Browsing by Author "Yao, HJ"

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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.
Effective induction, purification and characterization of Trichoderma koningii G-39 beta-xylosidase with high transferase activity
(2000-04-01)
Li, YK
;
Yao, HJ
;
Cho, YT
;
應用化學系
;
Department of Applied Chemistry
A beta-xylosidase was induced and purified from the culture filtrate of Trichoderma koningii G-39, grown in a medium containing 1% oat spelts xylan and 0.1% xylose, The presence of xylose unequivocally enhanced the induction of beta-xylosidase. The purified enzyme, which exhibited a significant alpha-arabinosidase activity, was obtained with high yield simply via ethanol precipitation and a single anion-exchange chromatography and was characterized as a monomeric glycoprotein with an estimated molecular mass of 104 kDa and a pl of 4.6. The K-m values towards P-nitrophenyl beta-D-xylopyranoside and p-nitrophenyl alpha-L-arabinopyranoside are 0.04 and 7.5 mM, respectively. It is stable at pH 2.5-7.4, 37 degrees C. The pH and temperature optima are in the range of 3.5-4.0 and 55-60 degrees C, respectively. Contrary to most beta-xylosidases from other sources, Hg2+ (up to 25 mM) has no effect on enzyme activity. Xylose was shown to inhibit the purified enzyme with a moderate Ki value of 5 mM, The enzyme exhibited transxylosylation activity and was characterized as a 'retaining' enzyme, catalysing the hydrolysis of substrate with the retention of anomeric configuration.
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.