Browsing by Author "Wu, TK"

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CMOS chip as luminescent sensor for biochemical reactions
(2003-06-01)
Lu, U
;
Hu, BCP
;
Shih, YC
;
Yang, YS
;
Wu, CY
We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-mum CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen. peroxide (H2O2) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H2O2 producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.
Conversion of a plant oxidosqualene-cycloartenol synthase to an oxidosqualene-lanosterol cyclase by random mutagenesis
(2002-07-02)
Wu, TK
;
Griffin, JH
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生物科技學系
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Department of Biological Science and Technology
A random mutacenesis/in vivo selection approach was applied to generate and identify mutations that alter the product specificity of oxidosqualene-cycloartenol synthase (CAS) from Arabidopsis thaliana. This work complements previous studies of triterpene cyclase enzymes and was undertaken to provide knowledge of the frequency and locations at which point mutations can alter cyclase product specificity. Random mutations were introduced by treatment with hydroxylamine or passage through a mutator strain of bacteria. Libraries of mutated plasmids carrying the cas1 gene were transformed into a cyclase-deficient strain of Saccharomyces cerevisiae (CBY57) bearing a complementing plasmid (pZS11) carrying an Erg7 gene that encodes wild-type yeast oxidosqualene-lanosterol cyclase and a URA3 marker that could be counterselected by growth in media containing 5-fluoroorotic acid (5-FOA). This allowed use of a plasmid shuffle to select for cas1 mutants that could substitute for ERG7 activity. Five of similar to73000 transformants were observed to grow in media containing 5-FOA but lacking ergosterol. pTKP5-derived plasmids isolated from these transformants were sequenced, revealing five distinct and unique point mutations: Tyr410Cys, Ala469Val, His477Tyr, Ile481Thr, and Tyr532His. Analysis of the nonsaponifiable lipids from CBY57 cells expressing these mutants suggests that the Tyr410Cys and His477Tyr mutants produce lanosterol as the dominant product, whereas the Ala469Val, Ile481Thr, and Tyr532His mutants produce a mixture of lanosterol and achilleol A, a product of monocyclization. Sequence and structural homology modeling of CAS indicate that the observed product specificity-altering mutations occur both within (Tyr410Cys, Ile481Thr, and Tyr532His) and outside of (Ala469Val and His477Tyr) the cyclase active site.
Detection of serum uric acid using the optical polymeric enzyme biochip system
(2004-07-15)
Huang, SH
;
Shih, YC
;
Wu, CY
;
Yuan, CJ
;
Yang, YS
An optical polymeric biochip system based on the complementary metal oxide semiconductor (CMOS) photo array sensor and polymeric enzyme biochip for rapidly quantitating uric acid in a one-step procedure was developed. The CMOS sensor was designed with N+/P-well structure and manufactured using a standard 0.5 mum CMOS process. The polymeric enzyme biochip was immobilized with uricase-peroxidase and used to fill the reacting medium with the sample. This study encompasses the cloning of the Bacillus subtilis uricase gene and expression in Escherichia coli, as well as the purification of uricase and measurement of its activity. The cloned uricase gene included an open reading frame of 1491 nucleotides that encodes a protein of approximately 55 kDa. The expression of the putative MBP-fusion protein involved approximately 98 kDa of the protein. The CMOS sensor response was stronger at a higher temperature range of 20-40degreesC, with optimal pH at 8.5. The calibration curve of purified uric acid was linear in the concentration range from 2.5 to 12.5 mg/dL. The results obtained for serum uric acid correlated quite closely with those obtained using the Beckman Synchron method. (C) 2004 Elsevier B.V All rights reserved.
Enzymatic formation of multiple triterpenes by mutation of tyrosine 510 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae
(2004-12-03)
Wu, TK
;
Chang, CH
;
應用化學系分子科學碩博班
;
Institute of Molecular science
Enzymatic formation of multiple triterpenes by mutation of tyrosine 510 of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.
(2005-03-13)
Chang, CH
;
Wu, TK
;
生物科技學系
;
Department of Biological Science and Technology
Functional analysis of alanine-scanning and site-directed mutations in the oxidosqualene-lanosterol cyclase gene of Saccharomyces cerevisiae
(2001-03-07)
Wu, TK
;
Wang, HJ
;
Chiu, FH
;
交大名義發表
;
National Chiao Tung University
Histidine residue at position 234 of oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae simultaneously influences cyclization, rearrangement, and deprotonation reactions
(2005-07-01)
Wu, TK
;
Liu, YT
;
Chang, CH
;
應用化學系分子科學碩博班
;
Institute of Molecular science
Histidine Residue at Position 234 of Oxidosqualene-Lanosterol Cyclase from Saccharomyces cerevisiae Simultaneously Influences Cyclization, Rearrangement, and Deprotonation Reactions
Molecular cloning and functional characterization of hemolysin gene from Vibrio furnissii
(2005-07-01)
Wang, YK
;
Wu, TK
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生物科技學系
;
Department of Biological Science and Technology
Polyacetylenic compounds and butanol fraction from Bidens pilosa can modulate the differentiation of helper T cells and prevent autoimmune diabetes in non-obese diabetic mice
(2004-11-01)
Chang, SL
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Chang, CLT
;
Chiang, YM
;
Hsieh, RH
;
Tzeng, CR
\Compelling evidence suggests that infiltrating CD4(+) type I helper T (Th1) cells in the pancreatic islets play a pivotal role in the progression of diabetes in non-obese diabetic (NOD) mice. We demonstrate in the present report that a butanol fraction of B. pilosa suppressed the development of diabetes, helped maintain levels of blood sugar and insulin in NOD mice in a dose-dependent manner and elevated the serum IgE levels regulated by Th2 cytokines in NOD mice. Moreover, the butanol fraction inhibited the differentiation of naive helper T (Th0) cells into Th1 cells but enhanced their transition into type II helper T (Th2) cells using an in vitro T cell differentiation assay. Two polyacetylenic compounds, 2-beta-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne and 3-beta-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne, identified from the butanol fraction also prevented the onset of diabetes like the butanol fraction. The latter compound showed a stronger activity for T cell differentiation than the former. In summary, the butanol fraction of B. pilosa and its polyacetylenes can prevent diabetes plausibly via suppressing the differentiation of Th0 cells into Th1 cells and promoting that of Th0 cells into Th2 cells.
Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver
(2004-01-01)
Wu, TK
;
Huang, CY
;
Ko, CY
;
Chang, CH
;
Chen, YJ
The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane fraction and purified to homogeneity by ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The purified protein required Triton X-100 to retain its highest activity. The cyclase had a molecular mass of similar to70 and similar to140kDa, as evidenced by a single protein band on silver-stained SDS-PAGE and Coomassie-stained PAGE, respectively. Results from Edman degradation of OSC suggested that it might have a blocked N-terminus. Further peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs. The purified cyclase showed pH and temperature optima at pH 7.4 and 37degreesC, respectively. The apparent K-M and k(cat)/K-M values were estimated to be 11 muM and 1.45 mM(-1) min(-1), respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC50 of 11 nM and 0.79 muM, respectively. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by diethyl pyrocarbonate near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for complete bovine liver OSC activity. (C) 2003 Elsevier Inc. All rights reserved.
Tryptophan 232 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences rearrangement and deprotonation but not cyclization reactions
(2006-03-30)
Wu, TK
;
Yu, MT
;
Liu, YT
;
Chang, CH
;
Wang, HJ
Oxidosqualene-lanosterol cyclases convert oxidosqualene to lanosterol in yeast and mammals. Site-saturated mutants' construction of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase, at Trp232 exchanges against proteinogenic amino acids, and product profiles are shown. All mutants, except Lys and Arg, produced protosta-12,24-dien-3 beta-ol, lanosterol, and parkeol. Overall, Trp232 plays a catalytic role in the influence of rearrangement process and determination of deprotonation position but does not involve intervention in the cyclization steps.