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J. Biol. Chem., Vol. 281, Issue 6, 3137-3144, February 10, 2006

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Exploration of Glycosyl Hydrolase Family 75, a Chitosanase from Aspergillus fumigatus^*

From the Center for Interdisciplinary Molecular Science and Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan

Received for publication, November 22, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
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 (GenBank^TM accession number AY190324 [GenBank] ), 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 ¹H 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 Asp¹⁶⁰ and Glu¹⁶⁹ are the essential residues for the action of A. fumigatus endo-chitosanase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Chitosanase (EC 3.2.1.132 [EC] ) is a hydrolytic enzyme acting on the -1,4-glycosidic linkage of chitosan, a linear biopolymer of -1,4-linked GlcN, to release chito-oligosaccharides. The oligomers GlcNAc and GlcN have interesting biological activities (1), including anti-tumor effects (2, 3), hypo-cholesterolemic effects (4), anti-microbial activities (5, 6), disease-resistance responses, and as phytoalexin elicitors in higher plants (7, 8). Hence chitosanase, chito-oligosaccharides and their derivatives have attracted interest from the food and pharmaceutical industries because they can be used as edible additives, agricultural immunity controls and promise many other prospective applications as prophylactic agents for liver diseases (4), atherosclerosis, and hypertension.

Chitosanases have been found and cloned from several organisms, such as viruses (9), bacteria (10), fungi (11–14), and plants (15). According to amino acid sequence homologies (16), chitosanases have been classified into five glycoside hydrolase families: GH-5,² GH-8, GH-46, GH-75, and GH-80. GH-5 and GH-8 contain a variety of glycoside hydrolases, such as chitosanase, cellulase, licheninase, and endo-1,4--xylanase, whereas, GH-46, GH-75, and GH-80 are currently exclusively classified for chitosanase. Among these families, GH-46, especially those from Bacillus (17) and Streptomyces strains (18), have been studied extensively for their catalytic features (18), enzymatic mechanisms (19), and protein structures (20), whereas GH-75 and GH-80 have only been studied for protein purification (11–14) and gene cloning (21, 22). An interesting finding is that all GH-75 chitosanases are fungal enzymes, such as those from Aspergillus (11, 14), Penicillium (12), Beauveria (GenBank^TM accession number AY008269 [GenBank] ), Cordyceps (GenBank^TM accession number AY008269 [GenBank] ), Fusarium (13), Hypocrea (GenBank accession number AY571342 [GenBank] ), Magnaporthe (GenBank^TM accession number AACU01000927), Metarhizium (GenBank^TM accession number AJ293219 [GenBank] ), and Neurospora (GenBank^TM accession number AABX01000003). Because the expression of these eukaryotic chitosanases is relatively inaccessible, studies on their catalytic details are scarce and limited.

Based on the anomeric configuration of the C1 proton of the reducing end sugar obtained from the enzymatic products, chitosanases from different families may involve different types of catalysis: either the retaining or the inverting mechanism. The retaining glycosidases catalyze hydrolysis via a two-step, double-displacement mechanism with one of the two essential amino acid residues functioning as a nucleophile and the other as a general acid/base. By contrast, the inverting glycosidases follow a one-step, single displacement mechanism with the assistance of a general acid and a general base. The general base polarizes a water molecule to develop a stronger nucleophile for attacking the anomeric carbon, whereas the general acid protonates the glycosidic oxygen to accelerate the reaction. In the five families containing chitosanase, the retaining configuration of the catalytic mechanism of GH-5 is derived from glucanase (23). The inverting configurations have been verified experimentally for GH-8 (24) and GH-46 (18, 19). GH-80 is also inferred to be an inverting enzyme. However, the catalytic stereochemistry of GH-75 enzyme remains unknown.

In our previous study, a powerful endo-chitosanase (further identified as belonging to the GH-75 family) was induced and purified from the culture medium of Aspergillus fumigatus (11). The purified chitosanase was used for the large scale preparation of chito-oligosaccharides (11). Here we describe the cloning of this gene, its overexpression in inclusion bodies, and protein refolding. In addition, we analyzed its enzymatic products and performed site-directed mutagenesis. This study is the first extensive investigation of GH-75.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Chitosans and Chito-oligosaccharides—Chitosans with different degrees of deacetylation (DDA) were obtained from local suppliers in Taiwan. The DDA values of chitosan were confirmed by quantitatively comparing the signals of the methyl group of the C2 acetyl moiety and the C1 proton in sugar ring with ¹H NMR spectroscopy. Most of the chitosans used in this study comprised at least 85% DDA unless otherwise specified. Chito-oligosaccharides (chitodimer, chitotrimer, chitotetramer, chitopentamer, and chitohexamer) were purchased from the Seikagaku Corporation (Tokyo, Japan).

Bacterial Strains, Plasmid, and Culture Conditions—The A. fumigatus Y2K strain screened from soil, with chitosan as the sole source of carbon (11), was used for gene cloning. For cultivation and enzyme induction, M9 medium with the following composition was used: 1.3 g of Na₂HPO₄, 3.0 g of KH₂PO₄, 0.5 g of NaCl, 1.0 g of NH₄Cl, 0.24 g of MgSO₄, and 0.01 g of CaCl₂ (in 1 liter). The medium also contained 1% chitosan and was maintained at a pH of 6.0. A spore suspension of 1 x 10¹⁰ was transferred to 1 liter of the culture medium at 28 °C and agitated at 120 rpm on a rotary shaker. The induced chitosanase and chromosomal DNA were prepared from 5-day cultures, and the total RNA extraction used a four-day culture. Escherichia coli strains TOP10 and pCR 2.1 TOPO-vector (TOPO TA Cloning kit; Invitrogen) were used for DNA cloning, and BL21 (DE3) and pRSET A vector (Stratagene, La Jolla, CA) were used as the protein expression systems.

Isolation of Chromosomal DNA and RNA—Mycelia of A. fumigatus were collected by filtration and ground to a fine powder with a mortar and pestle under liquid nitrogen. Chromosomal DNA was then extracted by the phenol-chloroform method, and total RNA was isolated through acidic guanidinium thiocyanate-phenol-chloroform extraction (25).

Construction of the cDNA Library—RNA isolated from the mycelium was used to construct a cDNA library. The method of cDNA synthesis was modified from a SMART PCR kit (Clontech Laboratories, Palo Alto, CA) (26). The set of smart primers, pT and pG, were designed and synthesized with the sequences 5'-AAA CAG TGG TAA CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN-3' and 5'-AAA CAG TGG TAA CAA CGC AGA GTA CGC GGG-3', respectively, and were used for reverse transcription-PCR amplification (ThermoScript^TM reverse transcription-PCR system; Invitrogen). The reverse transcription was performed at 65 °C for 1 h and subjected to PCR with the following parameters for 30 cycles: 94 °C for 30 s, 65 °C for 30 s, 68 °C for 2 min, an initial denaturing step at 94 °C for 5 min, and a final extension at 68 °C for 8 min. The freshly prepared cDNA was purified on elution through a GFX column (Amersham Biosciences), cloned into the pCR 2.1 TOPO vector, and transformed into competent E. coli TOP10 for the cDNA library construction.

Amplification of cDNA Fragments Containing csn—According to the N-terminal sequence of the mature CSN (11), two degenerate primers, p1 and p2, were designed. The p1 primer, 5'-TAY AAY YTR CCA CCY AAY YTR AAR C-3', was designed on the basis of the first eight amino acids of CSN, and the p2 primer, 5'-CNA AYA AYY TNA ARC ARA THT AYG AYG A-3', was synthesized according to the sequence obtained from the fourth to the twelfth amino acid. These primers were paired with pT for PCR using the cDNA library as the template for amplification of the csn gene fragment. These fragments were then cloned into pCR 2.1 TOPO and sequenced.

Amplification of the Genomic DNA of CSN—Genomic DNA of CSN was amplified using p2 and p3 (5'-GGA TCC GTT CTA GTT CGC TAT GCT TTC AA-3') as primers and chromosomal DNA as the template. The amplified DNA fragment was cloned into pCR 2.1 TOPO for sequencing.

Inverse PCR—The upstream nucleotide sequence containing information about the signal peptide was cloned using inverse PCR according to Ochman et al. (27). In general, chromosomal DNA was digested with restriction enzymes. DNA fragments of suitable size were then purified and self-ligated to form circular DNAs, which then served as the template for inverse PCR. Using a set of "outward going" primers, the circular DNA is then amplifiable. In this work, chromosomal DNA (5 µg) was completely digested with EcoRI. The digested fragments, ranging in size between 500 and 4000 bp, were isolated from the gels of DNA electrophoresis and further self-ligated with T4 DNA ligase (Hoffmann-La Roche) at 16 °C overnight. Using a GFX column, the ligated DNA fragments were purified and subjected to serve as a template for inverse PCR. Outward going primers were used to amplify the CSN-related gene function in pairs and were designed as p4 (5'-CTT GTG TTT GTC GTA GAT CTG TTT CAA GT-3') and p5 (5'-GGA AAA TGT TCC AAG GTA CTG GCA AAA G-3'). The cycling parameters of PCR amplification were as follows: 94 °C for 30 s, 53 °C for 30 s, 72 °C for 2 min (30 cycles), with an initial denatured step at 94 °C for 5 min, and a final extension at 72 °C for 10 min to complete the unfinished single-stranded products. The inverse PCR product was purified and inserted into pCR 2.1 TOPO for sequence analysis.

Construction of Expression System of Recombinant Chitosanase—For the reconstruction of csn in an expression vector, a p6 primer (5'-CAT ATG TAC AAT TTG CCA AAC AAC TTG AAA C-3') was synthesized with the insertion of a NdeI restriction site (CATATG) before the nucleotide sequence of the first eight residues of the mature protein. Using p3 and p6 as primers and the partial cDNA fragment of csn (described above) as template yielded the mature CSN gene. This PCR product was cloned into pCR 2.1 TOPO and further transferred into the pRSET A expression vector between the NdeI and BamHI sites. The resulting vector was named pRSET-csn and expressed in E. coli strain BL21 (DE3).

Chitosanase Activity Assays—Chitosanase activity was analyzed by estimating the amount of the reducing ends of sugars using the dinitrosalicylic acid method (28). The standard assay was prepared by mixing 0.3 ml of chitosan (1%, pH 6.0) and 0.3 ml of enzyme (at a suitable dilution) and incubated for 4 h at 37°C to allow for the completion of hydrolysis; 0.6 ml of dinitrosalicylic acid reagent was then added, and the resulting mixture was boiled for 15 min, chilled, and centrifuged to isolate the insoluble chitosan. The resulting adducts of reducing sugars were analyzed and measured spectrophotometrically at 540 nm. The absorption coefficient of the resulting adducts was determined to be 788 M^–1 cm^–1 when D-glucosamine was used as the control sample. One unit of chitosanase activity was defined as the amount of enzyme required to release 1 µmol of detectable reducing sugars at 37 °C in 1 min (29). Alternatively, the products could be analyzed using mass spectrometry with electrospray ionization as described below.

Protein Expression, Refolding, and Purification—E. coli strain BL21 (DE3) harboring the plasmid pRSET-csn was grown overnight in LB medium. This culture (2.5 ml) was freshly inoculated into the same medium (250 ml) supplemented with ampicillin at a final concentration of 0.1 mg/ml. The culture was incubated at 37 °C for 14 h, and the cells were then collected and resuspended in phosphate buffer (20 mM, pH 6.0) for sonication. The cell debris was then collected by centrifugation and treated with 5 M urea at 37 °C for 4 h. The supernatant was decanted and kept at 4 °C for at least 3 days to allow protein refolding. Without removing the urea, the supernatant was subjected to cation-exchanged chromatography (HiTrap^TM in 5 ml SP-Sepharose; Amersham Biosciences). The column was pre-equilibrated with phosphate buffer (pH 6.0, 20 mM) and eluted with a linear gradient of NaCl (from 170 to 240 mM; 10 mM/min) in the same buffer at a flow rate of 2 ml/min. The fractions with chitosanase activity were pooled and concentrated by ultrafiltration using Vivaspin 6 centrifugal concentrator with 3,000 molecular weight cut-off, (Vivascience, Edgewood, NY). For purification of inactive mutants, SDS-PAGE separation was used for analyzing the presence of target proteins in all of the fractions collected from column chromatography.

Protein Determination—The protein content of the enzyme preparation was determined either by the BCA method as described in the manufacturer's protocol (BCA-1 kit for protein determination; Sigma-Aldrich, St Louis, MO) or by UV absorption at 280 nm.

Nuclear Magnetic Resonance Study—The stereochemistry of enzymatic hydrolysis was determined by ¹H NMR spectroscopy at 40 °C (BioSpin AVANCE 500 spectrometer; Bruker, Rheinstetten, Germany). The chitosan mixture was prepared by dissolving 0.05 g of chitosan containing 98% DDA in 10 ml of 1% acetic acid. This solution was lyophilized and exchanged with D₂O repeatedly. The chitosanase for the NMR measurements was also exchanged with deuteriophosphate buffer (20 mM; pD 6.5) by ultrafiltration. After recording the spectrum of the substrate (450 µl), chitosanase (50 µl, 4 mg/ml) was added to the chitosan solution (0.5%). The spectra were recorded 5, 10 15, 20, 30, and 60 min after the addition of the enzyme. All the spectra were recorded over a 5000-Hz sweep width, with eight scans for a total recording duration of 25 s each.

Chemical Methylation of Chitosan Hydrolysate—The chito-oligosaccharides were obtained from enzymatic hydrolysis of chitosan containing 40% DDA. For identification of the structure of these oligosaccharides, a suitable amount of oligosaccharides was chemically methylated at the reducing end of the sugar. Methylation was performed in a solution consisting of an excess of methanol and 2% perchloric acid at 50 °C for 10 h. The methylated oligosaccharides were analyzed with a liquid chromatography (LC) tandem mass spectrometer (LC/MS/MS).

Electrospray Ionization Mass Spectrometric (ESI-MS) Analysis— Mass spectra were recorded with a quadrupole time-of-flight mass spectrometer (Micromass, Manchester, UK). This was used to scan with a ratio of mass to charge in the range 100–2500 units (m/z), with a scan of 2 s/step and an interscan duration of 0.1 s/step. In all the ESI-MS experiments, the quadrupole scan mode was used under a capillary needle at 3 kV, a source block temperature of 80 °C, and a desolvation temperature of 150 °C. The mass of the desalted form of proteins used for the MS measurements was normally within the range of 5–10 µg.

For quantitative analysis of enzymatic activity, chito-oligomers were used as substrates, and the multiple reaction-monitoring scan of a triple quadrupole MS system (Quattro Micro; Micromass) was used. Chitohexamer (100 ppm) and chitopentamer (100 ppm) were mixed with properly diluted chitosanase. The reaction time course was determined using 10 µl of the enzyme mixture injected into the quadrupole MS every 3 min by an auto-sampler. Hydrolysis product ion (m/z 502.2, chitotrimer) was chosen as the specific precursor ion for positive ionization tandem mass spectrometry. Based on its fragment ion signal of m/z 162.0, instrument optimization was performed to achieve more sensitive detection, and the optimum conditions of collision energy setting and cone voltage were 20 and 30 volts, respectively.

Site-directed Mutagenesis—Site-directed mutagenesis for mutations was performed according to the QuikChange method (Stratagene). The basic procedure involved PCR amplification with pRSET-csn as the template and two synthetic oligonucleotides containing the desired mutation as the primer. Table 1 lists the sequences of primers used for the mutagenic tests. The desired mutations were confirmed by DNA sequencing of the full gene.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Molecular Cloning and Structure of the csn Gene—To avoid introns in the gene, a cDNA library of A. fumigatus was constructed. Time course tests showed that extracellular chitosanase activity reached a maximum with 5 days of culture, so a 4-day culture of mycelium was used for RNA extraction. The complete molecular cloning of csn involved several steps, as summarized in Fig. 1. Step 1 involved the amplification of cDNA from RNA. Using pT and pG as primers, cDNAs of varied size were synthesized. To obtain the corresponding chitosanase gene, two degenerate primers, p1 and p2, were designed according to the N-terminal sequence of the mature CSN (as described under "Experimental Procedures"). When p1 and pT were used as primers, a major DNA fragment of 900 bp was amplified (Step 2). The amino acid sequence deduced from the major DNA fragment was analyzed using the BLAST search on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/). The deduced sequence was highly homologous with a putative conserved domain of the NADH oxidase family (data not shown). However, when the p2 and pT primers were used, a cDNA fragment corresponding to csn was obtained (Step 3). To obtain information on its structure, we amplified a DNA fragment using p2 and p3 as primers and the chromosomal DNA of A. fumigatus as the template (Step 4). Sequence analysis revealed that two introns with 67 and 82 bp were incorporated in the genomic csn. The upstream nucleotides containing the sequence of the signal peptide was unwound by inverse PCR (Step 5), and the sequence of the full-length genomic csn has been published in the National Center for Biotechnology Information GenBank^TM with accession number AY190324 [GenBank] .

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Procedure for the molecular cloning of csn. Step 1, construction of a cDNA library; Steps 2 and 3, amplification of a cDNA fragment containing csn; Step 4, amplification of the cDNA fragment of csn; Step 5, inverse PCR; Step 6, construction of the expression system of recombinant chitosanase. The primer pairs used in each PCR are displayed in parentheses.

Overexpression, Purification, and Refolding of Chitosanase—When pRSET-csn was expressed in BL21 (DE3), weak chitosanase activity was detected in the cytosol. In contrast, protein with a molar mass of 25 kDa was observed in cell debris using SDS-PAGE analysis (Fig. 2), indicating the formation of an inclusion body of chitosanase. Attempts to control the expression level of chitosanase by decreasing the culture temperature to 20–25 °C showed no significant improvement in obtaining active enzyme in the cytosol. The crude enzyme was therefore isolated from the inclusion body of cell debris via 5 M urea treatment and further purified by column chromatography as described under "Experimental Procedures." After cation exchange chromatographic separation, purified chitosanase with more than 90% homogeneity was obtained. All other mutants (discussed below) were purified using the same protocol and had similar protein homogeneity. SDS-PAGE analysis of the wild-type CSN at various stages of purification is shown in Fig. 2a. Although many fungal chitosanases GH-75 enzymes have been cloned, this is the first report on their first successful expression and purification of the corresponding recombinant enzyme.

Catalytic Features of A. fumigatus Chitosanase—It is believed that the molecular mechanism is conserved within the same family of glycosyl hydrolases (16, 30). Hence, a successful investigation of one particular enzyme is valuable for characterizing general features and the understanding of the whole family. Insight into the catalytic mechanism is best revealed from the study of the x-ray structure. Kinetic and stereochemical study of the enzymatic reaction provides information that enables conclusions about the catalytic mechanism (31). To date, information on anomeric specificity has been obtained for all other chitosanase families, but not for family 75. Therefore, this study set out to determine the anomeric form of hydrolytic products of Aspergillus chitosanase from GH 75.

¹H NMR spectra have been commonly used to investigate the stereochemistry of various glycosyl hydrolases such as -galactosidase (32), -mannanase (33), cellulases, xylanase (24), -xylosidase, -1,3-glucanase (34), and chitosanase (18). Using temporal NMR spectra on enzymatic hydrolysis of chitosan has provided much insight into the catalytic mechanism of A. fumigatus chitosanase. Time-dependent ¹H NMR spectra showing the product of enzymatic hydrolysis of chitosan were obtained to determine the anomeric form of the reaction products. These experiments were carried out and monitored by ¹H NMR at 40 °C. For clear demonstration of the stereochemistry of the catalysis of this chitosanase, high concentrations of enzyme (final concentration, 0.4 mg/ml) were used. Fig. 3 shows a series of partial ¹H NMR spectra (4.8–5.8 ppm) recorded within 1 h after the addition of enzyme. The ¹H NMR spectrum of chitosan was taken before the addition of chitosanase (labeled as 0min in Fig. 3). As shown in the chitosan ¹H NMR spectrum, a poorly resolved doublet signal centered at 5.0 ppm (J = 7.8 Hz) was observed. This peak corresponds to the C1 proton of the internal GlcN residues of chitosan. The poor resolution of NMR spectrum of chitosan is because of the high viscosity of sample and the temperature effect (40 °C). The spectra were subsequently taken at 5, 10, 15, 20, 30, and 60 min after the addition of chitosanase. The results showed that a distinguish doublet centered at 5.56 ppm (J = 3.6 Hz) was instantly evolved in the 5-min reaction, whereas another doublet, centered at 5.08 ppm (J = 8.4 Hz), emerged gradually. According to a previous study on GH-46 chitosanase (18), the peaks at 5.56 and 5.08 ppm were assigned as the C1 anomeric proton of the reducing end sugar in its -form and -form, respectively. The peaks in the range of 4.92–4.98 ppm were assigned to the internal C1 protons of various chito-oligosaccharides. The ratio of signal intensity corresponding to the - and -forms of the reducing end GlcN decreased when the reaction time was increased, indicating a mutarotation process between the forms of the sugar. The ratio of / approaches 65/35 after an hour of incubation. This is in a good agreement with that of GlcN anomers in equilibrium state. Based on these NMR results, this chitosanase can be identified positively as an inverting enzyme. It can thus be concluded that the catalytic mechanism of GH-75 enzymes involves the inversion of an anomeric configuration. With the exception of family 5 chitosanase, chitosanases from GH-8 (24), GH-46 (18, 19), GH-80, and GH-75 exhibit an identical stereochemical preference.

View larger version (46K): [in this window] [in a new window]   FIGURE 2. Analyses of molecular weight by SDS-PAGE (a) and LC-mass spectrometry (b). SDS-PAGE analysis of the supernatant of cell lysate (lane 1), cell debris (lane 2), supernatant of cell debris treated with 5 M urea (lane 3), and the purified protein (lane 4). The mass spectrum of the purified chitosanase (inset) and the deconvolution of the spectrum to give a molar mass of 23592 atomic mass units.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Time course 1H NMR study on the enzymatic hydrolysis of chitosan at 40 °C. Spectra were obtained over 0, 5, 10, 15, 20, 30, and 60 min in the presence of chitosanase. Note that only a partial spectrum (chemical shift 4.8–5.8 ppm) is shown, and the spectrum of the initial (chitosan) substrate is labeled as 0 min. Peak assignment is given in the text.

The activity assay showed that D59N, D76N, D78N, D80N, D112N, D114N, D194N, and D229N retained significant catalytic activity (>60%) compared with that of the wild-type enzyme. Similar to that of wild-type chitosanase, all of these recombinant enzymes hydrolyze chitosan to release (GlcN)₃, (GlcN)₄, and (GlcN)₅ as the major product (data not shown). In contrast, the activities of the mutant D160N and E169Q were significantly reduced to less than 0.1%. CD spectrometric analyses were used to examine the possible structural alteration of D160N and E169Q, which turned out to be similar to those of recombinant enzymes with significant activity, including the wild-type CSN, D194N (Fig. 6), and many other mutants (data not shown). This indicated the presence of structural conservation for both the mutants. Using azide for trying to rescue both mutants showed no significant effect. It is postulated that the residues Asp¹⁶⁰ and Glu¹⁶⁹ are likely to be the essential groups of CSN. The conserved motif DXDXE is the catalytic center of GH-18 chitinase (39), whereas the conserved motif DIDCD in GH-75 seems to play a less important role in comparison. The mutation in this region of CSN caused only 30–40% loss of activity. The precise function of the conserved motif DIDCD in chitosanase is still undefined.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. MS analysis of chito-oligosaccharides obtained from enzymatic hydrolysis of low content DDA chitosan. a, oligosaccharides with m/z values of 341, 383, 502, 544, and 705 correspond to a chitodimer, a monoacetyl chitodimer, a chitotrimer, a monoacetyl chitotrimer, and a monoacetyl chitotetramer, respectively. ESI/MS/MS analyses of the chemically methylated chito-oligosaccharides (b–d) and illustrations of structural fragmentations (e–g). b and e, the methylated monoacetyl chitodimer (m/z 397); c and f, for the methylated monoacetyl chitotrimer (m/z 558); d and g, methylated monoacetyl chitotetramer (m/z 719).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Multi-alignment of GH-75 fungal chitosanases. The deduced amino acid sequences from five gene sequences were aligned by Biology WorkBench 3.2 CLUSTALW (San Diego Supercomputer Center, San Diego, CA) software. GenBankTM accession details are: AY190324 [GenBank] for A. fumigatus (this study); AB038996 [GenBank] for A. oryzae; AY008269 [GenBank] for B. bassiana; AJ293219 [GenBank] for M. anisopliae; and D85388 [GenBank] for F. solani. Only partial sequences were shown. The conserved Glu and Asp are boxed and numbered based on A. fumigatus CSN sequence.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. CD spectra of recombinant CSN. Mutants of D160N () and E169Q () exhibit the similar CD spectra as other recombinant proteins with significant chitosanase activity (wild type, solid line; D194N, dotted line).

	FOOTNOTES

¹ To whom correspondence should be addressed: Dept. of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Rd., Hsinchu 30030, Taiwan. Tel.: 886-3-5731985; Fax: 886-3-5723764; E-mail: ykl{at}cc.nctu.edu.tw.

² The abbreviations used are: used: GH, glycosyl hydrolase family; CSN, chitosanase; DDA, degree of deacetylation; ESI, electrospray ionization; MS, mass spectroscopy; LC, liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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