Browsing by Author "Li, Yaw-Kuen"

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A highly sensitive system for urea detection by using CdSe/ZnS core-shell quantum dots
(2007-03-15)
Huang, Chin-Ping
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Li, Yaw-Kuen
;
Chen, Teng-Ming
;
應用化學系
;
Department of Applied Chemistry
An original and novel assay system with urease as a catalyst and CdSe/ZnS quantum dots (QDs) as an indicator has been developed for quantitative analysis of urea. By mixing urease and QDs, the determination of urea can be performed in a quantitative manner. The detection is based on the enhancement of QD photoluminescence (PL) intensity, which is correlated to the enzymatic degradation of urea. By controlling the buffer concentration and pH, PL enhancement due to the degradation of urea is linear in the urea concentration ranging from 0.01 to 100 mM. This property makes the urease/QDs system to be a promising urea-biosensing system. The newly developed system is a superior design and possesses many advantages, including its simple preparation, low cost, no enzyme immobilization required, high flexibility, and good sensitivity. (c) 2006 Elsevier B.V. All rights reserved.
A new approach for quantitative determination of glucose by using CdSe/ZnS quantum dots
(2008-03-14)
Huang, Chin-Ping
;
Liu, Shu-Wei
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Chen, Teng-Ming
;
Li, Yaw-Kuen
;
應用化學系
Quantitative detection of glucose is an important issue in food industry, various biotechnological processes and medication of metabolic disorders. Many ways have been developed to approach this target. In this study, we demonstrated a simple assay system containing glucose oxidase (GOD) and CdSe/ZnS quantum dots (QDs) for specific and quantitative detection of glucose, The role of QDs functioned as an "indicator" to reveal the acidity change of the assay system resulting from the enzymatic oxidation of glucose. By controlling the initial buffer conditions of the assay system at 10 mM or 30 mM phosphate, pH 8.0, the quench ratio of PL intensity of QDs at 586 nm was found to be linear to the tested glucose in the range of 0.2-10 mM or 2-30 mM, respectively. The assay system also provides a convenient way to determine the glucose concentration by visuahzing the color change of QDs fluorescence. As compare to most of the existing methods, this newly developed system possesses many advantages, including simplicity, low cost, high flexibility, and good sensitivity. Furthermore, no complicated chemical modification and enzyme immobilization were needed in this system. (C) 2007 Elsevier B.V. All rights reserved.
A Novel Metallo-beta-Lactamase Involved in the Ampicillin Resistance of Streptococcus pneumoniae ATCC 49136 Strain
(2016-05-23)
Chang, Chia-Yu
;
Lin, Hui-Jen
;
Li, Bor-Ran
;
Li, Yaw-Kuen
;
分子醫學與生物工程研究所
Streptococcus pneumoniae, a penicillin-sensitive bacterium, is recognized as a major cause of pneumonia and is treated clinically with penicillin-based antibiotics. The rapid increase in resistance to penicillin and other antibiotics affects 450 million people globally and results in 4 million deaths every year. To unveil the mechanism of resistance of S. pneumoniae is thus an important issue to treat streptococcal disease that might consequently save millions of lives around the world. In this work, we isolated a streptococci-conserved L-ascorbate 6-phosphate lactonase, from S. pneumoniae ATCC 49136. This protein reveals a metallo-beta-lactamase activity in vitro, which is able to deactivate an ampicillin-based antibiotic by hydrolyzing the amide bond of the beta-lactam ring. The Michaelis parameter (K-m) = 25 mu M and turnover number (k(cat)) = 2 s(-1) were obtained when nitrocefin was utilized as an optically measurable substrate. Through confocal images and western blot analyses with a specific antibody, the indigenous protein was recognized in S. pneumoniae ATCC 49136. The protein-overexpressed S. pneumonia exhibits a high ampicillin-tolerance ability in vivo. In contrast, the protein-knockout S. pneumonia reveals the ampicillin-sensitive feature relative to the wild type strain. Based on these results, we propose that this protein is a membrane-associated metallo-beta-lactamase (MBL) involved in the antibiotic-resistant property of S. pneumoniae.
A straightforward alpha-selective aromatic glycosylation and its application for stereospecific synthesis of 4-methylumbelliferyl alpha-T-antigen
(2009-03-10)
Chang, Shih-Sheng
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Lin, Chun-Cheng
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Li, Yaw-Kuen
;
Mong, Kwok-Kong Tony
;
應用化學系
A practical and efficient alpha-selective aromatic glycosylation with simple per-O-acetyl glycopyranosyl trichloroacetimidates is reported. The method is particularly effective for L-fucosyl and 2-azido-2-deoxy-D-galatosaminyl imidates, with which exclusive alpha-selectivity was achieved. The synthetic utility of this method was demonstrated in the stereoselective synthesis of 4-methylumbelliferyl alpha-T-antigen. (C) 2008 Elsevier Ltd. All rights reserved.
An antifouling peptide-based biosensor for determination of Streptococcus pneumonia markers in human serum
(2020-03-01)
Chang, Po-Han
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Weng, Chang-Ching
;
Li, Bor-Ran
;
Li, Yaw-Kuen
;
交大名義發表
We report a peptide-based sensor that involves a multivalent interaction with L-ascorbate 6-phosphate lactonase (UlaG), a protein marker of Streptococcus pneumonia. By integrating the antifouling feature of the sensor, we significantly improved the signal-to-noise ratio of UlaG detection. The antifouling surface was fabricated via electrodeposition using an equivalent mixture of 4-amino-N,N,N-trimethylanilinium and 4-aminobenzenesulfonate. This antifouling layer not only effectively reduces the non-specific adsorption on the biosensor but also decreases the charge transfer resistance (R-et) of the screen-printed carbon electrode. The aniline-modified 57 peptide, an UlaG-binding peptide, was pre-synthesized and further electrochemically modified to bind onto the antifouling layer. Bio-electrochemical analysis confirms that the antifouling 57-peptide sensor binds strongly to the UlaG with a dissociation constant (K-d) = 0.5 nM. This strong interaction can be attributed to a multivalent interaction between the biosensor and the heximeric form of UlaG. To demonstrate the potential for clinical application, further detection of Streptococcus pneumonia from 50 to 5 x 10(4) CFU/mL were successfully performed in 25% human serum.
An L-ascorbate-6-phosphate lactonase from Streptococcus pneumoniae ATCC 49136 strain reveals metallo-beta-lactamase activity
(2016-05)
Chang, Chia-Yu
;
Li, Bor-Ran
;
Li, Yaw-Kuen
;
分子醫學與生物工程研究所
;
應用化學系
Catalytic function of a newly purified exo-beta-D-glucosaminidase from the entomopathogenic fungus Paecilomyces lilacinus
(2013-04-02)
Chao, Cheng-Fu
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Chen, Yi-Yun
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Cheng, Chih-Yu
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Li, Yaw-Kuen
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應用化學系
An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-beta-D-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95 kDa; the optimum pH and temperature for activity are 6.0 and 45 degrees C, respectively. The purified exo-beta-D-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-D-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern confirmed using real-time mass spectrometry shows that exo-beta-D-glucosaminidase cleaves the glycosidic bonds between GlcN-GlcN and GlcN-GlcNAc but not between GlcNAc-GlcN or GlcNAc-GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-beta-D-glucosaminides were obtained, indicating that exo-beta-D-glucosaminidase is a retaining enzyme. (c) 2012 Elsevier Ltd. All rights reserved.
Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing--1, 3-glucanase
(2011-08-01)
Shrestha, Keshab Lal
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Liu, Sheng-Wen
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Huang, Chin-Ping
;
Wu, Hsin-Mao
;
Wang, Wen-Ching
Laminaripentaose-producing -1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of -1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to 90 homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residuesAsp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
Cloning and Expression of Chitinase A from Serratia marcescens for Large-scale Preparation of N,N-Diacetyl Chitobiose
(2009-08-01)
Wu, Yue-Jin
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Cheng, Chih-Yu
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Li, Yaw-Kuen
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應用化學系
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Department of Applied Chemistry
The gene of Serratia marcescens chitinase A (chiA) was cloned by PCR. The complete gene was constructed into a pRSET vector and expressed in Escherichia coli. The recombinant enzyme was purified to > 90% homogeneity by hydrophobic interaction chromatography followed by ion-exchange separation. Measured with an electrospray-ionization mass spectrometer, the molecular mass of the protein was 58,607 Da, consistent with a theoretical calculation of the deduced protein without the signal peptide. The recombinant enzyme was characterized and tested for the preparation of chitobiose. In general, the recombinant Chtinase A exhibited an exo-type catalytic activity toward colloidal chitin and released both N-acetylglucosamine and N,N-diacetyl chitobiose as products. After extensive testing, we produced N,N-diacetyl chitobiose as the predominant product when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100-g scale.
Cloning, expression and characterization of beta-xylosidase from Aspergillus niger ASKU28
(2015-11-01)
Choengpanya, Khuanjarat
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Arthomthurasuk, Siriphan
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Wattana-amorn, Pakorn
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Huang, Wan-Ting
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Plengmuankhae, Wandee
beta-Xylosidases catalyze the breakdown of beta-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 beta-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the alpha-mating factor signal sequence (alpha-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5\' end of the alpha-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70 degrees C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the beta-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 beta-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry. (C) 2015 Elsevier Inc. All rights reserved.
Controlling the electrostatic interaction using a thermal signal to structurally change thermoresponsive nanoparticles
(2019-09-20)
Akimoto, Jun
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Lin, Hsiu-Pen
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Li, Yaw-Kuen
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Ito, Yoshihiro
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應用化學系
The negatively charged thermoresponsive polymers poly(benzyl methacrylate)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide) (PBzMA-b-PAAc-b-PIPAAm) were prepared by reversible-addition fragmentation chain transfer polymerization. The polymer formed core-shell polymeric micelles possessing a thermoresponsive corona composed of PAAc-b-PIPAAm below the lower critical solution temperature (LCST) of the PIPAAm chain. The nonionic thermoresponsive polymeric micelle composed of PBzMA-b-PIPAAm exhibited extensive intermicellar aggregation after the hydrophobic phase transition of the corona-forming PIPAAm chains. In contrast, insertion of the PAAc chain into the thermoresponsive micelle maintained its dispersibility above the LCST of PIPAAm because of the electrostatic repulsion between the micelles. The micelles with short PAAc chains suppressed the effect of the negative charge derived from PAAc below the LCST because of the extended PIPAAm chain on the micellar surface. In contrast, the negative charge property appeared due to collapse of the PIPAAm layer above the LCST. This thermoresponsive change in its electrostatic behavior enables the micelle to control the adsorption and desorption of oppositely charged molecules in response to the temperature change across the LCST.
Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS
(2010-10-08)
Hsieh, Yin-Cheng
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Wu, Yue-Jin
;
Chiang, Tzu-Ying
;
Kuo, Chueh-Yuan
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Shrestha, Keshab Lal
Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-D-glucosamine (NAG)(n), into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67-Thr-69 and Ile-106-Val-112) to interact with (NAG)(n), generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of similar to 4.6 angstrom along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)(n) reveal (NAG)(2), (NAG)(2), and (NAG)(4) in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)(4); in an intermediate state, E145Q/Y227F with a boat-form NAG at the -1 subsite, -1-(NAG); after hydrolysis, E145Q with a chair form -1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between -1-(NAG) and -1-(NAG); Tyr-227 in the conformational change of -1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of -1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(L-His-L-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)(n) provide new insights into its substrate binding and the mechanistic action.
Crystal Structures of Bacillus cereus NCTU2 Chitinase Complexes with Chitooligomers Reveal Novel Substrate Binding for Catalysis A CHITINASE WITHOUT CHITIN BINDING AND INSERTION DOMAINS
(2010-10-08)
Hsieh, Yin-Cheng
;
Wu, Yue-Jin
;
Chiang, Tzu-Ying
;
Kuo, Chueh-Yuan
;
Shrestha, Keshab Lal
Chitinases hydrolyze chitin, an insoluble linear polymer of N-acetyl-D-glucosamine (NAG)(n), into nutrient sources. Bacillus cereus NCTU2 chitinase (ChiNCTU2) predominantly produces chitobioses and belongs to glycoside hydrolase family 18. The crystal structure of wild-type ChiNCTU2 comprises only a catalytic domain, unlike other chitinases that are equipped with additional chitin binding and insertion domains to bind substrates into the active site. Lacking chitin binding and chitin insertion domains, ChiNCTU2 utilizes two dynamic loops (Gly-67-Thr-69 and Ile-106-Val-112) to interact with (NAG)(n), generating novel substrate binding and distortion for catalysis. Gln-109 is crucial for direct binding with substrates, leading to conformational changes of two loops with a maximum shift of similar to 4.6 angstrom along the binding cleft. The structures of E145Q, E145Q/Y227F, and E145G/Y227F mutants complexed with (NAG)(n) reveal (NAG)(2), (NAG)(2), and (NAG)(4) in the active site, respectively, implying various stages of reaction: before hydrolysis, E145G/Y227F with (NAG)(4); in an intermediate state, E145Q/Y227F with a boat-form NAG at the -1 subsite, -1-(NAG); after hydrolysis, E145Q with a chair form -1-(NAG). Several residues were confirmed to play catalytic roles: Glu-145 in cleavage of the glycosidic bond between -1-(NAG) and -1-(NAG); Tyr-227 in the conformational change of -1-(NAG); Asp-143 and Gln-225 in stabilizing the conformation of -1-(NAG). Additionally, Glu-190 acts in the process of product release, and Tyr-193 coordinates with water for catalysis. Residues Asp-143, E145Q, Glu190, and Tyr-193 exhibit multiple conformations for functions. The inhibitors zinc ions and cyclo-(L-His-L-Pro) are located at various positions and confirm the catalytic-site topology. Together with kinetics analyses of related mutants, the structures of ChiNCTU2 and its mutant complexes with (NAG)(n) provide new insights into its substrate binding and the mechanistic action.
Detection of small molecule via the application of VH and VL fragments of scFv: An example of bisphenol A sensing
(2017-04-02)
Lu Yu-chun
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Chi, Chih-Jen
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Chao, Cheng-Fu
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Li, Yaw-Kuen
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Ueda, Hiroshi
Development of a novel engineered antibody targeting Neisseria species
(2017-03)
Liu, Chih-Yueh
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Weng, Chang-Ching
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Lin, Chih-Hsiang
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Yang, Chiou-Ying
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Mong, Kwok-Kong Tony
A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. The recombinant scFv could detect Neisseria strains at 10(6) CFU/ml.
Diversity of sugar acceptor of glycosyltransferase 1 from Bacillus cereus and its application for glucoside synthesis
(2016-05)
Chiu, Hsi-Ho
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Shen, Mo-Yuan
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Liu, Yuan-Ting
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Fu, Yu-Lieh
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Chiu, Yu-An
Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-beta-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are beta-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.
Effective Construction of a High-Capacity Boronic Acid Layer on a Quartz Crystal Microbalance Chip for High-Density Antibody Immobilization
(2019-01-01)
Lin, Pei-Heng
;
Huang, Sheng-Cih
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Chen, Kuang-Po
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Li, Bor-Ran
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Li, Yaw-Kuen
Boronic acids (BAs) provide strong potential in orientation immobilization of antibody and the modification method is crucial for efficiency optimization. A highly effective method has been developed for rapid antibody immobilization on gold electrodes through the electrodeposition of a BA-containing linker in this study. Aniline-based BA forms a condense layer while antibody could automatically immobilize on the surface of the electrode. Compare to traditional self-assembled monolayer method, the electrodeposition process dramatically reduces the modification time from days to seconds. It also enhances the immobilized efficiency from 95 to 408 (ng/cm(2)) with a strong preference being exhibited for shorter aniline-based linkers.
Expression, purification and characterization of a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidae from Trichoderma koningii G-39
(2007-02-01)
Wan, Chin-Feng
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Chen, Cheng-Ta
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Huang, Lina
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Li, Yaw-Kuen
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應用化學系
A gene of (alpha-L-arabinofuranosidase (Abf) from Trichoderma koningii G-39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation-exchanged chromatography. The purified enzyme exhibits both alpha-L-arabinofuranosidase and P-D-xylosidase (Xyl) activities with p-nitrophenyl-alpha-L-arabionfuranoside (pNPAF) and 2,4-dinitrophenyl-beta-D-xylopyanoside (2,4-DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 degrees C for 2 h, but retained 95% of Xyl activity, with 2,4-DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl-alpha-L-arabinofuranoside inhibition is noncompetitive towards 2,4-DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofaranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two-step, double displacement mechanism.
Expression, Purification and Characterization of Human alpha-L-Fucosidase
(2009-08-01)
Liu, Sheng-Wen
;
Li, Yaw-Kuen
;
應用化學系
;
Department of Applied Chemistry
alpha-L-Fucosidases (EC 3.2.1.51) are exo-glycosidases. On the basis of the multi-alignment of amino acid sequence, alpha-L-fucosidases were classified into two families of glycoside hydrolases, GH-29 and GH-95. They are responsible for the removal Of L-fucosyl residues from the non-reducing end of glycoconjugates. Deficiency of alpha-L-fucosidase results in Fucosidosis due to the accumulation of fucose-containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of alpha-L-fucosidases may relate to this abnormal cell physiology. Although the gene of human alpha-L-fucosidase (h-fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human alpha-L-fucosidases (h-Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion-exchange and gel-filtration chromatographic purification, the recombinant h-Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS-PAGE (similar to 50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h-Fuc was stable up to 55 degrees C with incubation at pH 6.8 for 2 h; the optimum temperature for h-Fuc is approximately 55 degrees C. The enzyme was stable at pH 2.5-7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K(m) and k(cat) values of the recombinant h-Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s(-1), respectively. The study of pH-dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h-Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h-Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two-step, double displacement mechanism.
Fabrication of a miniature CMOS-based optical biosensor
(2007-06-15)
Ho, Wei-Jen
;
Chen, Jung-Sheng
;
Ker, Ming-Dou
;
Wu, Tung-Kung
;
Wu, Chung-Yu
This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm x 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H2O2 and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 RL. Both optical H2O2 and glucose biosensors have an optimal operation temperature and pH of 20-25 degrees C and pH 8.4, respectively. The linear dynamic range of optical H2O2 and glucose biosensors is 0.05-20 mM H2O2 and 0.5-20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range. (c) 2007 Published by Elsevier B.V.