggesting possible future use as a potential adjuvant in chicken NDTC vaccine production.
Vaccine 11/2009; 28(5):1148-55. · 3.77 Impact Factor
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ABSTRACT: 2-Acetyl-1-pyrroline (2-AP) was identified as an important aroma compound of aromatic vegetable soybean. The level of 2-AP in 6 aromatic vegetable soybean lines was found to be positively correlated with popcorn-like aroma score. Comparison between aromatic and nonaromatic vegetable soybeans found that aromatic vegetable soybean contains higher concentration of methylglyoxal (MG) and Delta(1)-pyrroline-5-carboxylate (P5C) than a nonaromatic one. For MG formation-related genes, GapC was down-regulated and TPI was up-regulated in aromatic cultivar (Aromatic 7) as compared to nonaromatic control, which may contribute to the increase of MG level. Based on the data presented, a formation mechanism for 2-AP via interaction between MG and P5C in aromatic vegetable soybean was proposed.
Journal of Food Science 07/2009; 74(5):S192-7. · 1.66 Impact Factor
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ABSTRACT: The immunopharmacological activities of beta-glucans with a backbone of beta-1,3/beta-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for beta-1,3/beta-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate beta-glucan (p-beta-glucan) were examined. Results showed that p-beta-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-alpha/IL-10 production in all of three types of cell, whereas p-beta-glucan increased dectin-1/TLR4 and TNF-alpha/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of beta-1,3/beta-1,6-glucans with different structural features may direct different cellular responses.
Veterinary Immunology and Immunopathology 05/2009; 131(3-4):147-57. · 2.08 Impact Factor
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ABSTRACT: 2-Acetyl-1-pyrroline (2-AP) was identified as the major flavor compound in aromatic rice varieties Tainung 71 and 72. In order to understand the mechanism of 2-AP biosynthesis in aromatic rice, we studied the formation of putative precursors, Delta(1)-pyrroline-5-carboxylic acid and methylglyoxal. The endogenous Delta(1)-pyrroline-5-carboxylic acid contents of Tainung 71 and 72 calli reached 191 to 276%, compared to nonaromatic rice Tainung 67. In addition, calli of Tainung 71 and 72 contained 1.30- and 1.36-fold, respectively, higher methylglyoxal levels than that of Tainung 67. Specific enzyme activities of Delta(1)-pyrroline-5-carboxylic acid-synthetic enzyme including Delta(1)-pyrolline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) increased significantly in aromatic rice varieties. The expression levels of P5CS1 and P5CS2 genes were found to be significantly higher in aromatic rice than nonaromatic rice. Results of a tracer experiment with (15)N-labeled glutamic acid revealed that the nitrogen atom of 2-acetyl-1-pyrroline was derived from glutamic acid. Upregulation of P5CS in aromatic rice Tainung 72 may contribute to the increase of Delta(1)-pyrroline-5-carboxylic acid level and thus leads to the accumulation of an extra amount of 2-acetyl-1-pyrroline.
Journal of Agricultural and Food Chemistry 09/2008; 56(16):7399-404. · 2.82 Impact Factor
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ABSTRACT: This study demonstrated that ergocalciferol was able to inhibit leukemia cell growth in a concentration-dependent manner. Exploration of the acting mechanisms involved this event revealed that ergocalciferol induced DNA fragmentation and increased sub-G1 DNA contents in HL-60 cells, both of which are hallmarks of apoptosis. Analysis of the integrity of mitochondria demonstrated that ergocalciferol caused loss of mitochondrial membrane potential with release cytochrome c to cytosol, generation of reactive oxygen species (ROS), and depletion of glutathione (GSH), suggesting that ergocalciferol may induce apoptosis in HL-60 cells through a ROS-dependent pathway. Further results show that caspases-2, -3, -6, and -9 were all activated by ergocalciferol, together with cleavage of the downstream caspase-3 targets, DNA fragmentation factor (DFF-45), and poly(ADP-ribose) polymerase. In addition, ergocalciferol led to the increase in pro-apoptotic factor Bax accompanied with the decrease in anti-apoptotic member Mcl-1, and the reduced Mcl-1 to Bax ratio may be a critical event concerning mitochondrial decay by ergocalciferol. Furthermore, ergocalciferol also led to induction of Fas death receptor closely linked to caspase-2 activation, suggesting the involvement of a Fas-mediated pathway in ergocalciferol-induced apoptosis. Totally, these findings suggest that ergocalciferol causes HL-60 apoptosis via a modulation of mitochondria involving ROS production, GSH depletion, caspase activation, and Fas induction. On the basis of anticancer activity of ergocalciferol, it may be feasible to develop chemopreventive agents from edible mushrooms or hop.
Journal of Agricultural and Food Chemistry 06/2008; 56(9):2996-3005. · 2.82 Impact Factor
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ABSTRACT: The expression level of phase I (CYP1A1 and CYP1A2) and phase II (GST, and UGT) enzyme-coded genes were measured in liver microsomes of 30 Sprague-Dawley rats fed sea weed (Monostroma nitidum). Quantitative and qualitative analysis of the detoxifying enzymes were investigated using reverse transcription polymerase reaction (RT-PCR) and real time polymerase reaction (Real-time PCR) techniques. The antioxidative properties of seaweed were screened and investigated for its hepatoprotective activity in rat. There was no significant induction of GSTYa1, GSTYa2, and CYP1A2. However, an M. nitidum diet was found to significantly increase UGT1A1 and UGT1A6 mRNA levels and to decrease CYP1A1 mRNA levels in rat liver. Structural studies confirmed the presence of sulfated polysaccharides in the seaweed samples. The results demonstrate the potential of seaweed as a natural source of sulfated polysaccharide substances with potential use in chemoprevention medicine.
Food and Chemical Toxicology 01/2008; 45(12):2390-6. · 3.00 Impact Factor
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ABSTRACT: Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.
Journal of Agricultural and Food Chemistry 07/2007; 55(13):5097-102. · 2.82 Impact Factor
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ABSTRACT: The gene (lat) encoding L-lysine epsilon-aminotransferase (LAT) in Streptomyces clavuligerus was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of lat predicted a single open reading frame (ORF) of 1371 bp, encoding a polypeptide of 457 amino acids with calculated molecular mass of 49.89 kDa. S. clavuligerus LAT was grouped into aminotransferase subfamily II of alpha family on the basis of sequence homology. A model system composed of the recombinant LAT in phosphate buffer was set up to study the biosynthesis of 2-acetyltetrahydropyridine. Lysine was found to be transformed to 1-piperideine-6-carboxylic acid. 2-Acetyltetrahydropyridine was characterized from the mixture of 1-piperideine-6-carboxylic acid and methylglyoxal. For the first time, we demonstrated that the L-lysine epsilon-aminotransferase is responsible for the formation of 1-piperideine-6-carboxylic acid, which may react with methylglyoxal to generate the acylated N-heterocyclic odorant 2-acetyltetrahydropyridine.
Journal of Agricultural and Food Chemistry 04/2007; 55(5):1767-72. · 2.82 Impact Factor
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ABSTRACT: The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.
FEMS Microbiology Letters 04/2006; 256(1):119-25. · 2.04 Impact Factor
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FEMS Microbiology Letters 01/2006; 235(2):401 - 401. · 2.04 Impact Factor
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ABSTRACT: Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII). Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII). In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.
FEMS Microbiology Letters 04/2004; 232(1):61-6. · 2.04 Impact Factor
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ABSTRACT: A gene (AglyA) encoding serine hydroxymethyltransferase of Acinetobacter radioresistens CMC-1 was cloned and sequenced. Nucleotide sequence analysis of AglyA predicted a single open reading frame (ORF) of 1251 bp encoding a 417-amino acid polypeptide. Two putative MetR-like binding sites (5′-TGAAACATGAGCT) and (5′-TGAGCAAAGTTCA), centered at bp −123 and −95 relative to the +1 translation start site were found, which have six out of nine and eight out of nine nucleotides that match to the consensus sequence of Escherichia coli (5′-TGAANNT/ANNTTCA), respectively. The enzyme also showed a high level of homology to other sources of serine hydroxymethyltransferase proteins.
FEMS Microbiology Letters 12/1998; 170(2):413 - 418. · 2.04 Impact Factor
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ABSTRACT: The immunopharmacological activities of β-glucans with a backbone of β-1,3/β-1,6-linkages associated with anti-tumor, anti-viral, bacterial and fungal infections have been well documented. Dectin-1, a specific pattern recognition receptor for β-1,3/β-1,6-glucans, is expressed mainly on phagocytes, especially macrophages and dendritic cells (DCs). In this study, the encoding nucleotide for the carbohydrate-recognition domain (CRD) of porcine dectin-1 was sequenced for the first time, and the immunomodulatory functions of a synthetic particulate β-glucan (p-β-glucan) were examined. Results showed that p-β-glucan significantly enhanced cell activity and phagocytosis in porcine alveolar macrophages (AMs), immature DCs (imDCs) and mature DCs (mDCs), in a similar way to zymosan. Zymosan enhanced dectin-1/TLR2/TLR4 expression and TNF-α/IL-10 production in all of three types of cell, whereas p-β-glucan increased dectin-1/TLR4 and TNF-α/IL-12 production in AMs but inhibited IL-10 in mDCs. These results indicate that the complex collaborating interactions between dectin-1 and TLRs in the recognition of β-1,3/β-1,6-glucans with different structural features may direct different cellular responses.
Veterinary Immunology and Immunopathology.
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ABSTRACT: 一、Aeromonas hydrophila JP101幾丁?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?，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?，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。 二、直接檢測Vibrio vulnificus之聚合?連鎖反應方法之開發 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.
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ABSTRACT: 一、Aeromonas hydrophila JP101幾丁?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?，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?，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。 二、直接檢測Vibrio vulnificus之聚合?連鎖反應方法之開發 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.
