Browsing by Author "Huang, Wan-Ting"

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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.
Effects of mono-dentate and bi-dentate ligands on adsorption characteristics of Cu-ion-imprinted hybrids
(2019-12-01)
Chou, Chia-yin
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Chang, Sue-min
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Huang, Wan-Ting
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環境工程研究所
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Institute of Environmental Engineering
Two types of functional monomers, 3-(2-aminoethylamino)-propyl-trimethoxysilane (AAPTS) and 3-aminopropyl-triethoxysilane (APTES), with similar amino groups but different denticity (AAPTS: bi-dentate; APTES: mono-dentate) were used to prepare Cu-ion-imprinted hybrids (IIHs), and their influences on the imprinting quality, adsorption ability, and selectivity of the IIHs were symmetrically investigated. The AAPTS-IIHs exhibited the highest imprinting factor (IF) of 17.7 at the Cu/AAPTS = 1/1 and the highest Cu selectivity (k > 6.5) at the Cu/AAPTS = 0.5-1/1, whereas the two corresponding performances (IF = 22.6, k > 1.7) in the APTES-IIHs occurred at the Cu/APTES molar ratio of 0.124/1 and 0.5/1, respectively. Stronger complexation ability of the bidentate ligand led the AAPTS-IIH to exhibit 6.3-6.9 times higher Cu affinity than the APTES-IIH powder. In addition, it resulted in a higher degree of homogeneity among the binding sites and created more selective environment within the imprinted cavities than the mono-dentate APTES monomer did. Square-planar 4-coordinate and linear 2-coordinate Cu complexes were the major forms imprinted in the AAPTS-based and the APTES-based systems, respectively. The exclusion ability of the AAPTS-IIH for the reference ions was in the order of Pb2+similar to Zn2+ > Ni2+ > Hg2+, while it was Zn2+> Pb2+> Ni2+> Hg2+ in the APTES-IIH powder. The imprinted hybrids select ions mainly based on the coordinate geometries of the ion-complexes, and the complexation stability was the second priority.
Limb-girdle muscular dystrophy type 21 is not rare in Taiwan
(2013-08-01)
Liang, Wen-Chen
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Hayashi, Yukiko K.
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Ogawa, Megumu
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Wang, Chien-Hua
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Huang, Wan-Ting
Alpha-dystroglycanopathy is caused by the glycosylation defects of a-dystroglycan (alpha-DG). The clinical spectrum ranges from severe congenital muscular dystrophy (CMD) to later-onset limb girdle muscular dystrophy (LGMD). Among all alpha-dystroglycanopathies, LGMD type 21 caused by FKRP mutations is most commonly seen in Europe but appears to be rare in Asia. We screened uncategorized 40 LGMD and 10 CMD patients by immunohistochemistry for alpha-DG and found 7 with reduced a-DG immunostaining Immunoblotting with laminin overlay assay confirmed the impaired glycosylation of a-DG. Among them, five LGMD patients harbored FKRP mutations leading to the diagnosis of LGMD2I. One common mutation, c.948delC, was identified and cardiomyopathy was found to be very common in our cohort. Muscle images showed severe involvement of gluteal muscles and posterior compartment at both thigh and calf levels, which is helpful for the differential diagnosis. Due to the higher frequency of LGMD2I with cardiomyopathy in our series, the early introduction of mutation analysis of FKRP in undiagnosed Taiwanese LGMD patients is highly recommended. (C) 2013 Published by Elsevier B.V.
Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding
(2019-07-08)
Wang, Yu-Kuo
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Huang, Sheng-Cih
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Chang, Chin-Yuan
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Huang, Wan-Ting
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Liao, Man-Jun
Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P2(1)2(1)2, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of similar to 50 angstrom but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.
Mutated Thermostable Direct Hemolysin from Grimontia hollisae Affects Its Hemolytic Activity, Membrane Binding, and Protein Quaternary Structure
(2012-08-01)
Wu, Tung-Kung
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Huang, Wan-Ting
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交大名義發表
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National Chiao Tung University
"Mutated Thermostable Direct Hemolysin from Grimontia hollisae Affects Its Hemolytic Activity, Membrane Binding, and Protein Quaternary Structure"
(2012-08-01)
Wu, Tung-Kung
;
Huang, Wan-Ting
;
交大名義發表
;
National Chiao Tung University
Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants
(2015-04-01)
Huang, Sheng-Cih
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Wang, Yu-Kuo
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Huang, Wan-Ting
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Kuo, Tsam-Ming
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Yip, Bak-Sau
We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDHR46E/EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDHR46E. Cytotoxicity assay of Gh-TDHR46E/EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDHR46E or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDHR46E by the EGFR binding moiety. Further antitumor activity assay of Gh-TDHR46E/EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDHR46E with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect.