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• W2134735523 abstract "Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1–1 and Lxyl-p1–2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1–1, G-Lxyl-p1–2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s−1mm−1 against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases. Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1–1 and Lxyl-p1–2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1–1, G-Lxyl-p1–2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/Km of up to 1.07 s−1mm−1 against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases. The protection and sustainable utilization of natural resources are among the most important and global problems of the 21st century. Paclitaxel (Taxol®) is mainly isolated from slow-growing yew trees (genus Taxus, family Taxaceae) and is known as a “blockbuster drug ” showing unique active mechanisms (1Horwitz S.B. Taxol (paclitaxel): mechanisms of action.Ann. Oncol. 1994; 5: S3-S6PubMed Google Scholar), with prominent activity against various cancers (including ovarian, breast, lung, head, and neck carcinomas and the AIDS-related Kaposi sarcoma) (2Goldspiel B.R. Clinical overview of the taxanes.Pharmacotherapy. 1997; 17: 1105-1255Google Scholar). However, the source of paclitaxel has always been a top concern, because its content in the plant is extremely low, and it is isolated in “large ” amounts (∼0.02%) only from the bark of the tree (3Chattopadhyay S.K. Sharma R.P. Kumar S. Process for the production of important taxol analogues 10-deacetyl taxol A, B, and C.U.S. Patent 6028206. 2000; Google Scholar). A 100-year-old tree might yield 3 kg of bark, which provides enough paclitaxel for one 300-mg dose (4Horwitz S.B. How to make taxol from scratch.Nature. 1994; 367: 593-594Crossref PubMed Scopus (47) Google Scholar). To preserve the Taxus resource and alleviate some of the pressure on the source, several approaches have been employed to prepare paclitaxel or its analog Taxotere, including chemical semi-synthesis from the precursor 10-deacetylbaccatin III (DB), 1The abbreviations used are:DB10-deacetylbaccatin IIIDT10-deacetyltaxolGHglycoside hydrolasesL.LentinulaPNP-Arap-nitrophenyl-α-L-arabinopyranosidePNP-Galp-nitrophenyl-β-D-galactopyranosidePNP-Glcp-nitrophenyl-β-D-glucopyranosidePNP-Xylp-nitrophenyl-β-D-xylopyranosideRACErapid amplification of cDNA endsT.TaxusTLCthin layer chromatographyXDT7-β-xylosyl-10-deacetyltaxol. 1The abbreviations used are:DB10-deacetylbaccatin IIIDT10-deacetyltaxolGHglycoside hydrolasesL.LentinulaPNP-Arap-nitrophenyl-α-L-arabinopyranosidePNP-Galp-nitrophenyl-β-D-galactopyranosidePNP-Glcp-nitrophenyl-β-D-glucopyranosidePNP-Xylp-nitrophenyl-β-D-xylopyranosideRACErapid amplification of cDNA endsT.TaxusTLCthin layer chromatographyXDT7-β-xylosyl-10-deacetyltaxol. which is readily available from the twigs of yew trees such as Taxus baccata (5Ojima I. Habus I. Zhao M. Zucco M. Park Y.H. Sun C.M. Brigaud T. New and efficient approaches to the semisynthesis of taxol and its C-13 side chain analogs by means of β-lactam synthon method.Tetrahedron. 1992; 48: 6985-7012Crossref Scopus (330) Google Scholar, 6Denis J.N. Greene A.E. Guenard D. Gueritte-Voegelein F. Mangatal L. Potier P. A highly efficient, practical approach to natural Taxol.J. Am. Chem. Soc. 1988; 110: 5917-5919Crossref Scopus (583) Google Scholar); isolation from the twigs of nursery trees including T. chinensis var. mairei and T. media (hybrid); paclitaxel-producing endophytic strain fermentation (7Stierle A. Strobel G. Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew.Science. 1993; 260: 214-216Crossref PubMed Scopus (1402) Google Scholar, 8Jin R. Kang J.C. Wen T.C. He J. Lei B.X. A study on optimal fermentation of an endophytic fungus producing taxol.Mycosystema. 2011; 30: 235-241Google Scholar); and Taxus cell and tissue culture (9Bonfill M. Expósito O. Moyano E. Cusidó R.M. Palazón J. Piñol M.T. Manipulation by culture mixing and elicitation of Taxol and baccatin III production in Taxus baccata suspension cultures.In Vitro Cell. Dev. Biol. Plant. 2006; 42: 422-426Crossref Scopus (24) Google Scholar). The first two approaches might partially relieve this pressure, but they still cannot meet the growing market demand. 10-deacetylbaccatin III 10-deacetyltaxol glycoside hydrolases Lentinula p-nitrophenyl-α-L-arabinopyranoside p-nitrophenyl-β-D-galactopyranoside p-nitrophenyl-β-D-glucopyranoside p-nitrophenyl-β-D-xylopyranoside rapid amplification of cDNA ends Taxus thin layer chromatography 7-β-xylosyl-10-deacetyltaxol. 10-deacetylbaccatin III 10-deacetyltaxol glycoside hydrolases Lentinula p-nitrophenyl-α-L-arabinopyranoside p-nitrophenyl-β-D-galactopyranoside p-nitrophenyl-β-D-glucopyranoside p-nitrophenyl-β-D-xylopyranoside rapid amplification of cDNA ends Taxus thin layer chromatography 7-β-xylosyl-10-deacetyltaxol. 7-β-xylosyltaxanes are much more abundant and are extracted simultaneously with paclitaxel and DB from various species of yew (10Sénilh V. Blechert S. Colin M. Guénard D. Picot F. Potier P. Varenne P. Mise en evidence de nouveaux analogues du taxol extraits de Taxus boccata.J. Nat. Prod. 1984; 47: 131-137Crossref Scopus (167) Google Scholar, 11Rao K.V. Taxol and related taxanes. I. Taxanes of Taxus brevifolia bark.Pharm. Res. 1993; 10: 521-524Crossref PubMed Scopus (39) Google Scholar, 12Luo H. Nie Y.K. Fu Y.J. Zu Y.G. Li S.M. Liu W. Zhang L. Luo M. Kong Y. Li Z.N. Determination of main taxoids in Taxus species by microwave-assisted extraction combined with LC-MS/MS analysis.J. Sep. Sci. 2009; 32: 192-201Crossref PubMed Scopus (13) Google Scholar), but generally they are dealt with as byproducts. Among these analogues, 7-β-xylosyl-10-deacetyltaxol (XDT) can be obtained with a yield of as much as 0.5% (from dried stem bark) (13Chattopadhyay S.K. Sharma R.P. Kumar S. Madhusudanan K.P. A process for the production of taxol.EP Patent 0905130 B1. 2002; Google Scholar). These 7-β-xylosyltaxanes can be hydrolyzed via chemical or biological methods to give the corresponding 7-hydroxyltaxanes, including 10-deacetyltaxol (DT) and DB, for the semi-synthesis of paclitaxel. In contrast to the chemical approach, which utilizes periodate or other oxidizing agents and a substituted hydrazine in the reactions to remove the sugar, the biological approach is an enzymatic process that releases the d-xylose from 7-xylosyltaxanes through the specific β-xylosidase and is therefore considered to be environmentally friendly. β-xylosidases (EC3.2.1.37) belong to glycoside hydrolase (GH) or glycosidase (EC3.2.1.X) families 3, 30, 39, 43, 52, and 54 (14Cantarel B.L. Coutinho P.M. Rancurel C. Bernard T. Lombard V. Henrissat B. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics.Nucleic Acids Res. 2009; 37: D233-D238Crossref PubMed Scopus (4125) Google Scholar). However, the filamentous fungal β-xylosidases have hitherto been described as belonging only to GH families 3, 43, and 54 (15Knob A. Terrasan C.R.F. Carmona E.C. β-xylosidase from filamentous fungi: an overview.World J. Microbiol. Biotech. 2010; 26: 389-407Crossref Scopus (140) Google Scholar). Many kinds of β-xylosidases have been purified from different organisms, such as bacteria (16Quintero D. Velasco Z. Hurtado-Gómez E. Neira J.L. Contreras L.M. Isolation and characterization of a thermostable beta-xylosidase in the thermophilic bacterium Geobacillus pallidus.BBA Proteins Proteom. 2007; 1774: 510-518Crossref Scopus (22) Google Scholar, 17Brunzelle J.S. Jordan D.B. McCaslin D.R. Olczak A. Wawrzak Z. Structure of the two-subsite beta-D-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane.Arch. Biochem. Biophys. 2008; 474: 157-166Crossref PubMed Scopus (46) Google Scholar, 18Mattéotti C. Haubruge E. Thonart P. Francis F. De Pauw E. Portetelle D. Vandenbol M. Characterization of a new β-glucosidase/β-xylosidase from the gut microbiota of the termite (Reticulitermes santonensis).FEMS Microbiol. Lett. 2011; 314: 147-157Crossref PubMed Scopus (25) Google Scholar, 19Shao W. Xue Y. Wu A. Kataeva I. Pei J. Wu H. Wiegel J. Characterization of a novel β-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485.Appl. Environ. Microbiol. 2011; 77: 719-726Crossref PubMed Scopus (49) Google Scholar), fungi (20Saha B.C. Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides.J. Ind. Microbiol. Biotechnol. 2001; 27: 241-245Crossref PubMed Scopus (35) Google Scholar, 21Gargouri M. Issam S. Thierry M. Marie M.L. Nejib M. Fungus beta-glycosidases: immobilization and use in alkyl-beta-glycoside synthesis.J. Mol. Catal. B Enzym. 2004; 29: 89-94Crossref Scopus (47) Google Scholar, 22Kurakake M. Tomiko F. Masahiro Y. Takumi O. Toshiaki K. Characteristics of transxylosylation by beta-xylosidase from Aspergillus awamori K4.Biochim. Biophys. Acta. 2005; 1726: 272-279Crossref PubMed Scopus (31) Google Scholar, 23Suzuki S. Fukuoka M. Ookuchi H. Sano M. Ozeki K. Nagayoshi E. Takii Y. Matsushita M. Tada S. Kusumoto K. Kashiwagi Y. Characterization of Aspergillus oryzae glycoside hydrolase family 43 beta-xylosidase expressed in Escherichia coli.J. Biosci. Bioeng. 2010; 109: 115-117Crossref PubMed Scopus (28) Google Scholar), and plants (24Minic Z. Christophe R. Cao T.D. Patrice L. Lise J. Purification and characterization of enzymes exhibiting beta-D-xylosidase activities in stem tissues of Arabidopsis.Plant. Physiol. 2004; 135: 867-878Crossref PubMed Scopus (108) Google Scholar, 25Xiong J.S. Balland-Vanney M. Xie Z.P. Schultze M. Kondorosi A. Kondorosi E. Staehelin C. Molecular cloning of a bifunctional β-xylosidase/α-Larabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme.J. Exp. Bot. 2007; 58: 2799-2810Crossref PubMed Scopus (33) Google Scholar). Some β-xylosidase genes, such as those from bacteria (18Mattéotti C. Haubruge E. Thonart P. Francis F. De Pauw E. Portetelle D. Vandenbol M. Characterization of a new β-glucosidase/β-xylosidase from the gut microbiota of the termite (Reticulitermes santonensis).FEMS Microbiol. Lett. 2011; 314: 147-157Crossref PubMed Scopus (25) Google Scholar, 19Shao W. Xue Y. Wu A. Kataeva I. Pei J. Wu H. Wiegel J. Characterization of a novel β-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485.Appl. Environ. Microbiol. 2011; 77: 719-726Crossref PubMed Scopus (49) Google Scholar, 26Suryani Kimura T. Sakka K. Ohmiya K. Sequencing and expression of the gene encoding the Clostridium stercorarium beta-xylosidase Xyl43B in Escherichia coli.Biosci. Biotechnol. Biochem. 2004; 68: 609-614Crossref PubMed Scopus (19) Google Scholar, 27Morana A. Paris O. Maurelli L. Rossi M. Cannio R. Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation.Extremophiles. 2007; 11: 123-132Crossref PubMed Scopus (32) Google Scholar) or from fungi (23Suzuki S. Fukuoka M. Ookuchi H. Sano M. Ozeki K. Nagayoshi E. Takii Y. Matsushita M. Tada S. Kusumoto K. Kashiwagi Y. Characterization of Aspergillus oryzae glycoside hydrolase family 43 beta-xylosidase expressed in Escherichia coli.J. Biosci. Bioeng. 2010; 109: 115-117Crossref PubMed Scopus (28) Google Scholar, 28Reen F.J. Murray P.G. Tuohy M.G. Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding β-xylosidase from the thermophilic fungus Talaromyces emersonii.Biochem. Biophys. Res. Commun. 2003; 305: 579-585Crossref PubMed Scopus (23) Google Scholar, 29Wakiyama M. Yoshihara K. Hayashi S. Ohta K. Purification and properties of an extracellular β-xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene.J. Biosci. Bioeng. 2008; 106: 398-404Crossref PubMed Scopus (47) Google Scholar, 30Teng C. Jia H.J. Yan Q.J. Zhou P. Jiang Z.Q. High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli.Bioresour. Technol. 2011; 102: 1822-1830Crossref PubMed Scopus (52) Google Scholar), have been cloned and characterized. However, none of these enzymes have been reported to be active against 7-β-xylosyltaxanes. In fact, a lot of commercially available xylosidases, xylanases, and other glycosidases do not have any activity specific for removing xylose from 7-β-xylosyltaxanes (31Hanson R.L. Howell J.M. Brzozowski D.B. Sullivan S.A. Patel R.N. Szarka L.J. Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp.Biotechnol. Appl. Biochem. 1997; 26: 153-158Google Scholar). Some bacterial isolates, such as Moraxella sp. (ATCC 55475) (31Hanson R.L. Howell J.M. Brzozowski D.B. Sullivan S.A. Patel R.N. Szarka L.J. Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp.Biotechnol. Appl. Biochem. 1997; 26: 153-158Google Scholar, 32Hanson R.L. Patel R.N. Szarka J. Enzymatic hydrolysis method for the conversion of C-7 sugar to C-7 hydroxyl taxanes.U.S. Patent 5700669A. 1997; Google Scholar), Cellulosimicrobium cellulans XZ-5 (CCTCC No. M207130) (33Yang L. Luan H. Liu X. A Cellulosimicrobium cellulans, its hydrolase and in the use of transformation of taxanes.International Patent Application PCT/CN2008/000618. 2009; Google Scholar), and Enterobacter sp. (CGMCC 2487) (34Wang K. Wang T. Li J. Zou J. Chen Y. Dai J. Microbial hydrolysis of 7-xylosyl-10-deacetyltaxol to 10-deacetyltaxol.J. Mol. Catal. B Enzym. 2011; 68: 250-255Crossref Scopus (12) Google Scholar), have been reported to have the ability to convert XDT to DT. But these strains gave low yields of DT (0.23, 0.4, and 0.76 mg/ml, respectively (31Hanson R.L. Howell J.M. Brzozowski D.B. Sullivan S.A. Patel R.N. Szarka L.J. Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp.Biotechnol. Appl. Biochem. 1997; 26: 153-158Google Scholar, 32Hanson R.L. Patel R.N. Szarka J. Enzymatic hydrolysis method for the conversion of C-7 sugar to C-7 hydroxyl taxanes.U.S. Patent 5700669A. 1997; Google Scholar, 33Yang L. Luan H. Liu X. A Cellulosimicrobium cellulans, its hydrolase and in the use of transformation of taxanes.International Patent Application PCT/CN2008/000618. 2009; Google Scholar, 34Wang K. Wang T. Li J. Zou J. Chen Y. Dai J. Microbial hydrolysis of 7-xylosyl-10-deacetyltaxol to 10-deacetyltaxol.J. Mol. Catal. B Enzym. 2011; 68: 250-255Crossref Scopus (12) Google Scholar)), which is probably due to the ubiquitous low enzyme levels in the native organisms. The related β-xylosidases of these organisms have not yet been defined. Our lab discovered that a fungal species, Lentinula edodes, could transform XDT into DT, but a similarly low yield was also observed (supplemental Fig. S1). Thus, cloning and characterization of the specific enzyme from the fungus might lead to a new biocatalytic route of preparation for 7-hylosyltaxanes for the semi-synthesis of paclitaxel or its analogues. Here, we present a strategy in which we combine protein de novo sequencing with RT-PCR and the rapid amplification of cDNA ends (RACE) to mine the targeted β-xylosidase gene from this fungus. Moreover, yeast engineered with such a heterologous gene can robustly convert 7-β-xylosyltaxanes into 7-hydroxyltaxanes. The strain L. edodes M95.33 was grown in 100 ml wheat bran medium (contents per liter of distilled water: 50.00 g wheat bran (mixed with the appropriate amount of water, boiled for 30 min, and filtered to remove the solid residue), 20.00 g peptone, 1.50 g KH2PO4, 0.75 g MgSO4, final pH ∼6.3; inoculum amount: ∼1 cm2 of lawn picked from a mycelial slant) for 6 to 8 days at 25 °C to 26 °C at 160 rpm in an orbital shaker. For the natural enzyme purification, mycelium from 5 l culture was harvested via filtration, washed with sterile water, and then homogenized in liquid nitrogen. This was followed by suspension in three to five volumes of 50 mm Tris-HCl cell lysis buffer (pH 8.0) and sonication for 5 min (130 W, 10 s/10 s). The supernatant of the lysate underwent anion exchange column chromatography (DEAE Sepharose Fast Flow, 1.6 cm × 20 cm, GE Healthcare). Proteins were eluted with a phase gradient of 0, 0.1, 0.25, and 2.0 m NaCl (at a flow rate of 2 ml/min). Fractions with β-xylosidase activity at 0.1–0.25 M NaCl were collected, pooled, and added to 1 m (NH4)2SO4 for subsequent hydrophobic column chromatography (Phenyl Sepharose Fast Flow, 1.6 cm × 20 cm, GE Healthcare). Proteins were eluted with a linear gradient of 1.0–0 M (NH4)2SO4 in 1000 ml 50 mm Tris-HCl buffer, pH 8.0 (at a flow rate of 2 ml/min). Fractions with β-xylosidase activity were collected, pooled, dialyzed against 50 mm Tris-HCl buffer, pH 8.0, and applied to an anion exchange column (DEAE Sepharose Fast Flow, 1.6 cm × 20 cm, GE Healthcare) as previously described. Proteins were eluted with a linear gradient of 0.1–0.25 M NaCl in 1000 ml 50 mm Tris-HCl buffer, pH 8.0 (at a flow rate of 2 ml/min). Peak fractions with β-xylosidase activity were collected, pooled, concentrated, and subjected to gel filtration column chromatography (Sephacryl S200 High Resolution, 1.6 cm × 60 cm, GE Healthcare). Elution was performed with 50 mm Tris-HCl buffer containing 0.1 m NaCl, pH 8.0 (at a flow rate of 0.5 ml/min). Peak fractions with β-xylosidase activity were collected, pooled, and concentrated. During the purification process, the β-xylosidase activity was monitored with the substrate p-nitrophenyl-β-d-xylopyranoside (PNP-Xyl, Sigma) and examined with the substrate XDT. Protein concentration was determined using BCA (Thermo) or Bio-Rad protein assay kits. For the recombinant enzyme purification, 500 mg of freeze-dried recombinant yeast cells was mixed with 10 ml Yeast Protein Extraction Reagent (Merck) and 20 μl DNase I and incubated at room temperature for 30 min. The suspension was centrifuged for 10 min at 12,000 rpm, yielding about 8 ml of the supernatant. The recombinant protein was purified from the supernatant using an Amersham Biosciences HiTrap Chelating HP Kit (GE Healthcare) and concentrated via ultrafitration (Millipore, Billerica, MA). To remove the carbohydrates from the enzyme, 45 μl of the concentrated enzyme solution was mixed with 5 μl of 10× denatured glycoprotein buffer and boiled at 100 °C for 10 min. 5.5 μl of 10×G5 buffer and 2 μl Endo Hf enzyme (NEB) were added and incubated at 50 °C for 30 min. To preserve the deglycosylated enzyme activity, the glycoprotein was mixed with 5 μl 10×G5 buffer and 1 μl Endo Hf enzyme and incubated at 37 °C overnight. The β-xylosidase activity was assayed by measuring the amount of p-nitrophenol released from the substrate p-nitrophenyl-β-d-xylopyranoside (PNP-Xyl, Sigma) using spectrophotometry (NanoPhotometer® P300, IMPLEN, Munich, Germany) based on the absorbance at 405 nm. Assays were performed in a total volume of 125 μl (containing 25 μl of the enzyme solution) at 50 °C in 50 mm sodium acetate buffer, pH 5.0, containing 5 mm substrate for 20 min. Reactions were terminated by adding 2 ml saturated sodium tetraborate (Na2B4O7). One unit of activity was defined as the amount of enzyme that catalyzed the release of 1 nm p-nitrophenol per minute at 50 °C and pH 5.0. To observe the glycoside specificity of the enzyme, three other p-nitrophenyl glycosides (Sigma)—p-nitrophenyl-β-d-glucopyranoside (PNP-Glc), p-nitrophenyl-β-d-galactopyranoside (PNP-Gal), and p-nitrophenyl-α-l-arabinopyranoside (PNP-Ara)—were used as substrates with PNP-Xyl as the control using the same method as described above. To examine the enzyme activity against XDT (prepared in this lab), 1 ml of the enzyme solution was mixed with 10 μl XDT solution (dissolved in dimethyl sulfoxide, 20 mg/ml), and the reaction was carried out in a 45 °C water bath for 24 h. The suspension was extracted with ethyl acetate and assayed by means of thin layer chromatography (TLC) (mobile phase: petrolium ether:dichloromethane:methanol = 1.5:3.5:0.33, v/v/v), and the color developing agent was 10% sulfuric acid and ethanol solution. The protein samples were analyzed via SDS-PAGE on 10% (w/v) polyacrylamide gels and stained with silver or Coomassie Brilliant Blue R-250. For the reductive treatment of the samples, 5× loading buffer (0.2 m Tris-HCl, pH 6.8, 10% SDS, 10 mm β- mercaptoethanol, 20% glycerol, 0.05% bromphenol blue) was mixed with the sample and boiled for 10 min before SDS-PAGE. For the non-reductive treatment of the samples, 5× loading buffer without β-mercaptoethanol was added to the sample and directly subjected to SDS-PAGE without boiling. To determine the active protein band on the gel, the non-reductive samples were loaded into the two neighboring wells. After SDS-PAGE, one lane of the gel was cut for staining, and the other was cut for in situ xylosidase activity detection. To restore the enzyme activity, the latter was washed three times (10 min each time) with Triton X-100 (2.5%) in 50 mm sodium acetate buffer, pH 5.0, to replace the ionic detergent SDS with the non-ionic detergent Triton X-100 and to lower the pH (from 8.8 to 5.0). The SDS-free gel was submerged in 5 mm PNP-Xyl solution and incubated at 50 °C for 30 min; the process was then stopped by the addition of saturated sodium tetraborate solution. The targeted protein band was used for LC-MS/MS de novo peptide sequencing. Firstly, the optimal temperature and optimal pH were determined using PNP-Xyl as a substrate. To measure the optimal temperature, 100 μl of 5 mm PNP-Xyl solution in 50 mm sodium acetate buffer, pH 5.0, was mixed with 25 μl of the enzyme solution (0.01 mg/ml) at a reaction temperature in the range of 30 °C–65 °C. The reaction assay was performed as described above and in duplicate or triplicate. The optimal pH assay was conducted in a similar fashion, except that a temperature of 50 °C was constantly maintained. The pH was in the range of 3.0–5.5 (sodium acetate buffer) or 6.0–9.0 (potassium phosphate buffer). Additionally, the reaction time curve was plotted with a constant pH of 5.0 and a constant temperature of 50 °C in the time range of 6–84 min. The kinetic parameters of the enzyme were determined against the substrate PNP-Xyl in a concentration range of 0.39–50.0 mm. The kinetic parameters of the recombinant enzyme against PNP-Xyl and PNP-Glc were also examined under the same conditions. To measure the optimal temperature of the recombinant enzyme against XDT, 200 μl of 2.12 mm XDT solution in 50 mm sodium acetate buffer, pH 4.5, was mixed with 10 μl of the enzyme solution (0.1 mg/ml) at a reaction temperature in the range of 30 °C –65 °C for 1.5 h. The optimal pH assay was conducted in a similar fashion in the range from 3.0 to 8.0, except that a temperature of 45 °C was constantly maintained. The reaction time curve was plotted with the constant pH 4.5 and the constant temperature 45 °C and in a time range of 1–7 h. The kinetic parameters of the recombinant enzyme against XDT were determined in the XDT concentration range of 0.039–5.0 mm (XDT stock solution: 10 mg/ml or 10.6 mm, dissolved in dimethyl sulfoxide; the stock solution was diluted with 50 mm sodium acetate buffer, pH 4.5). The XDT conversion reaction was carried out in a volume of 300 μl (containing 100 μl of 0.01 mg/ml enzyme solution in 50 mm sodium acetate buffer, plus 200 μl XDT solution) at pH 4.5 and 45 °C for 40 min. 700 μl methanol was mixed with 300 μl of each reaction solution, and the mixtures were analyzed via HPLC for DT formation. The kinetic data were processed via a proportional weighted fit using a nonlinear regression analysis program based on Michaelis-Menten enzyme kinetics (35Leatherbarrow R.J. GraFit, Version 4.0. Ericathus Software, Horley, Surrey, UK1998Google Scholar, 36Lee R.C. Hrmova M. Burton R.A. Lahnstein J. Fincher G.B. Bifunctional family 3 glycoside hydrolases from barley with α-L-arabinofuranosidase and β-D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.J. Biol. Chem. 2003; 278: 5377-5387Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The stability of the recombinant enzyme was determined by measuring the released p-nitrophenol from PNP-Xyl as described previously. For determining the pH stability, the enzyme was properly diluted with various 50 mm buffers including sodium acetate (HAc, pH 2.0–5.0), potassium phosphate (PBS, pH 6.0–7.0), and Tris-HCl (pH 8.0–12.0) and incubated at 4 °C for 24 h. For testing the thermal stability, the enzyme was properly diluted with 50 mm Tris-HCl buffer (pH 8.0) and incubated at 4 °C, 25 °C, 37 °C, 45 °C, 60 °C, and 85 °C for 24, 48, and 72 h or at 25 °C for 1, 2, 3, 7, and 14 d and then cooled to 4 °C before analysis. For detecting the stability in the presence of metal ions (K+, Mn2+, Ag+, Cu2+, Fe2+, Co3+, Zn2+, Ca2+, Mg2+) and other reagents (EDTA and urea), the enzyme was properly diluted with 50 mm Tris-HCl buffer (pH 8.0) containing 5 nm of KCl, MnSO4, AgNO3, CuSO4, FeSO4, CoCl3, ZnCl2, CaCl2, MgSO4, EDTA, or urea and incubated at 4 °C for 24 h. The enzyme solution without metal ions or agents was used as the control, and the activity was recorded as 100%. The active protein bands (designated as LXYL-P1 and LXYL-P2) on the gel were cut and digested with trypsin. The digested samples were used for LC-MS/MS analysis. Accurate mass LC-MS and MS/MS data were collected in high-definition DDA (data-dependent) mode. LC-MS/MS data were processed using ProteinLynx Global Server Version 2.3 (Waters, Milford, MA), and the resulting peaklists were subjected to searches against the NCBInr protein database with the Mascot search engine. If the identical hit of the peaklists was not found in the NCBInr database, the spectra with the highest ion intensities were selected for de novo sequencing using Masslynx Pepseq 4.1 software. The fungal strain was cultured in the liquid wheat bran medium as mentioned above for 4 days, and the mycelia were filtered and ground into fine powder in liquid nitrogen. The genomic DNA was extracted via the genomic DNA isolation mini-prep method (37Zhao R.Y. Xiao W. Cheng H.L. Zhu P. Cheng K.D. Clo" @default.
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• W2134735523 title "Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues" @default.
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