Browsing by Author "Cheng, Chih-Yu"

Now showing 1 - 7 of 7

Catalytic function of a newly purified exo-beta-D-glucosaminidase from the entomopathogenic fungus Paecilomyces lilacinus
(2013-04-02)
Chao, Cheng-Fu
;
Chen, Yi-Yun
;
Cheng, Chih-Yu
;
Li, Yaw-Kuen
;
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.
Cloning and Expression of Chitinase A from Serratia marcescens for Large-scale Preparation of N,N-Diacetyl Chitobiose
(2009-08-01)
Wu, Yue-Jin
;
Cheng, Chih-Yu
;
Li, Yaw-Kuen
;
應用化學系
;
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.
Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis
(2011)
Huang, Chen-Ji
;
Peng, Hwei-Ling
;
Cheng, Chih-Yu
;
生物科技學系
;
Department of Biological Science and Technology
In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W(84), P(95), P(110), or V(129). The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W(84)S, P(110)S and V(129)L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 mu M(-1). Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.
Maximum contrast beamformer for electromagnetic mapping of brain activity
(2006-09-01)
Chen, Yong-Sheng
;
Cheng, Chih-Yu
;
Hsieh, Jen-Chuen
;
Chen, Li-Fen
;
資訊工程學系
Beamforming technique can be applied to map the neuronal activities from magnetoencephalographic/electroencephalographic (MEG/EEG) recordings. One of the major difficulties of the scalar-type MEG/EEG beamformer is the determination of accurate dipole orientation, which is essential to an effective spatial filter. This paper presents a new beamforming technique which exploits a maximum contrast criterion to maximize the ratio of the neuronal activity estimated in a specified active state to the activity estimated in a control state. This criterion leads to a closed-form solution of the dipole orientation. Experiments with simulation, phantom, and finger-lifting data clearly demonstrate the effectiveness, efficiency, and accuracy of the proposed method.
Novel Steroid-Sensing Model and Characterization of Protein Interactions Based on Fluorescence Anisotropy Decay
(2010-04-01)
Chang, Ko-Shing
;
Luo, Liyang
;
Chang, Chih-Wei
;
Huang, Yen-Chieh
;
Cheng, Chih-Yu
Intramolecular binding of a ligand with an alkyl link, (-CH(2))(3), covalently bound to a residue near the active site of the protein forms a novel steroid-sensing model. A genetically engineered Delta(5)-3-ketosteroid isomerase (KSI) was designed to conjugate uniquely with this ligand at its Cys-86 through the formation of a disulfide bond. The steady-state protein-ligand binding, mediated by hydrophobic interactions, was confirmed with fluorescence spectra, and the fluorophore-labeled peptide sequence was identified with tandem mass spectra. A comparison of steady-state fluorescence spectra of various fluorophore-labeled KS1 mutants reveals that the emission characteristics vary with environmental factors. An evaluation of the decay of the fluorescence anisotropy of the fluorophore indicates the existence of an intramolecular protein-ligand binding interaction. The measurement of time-resolved fluorescence anisotropy of various protein-ligand complexes yielded values of anisotropy decay representing the degrees of freedom of the fluorophore related to its location, inside or Outside the steroid-binding domain. When 19-norandrostenedione (19-NA) was added to this protein-ligand system, competitive binding between the ligand and the steroid was observed; this finding confirms the feasibility of the design of steroid detection with engineered KSI. On integration of this protein-ligand system with a silicon-based nanodevice (a p-type field-effect transistor with an Ultrathin body), it noncharged steroid, 19-NA, became detectable at it micromolar level (Biosens. Bioelectron. 2008, 23, 1883).
Stabilized linear model for neuromagnetic source localization
(2007)
Chen, Yong-Sheng
;
Cheng, Chih-Yu
;
Lin, Tzu-Jung
;
Hsieh, Jen-Chuen
;
Chen, Li-Fen
In MEG studies, head movement during data recording often causes problems in event-related averaging as well as source localization accuracy. We propose in this work a stabilized linear model which can solve this head movement problem by conceptually increasing the sensor number, under the condition that the head movement is known. This method is particularly beneficial for MEG studies in which head movement commonly Occurs, such as those studies requiring long experimental duration or those when patients/children are involved. Phantom studies and studies of somatosensory evoked field clearly show the effectiveness and accuracy of the proposed method.
Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase
(2008-09-01)
Yao, Yueh-Yun
;
Shrestha, Keshab Lal
;
Wu, Yue-Jin
;
Tasi, Huei-Ju
;
Chen, Chun-Chen
To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D(115) and T(222) of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a 'roof' over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the dominant product.