Browsing by Author "Pan, IH"

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Aqueous two-phase extraction as an effective tool for isolation of geniposide from gardenia fruit
(2002-11-22)
Pan, IH
;
Chiu, HH
;
Lu, CH
;
Lee, LT
;
Li, YK
Natural products are normally obtained by organic solvent extraction and many subsequent chromatographic separations. Compounds of interest are often isolated with very low yield and limited purity. An aqueous two-phase extraction process combined with a simple ethanol treatment, for removing excess inorganic salt, has been developed for preparation of geniposide from gardenia. The system was comprised of PE62, a random copolymer composed of 20% ethylene oxide and 80% propylene oxide, KH2PO4 and ethanol. To find optimal conditions, the partition behavior of geniposide under an aqueous two-phase system was investigated. Various factors were considered, including the concentration of salt, the concentration of polymer, the sample loading, and the addition of ethanol. The experimental results demonstrated that increasing salt concentration or decreasing PE62 concentration results in enhancement of the geniposide partition in the salt-rich phase. The addition of ethanol and higher sample loading also promoted the partition efficiency of geniposide. Based on this study, an optimized system containing 5% PE62, 7.5% KH2PO4, and 10% ethanol was tested on a large-scale extraction. A 39.0-g aliquot of final product (in powder form) with 77% purity of geniposide can be effectively extracted from 500 g of gardenia fruit. This process is proved to be useful for industrial application of geniposide preparation. (C) 2002 Elsevier Science B.V. All rights reserved.
Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning
(2001-02-01)
Pan, IH
;
Yao, HJ
;
Li, YK
;
應用化學系
;
Department of Applied Chemistry
Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta -xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH2PO4) in aqueous solution. beta -Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH2PO4 were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated. (C) 2001 Elsevier Science Inc. All rights reserved.
Mechanistic study of beta-xylosidase from Trichoderma koningii G-39
(2000-02-01)
Li, YK
;
Yao, HJ
;
Pan, IH
;
應用化學系
;
Department of Applied Chemistry
The catalytic mechanism of the beta-xylosidase purified from the culture filtrate of Trichoderma Koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4,dinitrophenyl and p-nitrophenyl-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration, Based on the k(cat) values of a series of arylxylosides with leaving group pK(a)s in the range of 4-10, an extended Bronsted plot was constructed with a slope (beta(Ig)) near zero. Enzymatic hydrolysis of aryl-beta-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the k(cat) values of p-cyanophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (k(H)/k(D)) measured for 2,4-dinitrophenyl-beta-D-xyloside was 1.02+/-0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is S(N)2-like.
Rapid process for purification of an extracellular beta-xylosidase by aqueous two-phase extraction
(2001-04-15)
Pan, IH
;
Li, YK
;
應用化學系
;
Department of Applied Chemistry
A rapid process for purification of an extracellular beta -xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended elude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na2HPO4. The aqueous two-phase extraction was performed with PE62 (10%)-NaH2PO4 (15%) as phase-forming reagent. SDS-PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography. (C) 2001 Elsevier Science B.V. All rights reserved.