Browsing by Author "Li, YK"

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A chitosanase from Paecilomyces lilacinus with binding affinity for specific chito-oligosaccharides
(2005-04-01)
Chen, YY
;
Cheng, CY
;
Haung, TL
;
Li, YK
;
應用化學系
A purple-spore, rhizosphere-inhabiting nematophagous fungus, further identified as Paecilomyces lilacinus, was found to grow on chitosanase-detecting plate. An induced endochitosanase having a molecular mass of 23 kDa was purified from the culture medium by a single cation-exchange column-chromatography step. Its optimum pH, optimum temperature and pi were found to be 6.0, 50 degrees C and 8.3 respectively. The N-terminal amino acid sequence of the purified enzyme was partially determined. On the basis of the partial sequence XQLPANLXXIYD and the BLAST results, the purified chitosanase was classified as a new member of the family 75 glycohydrolases. Complete hydrolysis of 95% deacetylated chitosan by the isolated chitosanase released chitotriose, chitotetraose and chitopentaose as the major hydrolytic products. Two oligosaccharides, which were further determined to be GlcN-GlcN-GlcNAc and GlcNAc-GlcN-GlcN-GlcNAc by chemical methylation followed by liquid chromatography-tandem MS analysis, were obtained after the denaturation of the purified chito-sanase. This is the first documented finding that chitosanase can be produced in a Paecilomyces strain and that it has binding affinity for specific N-acetylated oligosaccharides.
A new imidazoles as potent inhibitors of beta-glucosidase.
(1996-08-25)
Shu, HH
;
Li, YK
;
應用化學系
;
Department of Applied Chemistry
A new photo-sensing nano-device structure with CdSe and Au nanoparticles on silicon substrate
(2003-01-01)
Wu, CY
;
Li, YK
;
Tu, CC
;
電機工程學系
;
Department of Electrical and Computer Engineering
CdSe nanoparticles (NPs) have shown superior photoconductivity as the particle size is shrunk to nano-scale. This provides a potential application as a nano-photo-sensor. In this work, we choose the CdSe NPs with diameter of 25nm, which is comparable to the electron mean free path in bulk material, to get a high mobility in NPs. By using the DNA oligonucleotides as linkers through Au-S bonds, a new photo-sensing nano-device structure is proposed with Au NPs of 60 nm in diameter and CdSe NPs on a silicon substrate, where the CMOS interface circuits are designed to read signals of the sensor.
An Aspergillus chitosanase with potential for large-scale preparation of chitosan oligosaccharides
(2000-12-01)
Cheng, CY
;
Li, YK
;
應用化學系
;
Department of Applied Chemistry
A chitosan-degrading fungus, designated Aspergillus sp. Y2K, was isolated from soil. The micro-organism was used for producing chitosanase (EC 3.2.1.132) in a minimal medium containing chitosan as the sole carbon source. The induced chitosanase was purified to homogeneity from the culture filtrate by concentration and cationic SP-Sepharose chromatography. The purified enzyme is a monomer with an estimated molecular mass of 25 kDa by SDS/PAGE and of 22 kDa by gelfiltration chromatography. pi, optimum pH and optimum temperature values were 8.4, 6.5 and 65-70 degreesC, respectively. The chitosanase is stable in the pH range from 4 to 7.5 at 55 degreesC. Higher deacetylated chitosan is a better substrate. Chitin, xylan, 6-O-sulphated chitosan and O-carboxymethyl chitin were indigestible by the purified enzyme, By endo-splitting activity, the chitosanase hydrolysed chitosan to form chitosan oligomers with chitotriose, chitotetraose and chitopentaose as the major products. The enzyme hydrolyses chitohexaose to form chitotriose, while the chitopentaose and shorter oligomers remain intact. The N-terminal amino acid sequence of the enzyme was determined as YNLPNNLKQIYDDHK, which provides useful information for further gene cloning of this enzyme. A 275 g-scale hydrolysis of chitosan was performed,The product distribution was virtually identical to that of the small-scale reaction, Owing to the simple purification process and high stability of the enzyme, it is potential ly valuable for industrial applications.
An isolated Candida albicans TL3 capable of degrading phenol at large concentration
(2005-12-01)
Tsai, SC
;
Tsai, LD
;
Li, YK
;
應用化學系
;
應用化學系分子科學碩博班
An isolated yeast strain was grown aerobically on phenol as a sole carbon source up to 24 mm; the rate of degradation of phenol at 30 degrees C was greater than other microorganisms at the comparable phenol concentrations. This microorganism was further identified and is designated Candida albicans TL3. The catabolic activity of C. albicans TL3 for degradation of phenol was evaluated with the K-s and V-max values of 1.7 +/- 0.1 mm and 0.66 +/- 0.02 mu mol/min/mg of protein, respectively. With application of enzymatic, chromatographic and mass-spectrometric analyses, we confirmed that catechol and cis,cis-muconic acid were produced during the biodegradation of phenol performed by C. albicans TL3, indicating the occurrence of an ortho-fission pathway. The maximum activity of phenol hydroxylase and catechol-1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mm and 10 mm, respectively. In addition to phenol, C. albicans TL3 was effective in degrading formaldehyde, which is another major pollutant in waste water from a factory producing phenolic resin. The promising result from the bio-treatment of such factory effluent makes Candida albicans TL3 be a potentially useful strain for industrial application.
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.
Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247
(2001-05-01)
Li, YK
;
Chir, J
;
Chen, FY
;
應用化學系
;
Department of Applied Chemistry
A family 3 beta -glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta = -0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols > 7) indicates substantial bond cleavage at the transition slate. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta -D-glucopyanoside, o-nitrophenyl beta -3-D-glucopyranoside and p-cyanophenyl beta -D-glucopyanoside as substrates were 1.17 +/- 0.02, 1.19 +/- 0.02 and 1.04 +/- 0.02 respectively. Theseresults support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K-m) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20 % of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.
Characterization of an isozyme of beta-glucosidase from sweet almond
(1997-02-01)
Li, YK
;
Chang, LF
;
Shu, HH
;
Chir, J
;
交大名義發表
A sweet almond beta-glucosidase (EC 3.2.1.21) isozyme was purified from commercial crude product. The process of purification consisted of a Protein-Pak Q anion exchange chromatography following by a Superdex 75 HR gel filtration separation. The purified enzyme is a monomeric glycoprotein with molecular weight of 58 kDa and pI = 4.55 which is distinguished from reported isozymes. The enzyme has a pH optimum in the range of 5.2-5.6 when p-nitrophenyl-beta-D-glycopyranosides are used as substrate and is stable up to 50 degrees C at that pH range. The purified protein also exhibits profound beta-galactosidase and alpha-L-arabinosidase activity. The study of substrate specificity revealed that lacking of hydroxymethyl group at C-5 of glycosides resulted in higher affinity for substrate binding to enzyme, whereas the chemical step of hydrolysis (k(cat)) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-beta-D-glycopyranosides with apparent pK(1) and pK(2) values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by delta-gluconolactone (K-i = 160 mu M) and 4-phenylimidazole (K-i = 17.8 mu M) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-beta-D-glucosamine to the enzyme (K-i = 52 mM) was 6 times stronger than that of glucose and its epimers.
Characterization of an isozyme of beta-glucosidase from sweet almond
(1997-02-01)
Li, YK
;
Chang, LF
;
Shu, HH
;
Chir, J
;
應用化學系
A sweet almond beta-glucosidase (EC 3.2.1.21) isozyme was purified from commercial crude product. The process of purification consisted of a Protein-Pak Q anion exchange chromatography following by a Superdex 75 HR gel filtration separation. The purified enzyme is a monomeric glycoprotein with molecular weight of 58 kDa and pI = 4.55 which is distinguished from reported isozymes. The enzyme has a pH optimum in the range of 5.2-5.6 when p-nitrophenyl-beta-D-glycopyranosides are used as substrate and is stable up to 50 degrees C at that pH range. The purified protein also exhibits profound beta-galactosidase and alpha-L-arabinosidase activity. The study of substrate specificity revealed that lacking of hydroxymethyl group at C-5 of glycosides resulted in higher affinity for substrate binding to enzyme, whereas the chemical step of hydrolysis (k(cat)) was prevented significantly. The pH activity profile displayed a bell-shaped curve for all measured p-nitrophenyl-beta-D-glycopyranosides with apparent pK(1) and pK(2) values of 4.4-4.7 and 6.2-6.4, respectively. This isozyme was strongly inhibited by delta-gluconolactone (K-i = 160 mu M) and 4-phenylimidazole (K-i = 17.8 mu M) reversibly at pH 6.2. Among the tested glycoses, the binding affinity of N-acetyl-beta-D-glucosamine to the enzyme (K-i = 52 mM) was 6 times stronger than that of glucose and its epimers.
Cloning and expression of beta-glucosidase from Flavobacterium meningosepticum: A new member of family B beta-glucosidase
(1999-02-01)
Li, YK
;
Lee, JA
;
應用化學系
;
Department of Applied Chemistry
A beta-glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in Escherichia coli. The Entire nucleotide sequence was determined and analyzed. An open-reading frame of 2,178 bp encoding a polypeptide of 726 amino acids with a calculated M-r of 79,952 was derived from the sequence. The coding region is flanked by a putative promoter and transcription terminator sequences. The nucleotide sequence accession number of the complete gene sequence in Genbank is AF015915. Based on the comparison of amino acid sequences this enzyme is classified as a family B beta-glucosidase and is highly homologous with sequences from Clostridium thermocellum, Kluyvermyces fragilis, and Agrobacterium tumefaciens. The cloned enzyme was purified to near homogeneity by ammonium sulfate fractionation (35 similar to 75%) and chromatography an a cation-exchanged column at pH 6.9. This expressed beta-glucosidase has optimum activity at pH 4.2 similar to 5.0 and 50 degrees C and is stable in the pH range of 5.0 similar to 8.1 at 25 degrees C. it showed a high specificity on the glycone portion of aryl-beta-D-glycosides. All of these characteristics are highly consistent with those of the native beta-glucosidase. (C) 1998 Elsevier Science inc.
CMOS chip as luminescent sensor for biochemical reactions
(2003-06-01)
Lu, U
;
Hu, BCP
;
Shih, YC
;
Yang, YS
;
Wu, CY
We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-mum CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen. peroxide (H2O2) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H2O2 producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.
Design and fabrication of a photosensing nanodevice structure with CdSe and Au nanoparticles on a silicon chip
(2006-05-01)
Tu, CC
;
Li, YK
;
Chen, TM
;
Wu, CY
;
應用化學系
A newly developed tour-layered photosensing nanodevice was fabricated by integrating nanoparticles (NPs) on a silicon substrate. Through ionic interaction, negatively charged An NPs (similar to 15 nm) were assembled in alternate layers with positively charged CdSe NPs (similar to 5 nm) on the silicon oxide surface between the two Al electrodes. The silicon oxide surface after each step of the fabrication process was observed and evaluated by images obtained from the scanning electron microscope. By applying voltage biases across the electrodes, the currents were measured in the dark and under illumination using a 375-nm laser. It was found that a constant photocurrent increment can be obtained for different voltage biases, and the nanodevice structure with a longer length had less conductivity but a larger increment of photocurrent after illumination. In addition, the efficiency rate of photocurrent generation is much higher in comparison to that obtained from CdSe thin film. The fabrication process integrated a newly developed model of a diode-resistor array of semiconductor-metal junctions between CdSe and An NPs (nano-Schottky-diode structures), which can successfully explain the measured results. While nanotechnology has unprecedented advantages over the traditional silicon electronics, its technology presents physical challenges. However, the success of the fabrication of the multilayered photosensing nanodevice directly on the silicon chip paves the way for further applications and research.
Design and synthesis of activity probes for glycosidases
(2002-10-17)
Tsai, CS
;
Li, YK
;
Lo, LC
;
應用化學系
;
Department of Applied Chemistry
[GRAPHICS] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.
Detection of serum uric acid using the optical polymeric enzyme biochip system
(2004-07-15)
Huang, SH
;
Shih, YC
;
Wu, CY
;
Yuan, CJ
;
Yang, YS
An optical polymeric biochip system based on the complementary metal oxide semiconductor (CMOS) photo array sensor and polymeric enzyme biochip for rapidly quantitating uric acid in a one-step procedure was developed. The CMOS sensor was designed with N+/P-well structure and manufactured using a standard 0.5 mum CMOS process. The polymeric enzyme biochip was immobilized with uricase-peroxidase and used to fill the reacting medium with the sample. This study encompasses the cloning of the Bacillus subtilis uricase gene and expression in Escherichia coli, as well as the purification of uricase and measurement of its activity. The cloned uricase gene included an open reading frame of 1491 nucleotides that encodes a protein of approximately 55 kDa. The expression of the putative MBP-fusion protein involved approximately 98 kDa of the protein. The CMOS sensor response was stronger at a higher temperature range of 20-40degreesC, with optimal pH at 8.5. The calibration curve of purified uric acid was linear in the concentration range from 2.5 to 12.5 mg/dL. The results obtained for serum uric acid correlated quite closely with those obtained using the Beckman Synchron method. (C) 2004 Elsevier B.V All rights reserved.
Discovery of different types of inhibition between the human and Thermotoga maritima alpha-fucosidases by fuconojirimycin-based derivatives
(2006-05-09)
Ho, CW
;
Lin, YN
;
Chang, CF
;
Li, ST
;
Wu, YT
An efficient method for examining the selectivity of inhibitors on two alpha-fucosidases, one from Thermotoga maritima and the other from human, was established. The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human alpha-fucosidase, indicating the major difference between both enzymes in the periphery of the catalytic site. To investigate the difference at the molecular level, a variety of fuconojirimycin (FNJ) derivatives with substitution at Cl, C2, C6, or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha-fucosidases. Among the molecules that were tested, only the substitution at Cl can significantly enhance the inhibitory potency, in contrast to the control (no substitution) and compounds with substitution at other positions. The majority of Cl-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme, while acting as the reversible inhibitors of the human fucosidase. The best inhibitor exhibited 13700-fold difference in affinity between the two enzymes, which was attributed to the dissimilar aglycon binding site. Further investigations were carried out, including site-directed mutagenesis, the comparison of K-i values among the wild type and mutants, and the intrinsic fluorescence change upon inhibitor titration, all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors. The increased level of contact thus induced conformational change, leading to the observed slow tight-binding inhibition.
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.
Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus
(2006-02-10)
Cheng, CY
;
Chang, CH
;
Wu, YJ
;
Li, YK
;
交大名義發表
A powerful endo-chitosanase ( CSN) previously described for a large scale preparation of chito-oligosaccharides ( Cheng, C.-Y., and Li, Y.-K. ( 2000) Biotechnol. Appl. Biochem. 32, 197 - 203) was cloned from Aspergillus fumigatus and further identified as a member of glycosyl hydrolase family 75. We report here a study of gene expression, functional characterization, and mutation analysis of this enzyme. Gene cloning was accomplished by reverse transcription-PCR and inverse PCR. Within the 1382-bp Aspergillus gene ( Gen-Bank (TM) accession number AY190324), two introns ( 67 and 82 bp) and an open reading frame encoding a 238-residue protein containing a 17-residue signal peptide were characterized. The recombinant mature protein was overexpressed as an inclusion body in Escherichia coli, rescued by treatment with 5 M urea, and subsequently purified by cation exchange chromatography. A time course H-1 NMR study on the enzymatic formation of chito-oligosaccharides confirmed that this A. fumigatus CSN is an inverting enzyme. Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAc-GlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp(160) and Glu(169) are the essential residues for the action of A. fumigatus endo-chitosanase.
Exploration of glycosyl hydrolase family 75, a chitosanase from Aspergillus fumigatus
(2006-02-10)
Cheng, CY
;
Chang, CH
;
Wu, YJ
;
Li, YK
;
應用化學系
A powerful endo-chitosanase ( CSN) previously described for a large scale preparation of chito-oligosaccharides ( Cheng, C.-Y., and Li, Y.-K. ( 2000) Biotechnol. Appl. Biochem. 32, 197 - 203) was cloned from Aspergillus fumigatus and further identified as a member of glycosyl hydrolase family 75. We report here a study of gene expression, functional characterization, and mutation analysis of this enzyme. Gene cloning was accomplished by reverse transcription-PCR and inverse PCR. Within the 1382-bp Aspergillus gene ( Gen-Bank (TM) accession number AY190324), two introns ( 67 and 82 bp) and an open reading frame encoding a 238-residue protein containing a 17-residue signal peptide were characterized. The recombinant mature protein was overexpressed as an inclusion body in Escherichia coli, rescued by treatment with 5 M urea, and subsequently purified by cation exchange chromatography. A time course H-1 NMR study on the enzymatic formation of chito-oligosaccharides confirmed that this A. fumigatus CSN is an inverting enzyme. Tandem mass spectrum analysis of the enzymatic hydrolysate revealed that the recombinant CSN can cleave linkages of GlcNAc-GlcN and GlcN-GlcN in its substrate, suggesting that it is a subclass I chitosanase. In addition, an extensive site-directed mutagenesis study on 10 conserved carboxylic amino acids of glycosyl hydrolase family 75 was performed. This showed that among these various mutants, D160N and E169Q lost nearly all activity. Further investigation using circular dichroism measurements of D160N, E169Q, wild-type CSN, and other active mutants showed similar spectra, indicating that the loss of enzymatic activity in D160N and E169Q was not because of changes in protein structure but was caused by loss of the catalytic essential residue. We conclude that Asp(160) and Glu(169) are the essential residues for the action of A. fumigatus endo-chitosanase.
Facile synthesis toward the construction of an activity probe library for glycosidases
(2006-03-20)
Shie, TH
;
Chiang, YL
;
Lin, JJ
;
Li, YK
;
Lo, LC
Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based oil the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-D-Glc, alpha- and beta-D-Gal, alpha-D-Man and alpha-L-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-D-glucosidase, alpha-D-galactosidase, and alpha-L-fucosidase, respectively. (c) 2006 Elsevier Ltd. All rights reserved.