Purification, Characterization, Cloning, and Expression of a Novel Xyloglucan-specific Glycosidase, Oligoxyloglucan Reducing End-specific Cellobiohydrolase

Abstract

A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed ∼35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed inEscherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained. A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed ∼35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed inEscherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained. Xyloglucan is a major hemicellulose polysaccharide found in the primary cell wall of plants. It constitutes ∼20% of the total cell wall in dicot and non-graminaceous monocot plants and 5–10% of Gramineae cell walls. Xyloglucan associates with cellulose microfibrils by hydrogen bonding, forming a cellulose-xyloglucan network (1Hayashi T. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 139-168Google Scholar, 2Fry S.C. Smith R.C. Renwick K.F. Martin D.J. Hodge S.K. Matthews K.J. Biochem. J. 1992; 282: 821-828Google Scholar, 3Carpita N.C. Gibeaut D.M. Plant J. 1993; 3: 1-30Google Scholar). Xyloglucan consists of a cellulose-like backbone chain of β-1,4-linked β-d-Glcp residues that are frequently substituted at C-6 with side chains of α-d-Xylp-(1→6)-, 1The abbreviations used are: Xyl, xylose; OXG-RCBH, oligoxyloglucan reducing end-specific cellobiohydrolase; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; CAPS, 3-(cyclohexylamino)propanesulfonic acid; RACE, rapid amplification of cDNA ends; ORF, open reading frame. β-d-Galp-(1→2)-α-d-Xylp-(1→6)-, or α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp-(1→6)- residues. Each segment has a specific code letter depending on the side chains. The letters G, X, L, and F refer to an unbranching β-d-Glcp residue, an α-d-Xylp-(1→6)-β-d-Glcpsegment, a β-d-Galp-(1→2)-α-d-Xylp-(1→6)-β-d-Glcpsegment, and an α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp-(1→6)-β-Glcpsegment, respectively (4Fry S.C. York W.S. Albersheim P. Darvill A. Hayashi T. Joseleau J.P. Kato Y. Lorences E.P. Maclachlan G.A. McNeil M. Mort A.J. Reid J.S.G. Seitz H.U. Selvendran R.R. Voragen A.G.J. White A.R. Physiol. Plant. 1993; 89: 1-3Google Scholar). Structural studies suggest that most xyloglucan consists of XXXG or XXGG repeating units (5Vincken J.P. York W.S. Beldman G. Voragen A.G.J. Plant Physiol. 1997; 114: 9-13Google Scholar). In dicots, treatment of xyloglucan with endo-β-1,4-glucanase results in the cleavage of unbranched, 4-linked Glc residues in the backbone chain, generating mainly oligosaccharide subunits (XXXG, XLXG, XXLG, XLLG, XXFG, XLFG, and so on) (6Kato Y. Matsuda K. BeMiller J.N. Manners D.J. Sturgeon R.J. Whistler R.L. Methods in Carbohydrate Chemistry. X. John Wiley & Sons, Inc., New York1994: 207-216Google Scholar). Disassembly of the cellulose-xyloglucan network is required for cell expansion and development. Xyloglucan metabolism is thought to have an important role in cell definition, cell expansion, and regulation of plant growth and development (7York W.S. Darvill A.G. Albersheim P. Plant Physiol. 1984; 75: 295-297Google Scholar, 8Fry S.C. J. Exp. Bot. 1989; 40: 1-11Google Scholar, 9McDougall G.J. Fry S.C. Planta. 1988; 175: 412-416Google Scholar, 10McDougall G.J. Fry S.C. Plant Physiol. 1990; 93: 1042-1048Google Scholar, 11Augur C. Yu L. Sakai K. Ogawa T. Sinay P. Darvill A.G. Albersheim P. Plant Physiol. 1992; 99: 180-185Google Scholar). Recently, it was reported that xyloglucan integration by xyloglucan endotransglycosylase in the cell wall suppresses cell elongation, whereas that of the xyloglucan oligosaccharide XXXG accelerates elongation (12Takeda T. Furuta Y. Awano T. Mizuno K. Mitsuishi Y. Hayashi T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9055-9060Google Scholar). It was proposed that xyloglucan metabolism controls plant cell elongation. In the growing plant cell wall, xyloglucan oligosaccharides may provide positive or negative feedback control during cell elongation. Consequently, compositional analysis of oligosaccharide units in xyloglucan polymers is very important. Glycosidases are useful tools for analyzing the fine structure of complex carbohydrates, and screening and characterizing novel glycosidases are important for ensuring progress in glycotechnology. In this study, we report the purification, characterization, cloning, and expression of a novel oligoxyloglucan-specific β-glycosidase produced by Geotrichum sp. M128 fungus, which was isolated from soil in Tsukuba, Japan. The enzyme recognized the reducing end of the oligoxyloglucan heptasaccharide XXXG and produced the tetrasaccharide XX and the trisaccharide XG. This enzymatic activity is very different from the activities of known glycosidases and constitutes a potentially powerful tool for the structural analysis of cell wall xyloglucan. It is also shown that the enzyme had minor activity for the reducing end of cello-oligosaccharides, releasing cellobiose. Based on these results, we propose oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH) as the trivial name of this new enzyme. Geotrichum sp. M128 was isolated from soil in Tsukuba, Japan, and deposited in the International Patent Organism Depository, National Institute of Advanced Industrial Science and Technology (Ibaraki, Japan) under FERM P-16454. Enzymatic activity was assayed by normal phase HPLC. The Geotrichum sp. M128 culture supernatant was incubated with 1% oligoxyloglucan heptasaccharide XXXG prepared using a previously described method (12Takeda T. Furuta Y. Awano T. Mizuno K. Mitsuishi Y. Hayashi T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9055-9060Google Scholar). The reaction mixture (10 μl) was loaded onto a TSKgel Amide 80 column (Tosoh Co., Tokyo, Japan) and eluted with 58% acetonitrile (isocratic) at a flow rate of 0.6 ml/min to analyze the degradation products. Product structures were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and 13C NMR spectroscopy. MALDI-TOF mass spectra were recorded using a Kompact MALDI IV instrument (Shimadzu Co., Kyoto, Japan) according to previously described methods (13Yamagaki T. Mitsuishi Y. Nakanishi H. Biosci. Biotechnol. Biochem. 1998; 62: 2470-2475Google Scholar). NMR spectra were obtained with a JNM-GX270 apparatus (Jeol Ltd., Tokyo) at 68.5 MHz in the SGBCM mode, with complete proton decoupling at 24.5 °C, using D2O as a solvent in a 0.5-cm inner diameter tube. Chemical shifts were reported as δ (ppm) relative to the resonance of sodium 4,4-dimethyl-4-silopentano-1-sulfonate, which was used as an internal standard (δ = 0). The spectral width was 200 ppm. Geotrichum sp. M128 was cultivated in 4-liters cultures consisting of 0.8% xyloglucan from Tamarindus indica (Glyloid, Dainippon Pharmaceutical, Osaka, Japan), 0.8% Bactopeptone, 0.2% KH2PO4, 0.05% MgSO4, and 0.05% yeast extract (pH 6.0) at 30 °C for 6 days. Cells were removed by centrifugation, and the broth supernatant was concentrated by ultrafiltration. The concentrated supernatant was diluted with 25 mm imidazole HCl buffer (pH 7.4), loaded onto a chromatofocusing column containing Polybuffer Exchanger PBE media, PBE 94 (Amersham Biosciences), equilibrated with 25 mm imidazole HCl buffer (pH 7.4), and then eluted with a linear gradient of NaCl (0–0.5 m). Each separated fraction was assayed for XXXG hydrolytic activity by the method of Nelson (14Nelson N. J. Biol. Chem. 1944; 153: 375-380Google Scholar) and Somogyi (15Somogyi M. J. Biol. Chem. 1945; 160: 61-68Google Scholar, 16Somogyi M. J. Biol. Chem. 1952; 195: 19-23Google Scholar). The active fractions were pooled and then applied to the same column with a pH gradient using Polybuffer 74 (Amersham Biosciences). The active fractions were gel-filtrated twice with an HW55F column (Tosoh Co.). The final active fraction was subjected to SDS-PAGE. Mcllvaine buffer solutions (pH 2.5–9.0), prepared from 0.2 m disodium hydrogen phosphate and 0.1m citric acid, were used to identify the optimum pH and to determine pH stability. The optimum pH was examined by incubation with the oligoxyloglucan heptasaccharide XXXG at 45 °C for 30 min in Mcllvaine buffer solutions. The pH stability was assayed by incubating the enzyme, in the absence of substrate, at 45 °C for 30 min in buffer solutions. The buffer solutions were then adjusted to pH 4.0, and the remaining activity was assayed. The optimum temperature was determined by incubation in 50 mm sodium acetate (pH 4.0) at various temperatures. To analyze thermostability, the enzyme was incubated in the same buffer, without substrate, for 10 min at various temperatures. The remaining activity was then assayed at 45 °C. The substrate specificity of purified OXG-RCBH was analyzed with various, well defined xyloglucan oligosaccharide structures prepared from tamarind seed using previously described methods (17Konishi T. Mitsuishi Y. Kato Y. Biosci. Biotechnol. Biochem. 1998; 62: 2421-2424Google Scholar). The resulting products were analyzed by normal phase HPLC. The purified protein was separated by SDS-PAGE and transferred from the gel onto a polyvinylidene difluoride membrane filter (Millipore Corp.) in transfer buffer (10 mm CAPS (pH 11) and 10% methanol). The amino acid sequence was determined by automatic sequential Edman degradation using a Procise 494 HT protein sequencing system (Applied Biosystems). To determine internal amino acid sequences, the purified protein was digested with lysyl endopeptidase (Wako Pure Chemical Industries, Ltd., Osaka). The resulting peptide fragments were fractionated by reverse phase HPLC on a TSKgel ODS-80Ts QA column (Tosoh Co.), and their N-terminal amino acid sequences were analyzed. Geotrichum sp. M128 total RNA was obtained from 3 ml of culture using a FastRNA RED kit (Bio 101, Inc.). The mRNA was prepared with an oligo(dT) column using a QuickPrep mRNA purification kit (Amersham Biosciences). The cDNA was synthesized from mRNA with an oligo(dT) primer using a TimeSaver cDNA synthesis kit (Amersham Biosciences). First, the cDNA fragment was amplified by PCR with degenerate primers designed from the N-terminal and internal (peptide 2) amino acid sequences: F1, 5′-GA(AG)CA(CT)TA(CT)GA(AG)TT(CT)AA(AG)AA(CT)GT-3′; and R1, 5′-GT(ACGT)CCCCA(AGT)AT(AG)AA(ACGT)AC(ACGT)GC-3′. The amplified fragment was subcloned into the T-overhang vector pGEM-T-Easy (Promega). Dideoxy double-stranded sequencing of the cDNA insert was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions, and the expected amplified fragment was obtained and confirmed. The complete nucleotide sequences were determined by 5′- and 3′-RACE using a SMART RACE cDNA amplification kit (Clontech). For 5′-RACE, the following gene-specific primers were designed: R2, 5′-CGTACAGCAGGTCCTTGGTCTTTGG-3′; and R3 (nested primer), 5′-TAATGTACCCGCCGCCGCCGAT-3′. For 3′-RACE, F2 (5′-GGCAAGTTCTTCGTCTCGACCGAC-3′) and F3 (nested primer, 5′-CCAAGTCGGACGGCAAGAAGGCTA-3′) were used as the gene-specific primers. The cDNA fragment encoding the mature region of OXG-RCBH was amplified by PCR with primers designed from 5′- and 3′-sequences corresponding to the N- and C-terminal regions of the mature protein: F4, 5′-TCGCTCGCGCATATGAAGGAGCACTACGAGTTCAAGAATG-3′; and R4, 5′-TGGAAGATCTTTACTTTTTCACATAGCAGTGCGTGCCC-3′. TheNdeI and BglII sites (underlined) were added to the forward and reverse primers, respectively. The amplified DNA was digested with NdeI and BglII, subcloned into the pET-29a(+) expression vector (Novagen), digested with the same restriction enzymes, and transfected into E. coliBL21-CodonPlus(DE3) RP (Stratagene). The transfected cells were cultured, and expression was induced with 1 mmisopropyl-β-d-thiogalactopyranoside for 6 h at 37 °C. The recombinant protein was produced as an inclusion body that was extracted and partially purified with BugBuster protein extraction reagent (Novagen). The protein was solubilized at a concentration of 5 mg/ml in 50 mm Tris-HCl (pH 8.0) containing 8 m urea and 1 mm EDTA for 1 h at room temperature and renatured by removing the urea by dialysis against 25 mm imidazole HCl buffer (pH 7.4). The oligoxyloglucan heptasaccharide XXXG was incubated with the Geotrichum sp. M128 culture supernatant, and the reaction products were separated by normal phase HPLC (Fig. 1B). Two products (a and b) were observed, and MALDI-TOF mass spectrometry and 13C NMR spectroscopy were used to identify their structures. The MALDI-TOF mass spectra of the reaction products revealed the presence of two main components with m/z 497 andm/z 629, indicating that products a and b were the trisaccharide XG and the tetrasaccharide XX, respectively (Fig. 1D). The results of further structural analysis by13C NMR spectroscopy are shown in Fig. 2. The anomeric carbon chemical shifts were compared with those of xyloglucan oligosaccharides shown in Ref.18York W.S. van Halbeek H. Darvill A.G. Albersheim P. Carbohydr. Res. 1990; 200: 9-31Google Scholar. The results suggested that product a was the trisaccharide XG (Fig. 2A). Although XX dose data was not available in Ref. 18York W.S. van Halbeek H. Darvill A.G. Albersheim P. Carbohydr. Res. 1990; 200: 9-31Google Scholar, it seemed reasonable to conclude that product b was the tetrasaccharide XX (Fig. 2B). These results suggest that Geotrichumsp. M128 produced a glycosylhydrolase (OXG-RCBH in this study) that hydrolyzed XXXG to produce XX and XG in the culture supernatant.Figure 213 C NMR spectroscopy of products a (A) and b (B). Chemical shifts are shown in the insets.View Large Image Figure ViewerDownload (PPT) OXG-RCBH activity was observed in the Geotrichum sp. M128 culture supernatant, indicating that it was expressed as a secreted protein in the culture supernatant. OXG-RCBH was purified from the culture supernatant by liquid chromatography (Table I). Each separated fraction was assayed for XXXG hydrolytic activity. Throughout these steps, enzymatic activity always appeared as a single peak. The final purified fraction had an XXXG hydrolytic activity of 15.31 units/mg of protein (Table I). This fraction was subjected to SDS-PAGE, revealing a single 97-kDa band (Fig. 3). Chromatofocusing showed that its pI was 6.1 (data not shown).Table IPurification of OXG-RCBH from Geotrichum sp. M128StepActivityaOne unit of activity is defined as the amount of enzyme that released 1 μmol of glucose eqs/min as reducing sugars from the substrate.Total proteinSpecific activity(units)(mg)(units/mg protein)Culture supernatant216.7Ultrafiltration197.21,158.30.17PBE94Salt gradient97.9128.70.76pH gradient42.514.32.97HW55F1st gel filtration27.82.89.932nd gel filtration19.91.315.31a One unit of activity is defined as the amount of enzyme that released 1 μmol of glucose eqs/min as reducing sugars from the substrate. Open table in a new tab The effect of pH and temperature on purified OXG-RCBH was analyzed. The optimum pH was between 3.5 and 5.0, and the optimum temperature was between 50 and 60 °C. The pH stability was determined at 45 °C using Mcllvaine buffer solutions at various pH values. OXG-RCBH was stable between pH 4.0 and 8.0, where >90% of the activity was retained. Thermostability was analyzed at pH 4.0 by incubation at various temperatures for 10 min. More than 90% activity was retained at 50 °C. Various oligosaccharide structures were used to analyze the substrate specificity of OXG-RCBH, and the results are shown in Table II. OXG-RCBH had very unique substrate specificity; the results indicated that it was an exoglucanase that recognized the structure of the reducing end of oligoxyloglucan and that released the two glucosyl main chain residues (GG, XG, or LG). When there were less than three Glc residues on the main chain, the activity decreased significantly, suggesting that OXG-RCBH has at least four subsites (−2 to +2). It was very important that the reducing end of the Glc residue (site +2) was unbranched. No OXG-RCBH activity was seen for substrates with reducing ends branched with Xyl residues. It was also important that the third Glc residue from the reducing end (site −1) was branched with Xyl unmodified by a Gal residue. If this Glc residue was branched with Xyl modified with a Gal residue, OXG-RCBH was not active; and if it was unbranched, the activity decreased significantly. Based on these results, we propose the subsite image shown in Fig. 4.Table IISubstrate specificity of OXG-RCBHSubstrateRelative activityFinal products(%)XXXGXXXG96.7XXXGXX, XGXXXGG98.2XXX, GGXXXG100.0XX, XGXXLG96.5XX, LGXLXG0.0XLLG0.0GXXG60.2GX, XGXXGG64.8XX, GGGGGG5.7GG, GGXXX0.0XXG28.3X, Open table in a new tab OXG-RCBH was analyzed to determine its N-terminal amino acid The following amino were To analyze the internal amino acid sequences, OXG-RCBH was digested with lysyl and the resulting peptide fragments were separated by reverse phase HPLC. peptide fragments were analyzed to their N-terminal amino acid The amino acid sequences were as peptide peptide and peptide PCR was using cDNA from Geotrichum sp. M128 as a and degenerate primers and designed from the N-terminal and internal (peptide 2) amino acid The resulting DNA fragment of was subcloned and The complete cDNA nucleotide sequences were determined by 5′- and 3′-RACE, and the full-length cDNA encoding OXG-RCBH was The cDNA sequence a 2436-bp open reading frame encoding a acid protein (Fig. The for a M. Res. Scholar, M. Proc. Natl. Acad. Sci. U. S. A. 1990; were The deduced amino acid sequence the amino acid sequences obtained from the purified protein. were sites The N-terminal amino acid sequence of the mature protein at the amino acid residue, indicating that amino were from the of the mature These amino to a N-terminal cleavage in an protein containing amino acid residues. the enzyme isolated from the Geotrichum sp. M128 culture had a molecular mass of 97 the in molecular mass between these two may from the presence of a or The deduced amino acid identity to in the hydrolase 74 family (Fig. M. A. J. H. Mol. Scholar, R.J. Nelson L. J. White Smith Scholar, P. 40: Scholar, J. G. J. J. R.L. L. P. Smith J. Scholar, G. J. H. A. J. A. C. White J.P. 2002; Scholar, E. van M. van 2002; indicating that OXG-RCBH in this The of most of the in this family are was proposed as an analysis has not has and it was reported that it activity for and and xyloglucan E. van M. van 2002; Scholar). the OXG-RCBH had exoglucanase recognized the reducing end of and produced two glucosyl residue as described This is to that of cellobiohydrolase C. J. T. L. G. which cellulose at the reducing end to produce cellobiose. sequence was observed between these To that the protein by the cloned cDNA had enzymatic cDNA was expressed in E. This has of the which that the and and the respectively. of are found in OXG-RCBH Although we OXG-RCBH in the expression was than in RP (data not A protein of kDa was expressed in the cells (Fig. the we recombinant OXG-RCBH was expressed as an inclusion body (Fig. were temperature of °C and concentration of mm or a different cell OXG-RCBH activity was observed in the fraction (data not to recombinant inclusion body (Fig. was solubilized in 8 m urea and renatured by this renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained units/mg of The of recombinant OXG-RCBH was from 1 of the of pH and temperature on recombinant OXG-RCBH revealed that were very to those on the recombinant OXG-RCBH found in this useful for further as studies of molecular of and structural In this study, we described a novel oligoxyloglucan-specific OXG-RCBH, sp. M128 that was and expressed in E. OXG-RCBH recognized the reducing end of oligoxyloglucan and produced two glucosyl residue Although with specific activity for xyloglucan have reported E. van M. van 2002; Scholar, J.P. Beldman G. Voragen A.G.J. Carbohydr. Res. 1997; Scholar, M. S. York W.S. Albersheim P. Darvill A. OXG-RCBH activity was very different from known OXG-RCBH to a powerful tool for the fine structure of