INTRODUCTION

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Chitosan, a partly acetylated or nonacetylated counterpart (4-linked 2-amino-2-deoxy--D-glucopyranan) of chitin, is present in the mycelial and sporangiophore walls of fungi and the exoskeletons of insects and crustacea (9, 27). It is usually obtained by the artificial deacetylation of chitin in the presence of alkali. Chitosan is a copolymer consisting of -(14)-2-acetamido-D-glucose and -(14)-2-amindo-D-glucose units, with the latter usually exceeding 80% (6). Chitosanase (EC 3.2.1.99) hydrolyzes polymers of (1-4)--D-linked glucosamine (GlcN) residues to chitosan oligomers. Over the last decade, some chitosanolytic enzymes with different substrate specificities have been characterized (9, 10, 12, 25, 28, 35), and most of them catalyze the endo-type cleavage of chitosan with a narrow range of deacetylation degrees (10, 11, 26). Recently, chitosan and its partially degraded oligosaccharides have become important because of their potential applications as medical and agricultural agents (2, 36). Thermostable chitosanases active between 60 and 100°C and specifically attacking the -D-glucosaminidic bonds are of special interest (34, 35). Several chitosanases from mesophilic bacteria have been cloned and sequenced to date (1, 4, 21, 22, 26). Most of them belong to the thermolabile chitosanases, whereas little information is available on thermostable chitosanases. Thermostability is presumably based on the protein structure. To elucidate the thermostable character of the enzyme, information on its molecular structure, including the entire amino acid sequence and three-dimensional structure, is needed.

We have screened bacteria producing thermostable chitosanases and found a strain, Bacillus sp. strain CK4, producing a thermostable chitosanase. Here we analyzed the homology of the 16S rRNA genes in the strains reported as the chitosanase producers, including Bacillus sp. strain CK4, in order to differentiate between them based on a phylogenetic analysis of the 16S rRNA genes. We performed the cloning, expression, and nucleotide sequencing of a novel chitosanase gene from Bacillus sp. strain CK4. We also compared the sequences of several chitosanases and predicted possible amino acid residues related to catalytic activity and thermostability.

	MATERIALS AND METHODS

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Materials. Chitin, chitosan, glycol chitin, and glycol chitosan were purchased from Sigma Chemical Co. (St. Louis, Mo.). Colloidal chitosan was prepared by the method of Uchida and Ohtakara (31). Colloidal chitin was also prepared by Hsu and Lockwood's methods (14). Partially N-acetylated chitosan (25 to 83% acetylated) prepared from practical-grade chitin was purchased from Sigma Chemical Co. Chitosanase of Bacillus sp. strain PI-7S was obtained from PIAS Inc. (Osaka, Japan). Chitosanase of a Streptomyces sp. was purchased from Sigma Chemical Co. Other reagents were of analytical grade.

Bacterial strains, plasmids, and culture conditions. The thermophilic bacterium Bacillus sp. strain CK4 was isolated as a potent thermostable chitosanase producer from a hot spring in Korea and was used as the source of chromosomal DNA to clone the enzyme gene. The transformants were screened on CY medium (1.0% glycol chitosan, 0.1% yeast extract, 0.05% tryptone, 0.15% K₂HPO₄, and 0.05% KH₂PO₄; pH 7.0) with or without 2.0% agar, containing appropriate antibiotics (50 µg/ml). The plasmids pUC18, pUC19 (Pharmacia Biotech, Uppsala, Sweden), and pBluescript II SK() and SK(+) (Stratagene, La Jolla, Calif.) were used as the cloning vectors. Escherichia coli DH5 [supE44 lacU169 (80 lacZM15) hsd-17 recA1 endA1 gyrA96 thi-1 relA1] was used as the cloning host for recombinant plasmids. E. coli BL21(DE3) [hasS gal(cIts857 ind-1 Sam7 nin5 lacUV5-T7 gene 1)] was used as the host for pET 28a(+) (Novagen, Inc., Madison, Wis.) to overproduce chitosanase. All recombinant strains were grown at 37°C on Luria-Bertani (LB) medium containing 50 µg of ampicillin/ml for the production of chitosanase.

Analysis of biochemical and physiological properties of strain CK4. The morphological characteristics of strain CK4 were determined by using Bergey's Manual of Systematic Bacteriology (13) and the method of Priest et al. (29). Physiological tests were carried out by using the Bacillus Biochemical Card of the Vitek system and API 50CHB (both from Biomérieux, Inc., St. Louis, Mo.). Fatty acid composition was analyzed by the microbial identification system (Sherlock; MIDI Co., Newark, N.J.), and the G+C content was determined by high-performance liquid chromatography (HPLC) by the method of Kumura et al. (18).

PCR amplification of the 16S rRNA gene. PCR was performed to amplify the 16S rRNA coding region, using two oligonucleotide primers, 5'-GGCTGCAGAACACATGCAAGTCGAACGGT-3' (positions 50 to 70 relative to E. coli 16S rRNA) and 5'-GGCTTAAGTGTTCCGGGCCCTTGCATAAG-3' (positions 1374 to 1394 relative to E. coli 16S rRNA). The initial denaturation step was 2 min at 94°C; this was followed by an annealing step at 48°C for 2 min and an extension step at 72°C for 3 min. The thermal profile then consisted of 29 cycles of denaturation at 94°C for 1 min, annealing at 48°C for 2 min, and extension at 72°C for 3 min, followed by a final extension step at 72°C for 10 min. The PCR products of the expected sizes were subcloned into pBluescript II SK(+).

Construction of the gene library and screening of chitosanase-producing recombinants. Chromosomal DNA was prepared from Bacillus sp. strain CK4 by using Marmur's method (23). The DNA was partially digested with Sau3AI and electrophoresed on a 1.0% agarose gel. Fragments measuring 4 to 10 kb were collected using a Prep-A Gene DNA Purification kit (Bio-Rad, Hercules, Calif.). pUC18 was cleaved at the BamHI site and treated with calf intestinal alkaline phosphatase. The Sau3AI fragments from the chromosomal DNA were ligated into the dephosphorylated BamHI site of pUC18. E. coli DH5 was transformed with the ligation mixture by electroporation. Transformed cells were grown on a 0.5% glycol chitosan-0.1% Congo red agar medium containing ampicillin (50 µg/ml) at 37°C. The colonies of the enzyme-positive transformants developed clear orange haloes on the red background of the medium.

Analysis of the cloned thermostable chitosanase gene. The recombinant plasmid was digested with BamHI, and the inserted DNA was isolated by using agarose gel electrophoresis. The inserted DNA was used for restriction mapping and subcloning. Various lengths of the DNA fragments of pKCO4 were unidirectionally detected from each side. The deletion mutants of pKCO4 were introduced into E. coli DH5. The chitosanase activity of each transformant was assayed. Plasmid DNAs from the recombinants were prepared using an alkaline lysis procedure (15).

DNA sequencing. The plasmid DNA of the subclones was prepared for sequencing using a Wizard Plus SV DNA purification kit (Promega Co., Madison, Wis.). Dideoxy DNA sequencing reaction was performed with an ALFexpress Autoread sequencing kit (Pharmacia Biotech) as specified by the manufacturer. The DNA fragments were analyzed on an ALFexpress Autoread sequencer (Pharmacia Biotech). Nucleotide and amino acid sequence analysis, including an open reading frame search, molecular weight calculation, and homology search, was performed using Lasergene software (DNASTAR, Inc., Madison, Wis.).

Construction of expression vectors and expression in E. coli. The open reading frame of the cloned chitosanase gene was amplified by PCR with 5' and 3' primers harboring NcoI and BamHI restriction sites. The amplified DNA fragment and the vector, pET 28a(+), were treated with NcoI and BamHI, ligated, and transformed into E. coli BL21(DE3) cells. The transformed cells were grown at 37°C in LB medium containing 20 µg of kanamycin/ml to an A₆₀₀ of 0.6 with vigorous shaking. Protein expression was induced by adding 1 mM isopropyl--D-thiogalactopyranoside (IPTG) for 4 h. Cells were harvested by centrifugation at 13,000 × g for 5 min.

Localization of chitosanase in recombinant E. coli. The enzyme product from the E. coli transformant was subcellularly fractionated (17). Two milliliters of the culture broth was sonicated three times at 20 kHz for 20 s in an ice-water bath. This sonicated lysate was used as an enzyme source. Five milliliters of the culture broth was centrifuged at 13,000 × g for 10 min at 4°C. The supernatant was used to detect the extracellular activity of the enzyme, and the precipitate was suspended in 5 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 25% (wt/vol) sucrose and 1 mM EDTA. One milliliter of the suspension was diluted threefold with water and sonicated as described above. This sonicated lysate was used to detect intracellular chitosanase activity. The remaining suspension (4 ml) was incubated at 30°C for 1 h after the addition of egg white lysozyme (0.3 mg). One milliliter of the spheroplast suspension was diluted threefold with 10 mM Tris-HCl buffer (pH 7.3) containing 25% (wt/vol) sucrose and 30 mM MgSO₄ and then centrifuged. The supernatant was used to detect periplasmic enzyme activity. The precipitate was washed with a high-osmotic-strength buffer, suspended in 0.5 mM MgCl₂, and sonicated briefly. This sonicated lysate was then used to detect cytoplasmic enzyme activity.

Purification of thermostable chitosanase. To construct the glutathione S-transferase (GST)-chitosanase fusion protein, two oligonucleotide primers, 5'-GGGGATCCATGCGGGAAGCAGA-3' and 5'-GGGAATTCTTATTTGATTACAC-3', were synthesized by Bioneer Co. (Seoul, Korea). These primers were modified to contain BamHI and EcoRI recognition sites in order to facilitate cloning into the GST fusion protein expression vector pGEX 4T-2 (Pharmacia Biotech). When these primers are used, the PCR product corresponds to the bases from 163 to 984 of the choK gene. The PCR mixture included 100 pmol of primer, 200 ng of template DNA, 20 mM each deoxynucleoside triphosphate, and 1.0 U of Taq DNA polymerase in a 50-µl reaction volume. Thirty rounds of amplification were done with the following cycles: 96°C for 1 min, 72°C for 2 min, and 50°C for 3 min. The amplified DNA was digested with BamHI and EcoRI and then cloned into pGEX 4T-2 digested with BamHI and EcoRI. The fusion protein was purified from the E. coli DH5 lysate by affinity chromatography with glutathione-Sepharose 4B (Pharmacia Biotech). The purified fusion protein was treated with thrombin for 12 h at room temperature to obtain the thermostable chitosanase. The desired protein was purified by HPLC with a Protein Pak 300SW semipreparative column (Waters Co., Franklin, Mass.) at a flow rate of 0.7 ml/min with a 10 mM potassium phosphate buffer (pH 7.5).

Enzyme assay and protein determination. The reaction mixture containing 250 µl of 1.0% soluble chitosan, 50 µl of 1.0 M potassium phosphate buffer (pH 7.5), and the enzyme solution in a final volume of 1 ml was incubated at 55°C for 30 min with shaking. The reaction was stopped by heating at 100°C for 10 min, followed by centrifugation. The amount of reducing sugar in the supernatant was determined using the modified dinitrosalicyclic acid (DNS) method (24). One unit of enzyme was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per min. D-Glucosamine was used as a standard. The protein concentration was determined by using the Lowry method (20) with bovine serum albumin as a standard.

Analysis of hydrolysis product. The substrate, soluble chitosan, was dissolved in 10 mM potassium phosphate buffer (pH 7.5) to give a 0.5% solution. The enzyme (0.1 mg/ml) was added to 1.0 ml of the substrate solution, and the reaction mixture was incubated at 55°C. After an appropriate reaction time, a portion of the reaction mixture was withdrawn and boiled for 10 min in order to terminate the enzymatic reaction. In order to analyze the chitosan oligosaccharide by thin-layer chromatography (TLC), the supernatants prepared under the conditions described above were spotted onto silica gel plate (Kieselgel 60; Merck, Darmstadt, Germany) and developed with n-propanol-30% ammonia water (2:1). The sugars on the TLC plate were visualized by spraying 0.1% ninhydrin dissolved with 99% ethanol. HPLC analysis was carried out with a TSK-Gel NH₂-60 column (Toso Co., Tokyo, Japan). The products were eluted with an acetonitrile-water mixture (60:40) at a flow rate of 0.8 ml/min and detected with a refractive index (RI) detector. D-Glucosamine and a chitosan dimer, trimer, tetramer, pentamer, and hexamer (Seikagaku Co., Tokyo, Japan) were used as authentic standards. (GlcN)_n product concentrations were calculated from peak areas in the HPLC profiles using the standard curves obtained from pure saccharide solutions.

N-terminal amino acid sequence analysis. The purified thermostable chitosanase (about 0.1 nmol of protein in 10 mM potassium phosphate buffer [pH 7.0]) was used directly for automated Edman degradation with an Applied Biosystems 470A gas-liquid phase protein sequencer. The phenylthiohydantoin (PTH) amino acid derivatives were separated and identified using an on-line PTH analyzer, model 120A (Applied Biosystems), with a PTH C₁₈ column.

Nucleotide sequence accession numbers. The nucleotide sequences of the choK and 16S rRNA genes reported in this article have been assigned GenBank accession numbers AF160195 and AF165188, respectively.

	RESULTS

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Strain properties and identification. The strain used in this study, strain CK4, is one of the thermophilic bacterial strains isolated from a hot spring in Korea (34, 35). Strain CK4 is a gram-positive rod-shaped bacterium, motile by a polar flagellum; it is also obligately aerobic, catalase and esculin positive, and indole and oxidase negative. It does not require sodium ions for growth, and it cannot utilize galactose and arabinose, as opposed to most Bacillus species, which can utilize arabinose as a carbon source (13). Strain CK4 can also be distinguished from Sporolactobacillus, Desulfotomaculum, and Sporosarcina spp. by its high G+C content, growth at 55°C, and gas production from glucose. Although several characteristics, such as growth temperature and carbon utilization, were not consistent with those of most Bacillus species, the analysis of fatty acid composition in cell walls using a microbial identification system revealed that strain CK4 showed high levels of homology to Bacillus species (data not shown). We also determined the partial nucleotide sequence of the 16S rRNA gene from strain CK4, corresponding to the region between positions 50 and 1394 of the gene in E. coli (8). The rRNA sequence of strain CK4 was compared to sequences available from GenBank. Figure 1 shows a phylogenetic tree of the Bacillus species and other endospore-forming bacteria. Strain CK4 and Bacillus subtilis formed a robust clade but were not exactly identical with each other. Based on these data, we propose the assignment of our strain as Bacillus sp. strain CK4.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree of 16S rRNA genes of the genus Bacillus and other endospore-forming bacteria. The sequences used for comparison with the 16S rRNA gene of strain CK4 were obtained from GenBank. The origins and accession numbers of the sequences are as follows: Bacillus sp. strain BDID723, AF027659; Bacillus anthracis, X5509; Bacillus cereus, D16266; Bacillus thuringiensis, D16281; Bacillus megaterium, D16273; Bacillus sp. strain CK4, AF165188; B. subtilis, AB018595; Bacillus sp. strain JJ#1, Y15466; Bacillus coagulans, D16267; Sporosarcina ureae, X62175; Lactobacillus sp. Y16329; Desulfotomaculum nigrificans, AB026550; Clostridium sp., Y12289. The phylogenetic tree was constructed by the Clustal method with a weighted residue weight table using Lasergene software. The numbers on the baseline refer to the divergence between species.

Cloning of the chitosanase gene. The recombinant E. coli DH5 containing the chitosanase gene from the Bacillus sp. strain CK4 genomic DNA was screened as a colony forming an orange halo on glycol chitosan-Congo red agar medium. Of approximately 10,000 ampicillin-resistant colonies, 1 colony exhibited the orange halo formed by the action of chitosanase. The DNA insert of the plasmid (designated pKCO4) was analyzed by digestion with restriction enzymes. The resulting physical map showed that the plasmid insert size was 5.1 kb, containing PstI, EcoRI, SacII, EcoRV, and BglI restriction enzyme sites.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Restriction map and deletion analysis of the 5.1-kb BamHI-BamHI fragment of pKC04. The thick arrow indicates the region necessary for the expression of chitosanase. +, chitosanase activity detected; , chitosanase activity not detected.

Nucleotide sequencing of the thermostable chitosanase gene. The DNA sequence of the 1.1-kb fragment contains an open reading frame of 822 nucleotides starting with the initiation codon ATG and ending with the termination codon TAA at position 984. The ATG codon was chosen as the translation initiation site because its location was close to the possible ribosome binding site. Six bases upstream of the ATG codon, there is a 5-base sequence, 5'-AAGGA-3', that is considerably complementary with the 3' end of 16S rRNA. The A+T content of the region upstream of the initiation codon is 61.8 mol%, which is higher than those of the total Bacillus sp. strain CK4 chromosomal DNA (42 to 48 mol%) and the reading frame of the thermostable chitosanase (48.4 mol%). This region contains a putative promoter that displays some sequence homology to the TATAAT (10) and TTGACA (35) of the E. coli promoter consensus sequence (Fig. 3). Downstream from the TAA stop codon, there is a G+C-rich region of dyad symmetry, capable of forming a stem-and-loop structure. However, the sequence is not followed by a stretch of T residues, unlike the E. coli -independent transcription terminators.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence of the choK gene and deduced amino acid sequence of the gene product. The coding region starts at position 163 and ends at position 984. The 35 and 10 regions of a putative promoter sequence and a possible Shine-Dalgarno (SD) sequence for the ribosome binding site are underlined. In the 3'-flanking region from the coding sequence, a sequence capable of forming a stem-and-loop structure, which may be involved in transcription termination, is indicated by arrows. The N-terminal amino acids determined by Edman degradation are also indicated (broken underline). The amino acid residues that seem to be essential for chitosanase activity are marked by asterisks. Cysteine residues, potential sources of thermostability, are circled.

Comparison of the deduced amino acid sequence of the choK gene product with those of other chitosanases. The deduced amino acid sequence of the thermostable chitosanase from Bacillus sp. strain CK4 was compared with the sequences of eight available bacterial chitosanases. The nine sequences were linearly aligned by the Clustal method (Lagergene software) as shown in Fig. 4A. The ChoK sequence showed similarities of 76.6, 18.2, 16.8, 15.3, and 14.2% to the sequences of B. subtilis, Bacillus ehemensis, Streptomyces sp. strain N174, Nocardioides sp. strain N106, and Bacillus circulans chitosanases, respectively. Linear alignment of two sequences, of the Bacillus sp. strain CK4 and B. subtilis chitosanases, revealed a marked similarity between the two enzymes (Fig. 4). The overall sequence homology is calculated as 76.6%, which is considerably high for interspecies sequence homology between thermostable and thermolabile enzymes, strongly suggesting that the two chitosanases may have very similar three-dimensional structures. C-terminal sequence homologies between pairs of chitosanases are calculated as 93% (Bacillus sp. strain CK4 and B. subtilis), 96% (B. circulans and B. ehemensis), and 95% (Streptomyces sp. strain N174 and Nocardioides sp. strain N106). The 93% similarity between B. subtilis and Bacillus sp. strain CK4 means that they belong to the same group, that is, cluster III (Fig. 4B). Since only eight nucleotide sequences of bacterial chitosanases have been reported so far, the essential catalytic residues have not been studied clearly yet. Although some homologies were found in N-terminal segments (between positions 37 and 78 of the chitonase gene in Bacillus sp. strain CK4), ChoK has no extensive similarity with other chitosanases in other parts (except for the B. subtilis chitosanase). The N-terminal segments of the nine chitosanases sequenced have 3 amino acid residues in common, which were thought to be putative catalytic sites of chitosanase.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   (A) Alignment of putative catalytic N-terminal segments of bacterial chitosanases. Asterisks, essential catalytic residues of Streptomyces sp. strain N174; arrows, the amino acid residues which seem to be essential for chitosanase activity. (B) Amino acid sequence alignment of C-terminal regions of bacterial chitosanases. The origins and accession numbers of the sequences are as follows: Bacillus sp. strain CK4, AF160195; B. ehemensis, AB008788; B. subtilis, Z99117; B. circulans, D10624; Amycolatopsis sp. strain CsO-2, AB041775; Streptomyces sp., L07779; Matsuebacter chitosanotabidus, AB006851; Sphingobacterium multivorum CsoA, AL109849; Nocardioides sp., L40408. The homology search was performed with Lasergene software (DNASTAR Inc.).

Overexpression of thermostable chitosanase and subcellular fractionation. Plasmid pETCOK was transformed into E. coli BL21(DE3) so that thermostable chitosanase could be overexpressed. A cell extract was prepared as described in Materials and Methods. Indeed, more than 50% of the soluble protein in the E. coli cell extract was estimated to be chitosanase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The activities of the enzyme from the periplasmic layer of this overproducer were compared with that of the extracellular enzyme from wild-type Bacillus sp. strain CK4. The activities of the enzymes produced by E. coli BL21(DE3)/pETCOK and E. coli DH5/pKCO4 were about 30- and 5-fold higher, respectively, than that of the enzyme from Bacillus sp. strain CK4 (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   (A) Location of chitosanase activity in E. coli carrying pETCOK. E. coli BL21(DE3) carrying pETCOK was grown in LB medium containing 20 µg of kanamycin per ml. Cultivation was done at 37°C on a rotary shaker. , bacterial growth; , periplasmic fraction (lane 1 on inset gel); , extracellular fraction (lane 2); , cytoplasmic fraction (lane 3). (B) Purification of chitosanase from the GST-chitosanase fusion protein. Lane S, standards; lane 4, insoluble fraction; lane 5, soluble fraction after 6 h of induction at 37°C; lane 6, soluble fraction after 20 h of induction at 20°C; lane 7, GST-chitosanase following fusion protein adsorption; lane 8, results of semipreparative HPLC following thrombin elution.

Purification of chitosanase from the GST-chitosanase fusion protein. The thermostable chitosanase was purified using glutathione affinity chromatography (3) and a semipreparative HPLC column. A substantial portion of the GST-chitosanase fusion protein (M_r, 57,000) was in the insoluble pellet in the form of inclusion bodies. To maximize the yield of the soluble chitosanase, we examined enzyme induction conditions and found that enzyme induction with 0.2 mM IPTG at 20°C for 20 h produced the maximum level of soluble active fusion protein. Thus, we routinely used these conditions. In the first purification step, thrombin cleavage and elution of the full-length chitosanase from glutathione-Sepharose beads gave one major band of 32 kDa and several minor bands. To remove minor bands, thrombin-eluted chitosanase was applied to a semipreparative HPLC column. The enzyme, purified 400-fold with a 28% final yield, appeared to be homogenous by the criteria of PAGE (Fig. 5B). The molecular size of the protein was estimated to be approximately 32 kDa, based on its motility calculated by use of standard calibration proteins. These results show that the enzyme is a monomeric enzyme and is considered to correspond to the intact gene product not generated by proteolytic processing.

Stability of chitosanase. The optimal temperature and pH for chitosanase activity were examined. The enzyme was most active at 55°C and pH 7.5 under the standard assay conditions (data not shown). The thermostability of the expressed protein was examined by measuring the remaining activity after incubation at various temperatures. The remaining activities after treatment of the enzyme at 80°C for 30 and 60 min were 85 and 66%, respectively. The enzyme (0.5 mg per ml of 50 mM potassium phosphate buffer, pH 7.5) retained its full activity after treatment at 60°C for 30 min, and 92% initial activity remained even after incubation at 70°C for 30 min, although enzyme activity was completely lost after 60 min at 90°C (Fig. 6A). We also found that the enzyme is quite stable in a high concentration of chemical denaturants such as ethanol and SDS. For example, the enzyme was not inactivated at all when incubated with 50% ethanol at 55°C, and it retained about 81% of its activity after incubation with 5% SDS at 55°C for 1 h. The enzyme was resistant to urea and guanidine HCl as well; it retained full activity after incubation with 6 M urea or 2 M guanidine HCl at 37°C for 30 min (Fig. 6B). It is noteworthy that the enzyme is quite stable even in 8 M urea, which causes complete denaturation of ordinary proteins.

View larger version (12K): [in this window] [in a new window]   FIG. 6.   (A) Thermostability. After the enzyme was preincubated at 50, 60, 70, 80, or 90°C for 30 () or 60 () min, remaining activities were measured to determine the thermostability of the enzyme. (B) Effects of protein denaturants on the stability of chitosanase. The enzyme (0.5 mg/ml) was incubated in 10 mM potassium phosphate buffer (pH 7.5) containing urea () or guanidine HCl () at 37°C for 30 min, and then the remaining activity was assayed after ultrafiltration with Centricon-10.

Substrate specificity. The activities of the purified chitosanase upon chitosan, chitosan derivatives, and other polysaccharides are presented in Table 1. Soluble chitosan, colloidal chitosan, and glycol chitosan served as good substrates. The K_m values for soluble chitosan and colloidal chitosan were 0.8 and 8.7 mg/ml, respectively, and the V_max values were 173 and 71.5 U/mg, respectively. Soluble chitosan was hydrolyzed 6.2 times faster than glycol chitosan. The enzyme was specific for chitosan but attacked neither chitin, cellulose, amylose, nor starch. The substrate specificity of chitosanase on chitosan with different degrees of deacetylation (DDA), prepared by different procedures for N-acetylation, was examined. The relative activity increased when the DDA of soluble chitosan increased but decreased when the DDA of colloidal chitosan increased (Table 2). This indicates that the physical form and DDA of the substrate affect the rate of hydrolysis. However, no great difference was found among the hydrolysates of soluble chitosan and colloidal chitosan with different DDA (unpublished data).

Analysis of hydrolysis products. The catalytic pattern of chitosanase was examined by using soluble chitosan as the substrate. A change in the hydrolysis products from soluble chitosan was observed during incubation with the recombinant purified enzyme at 55°C for 12 h. At the initial stage, soluble chitosan was hydrolyzed to (GlcN)₄ to (GlcN)₅ (80% of total products) and small amounts of the dimer and trimer. After 12 h of incubation, the amount of the pentamer in the hydrolysate decreased, while dimer, trimer, and tetramer levels increased, but there was still no monomer (Fig. 7). The hydrolysate profile of the chitosanase of Bacillus sp. strain CK4 was compared with those of other bacterial chitosanases. The chitosanase of Bacillus sp. strain PI-7S produced oligosaccharides ranging from a monomer through a pentamer, with the trimer as the main product. In the case of Streptomyces sp., the main product was the monomer (about 30% of the total yield). Both enzymes produced a monomer and a dimer, with a high rate of about 40 to 60% of the total product.

View larger version (27K): [in this window] [in a new window]   FIG. 7.   (A) Hydrolysate profiles of bacterial chitosanases. Samples were incubated at 37°C for 12 h and analyzed on a TSK-Gel NH2-60 column for the chitosan oligosaccharide. Standards G1 through G6 indicate standard (GlcN)n where n is 1 through 6, respectively. (B) Time course of soluble chitosan hydrolysis by Bacillus sp. strain CK4 chitosanase. The enzyme (0.1 mg/ml) was added to 0.5 ml of 1% soluble chitosan dissolved in 10 mM potassium phosphate buffer, pH 7.5. The reaction was carried out at 55°C.

	DISCUSSION

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We described here the characterization of a thermostable chitosanase-producing bacterium isolated from a hot spring in Korea. This strain was classified into the genus Bacillus by virtue of its morphological and physiological properties and by phylogenetic studies based on analysis of the 16S rRNA gene sequences. The 16S rRNA sequence of strain CK4 showed high similarity (95.7%) to that of B. subtilis. However, several characteristics such as growth temperature, carbon utilization, thermolabile enzyme secretion, and chitinase production were not consistent between the two strains. Therefore, we classified strain CK4 as a new member of the Bacillus genus. A gene (choK) coding for chitosanase from Bacillus sp. strain CK4 was cloned, and the complete nucleotide sequence was characterized. The open reading frame of choK encodes a protein consisting of 242 amino acids, and the molecular size of the protein calculated from the open reading frame is 29,926 Da, which corresponds to that determined by SDS-PAGE and high-performance gel permeation chromatography. The thermostable chitosanase was purified to homogeneity from E. coli DH5 by using the GST fusion protein purification system and semipreparative HPLC. The expressed fusion protein was present as a form of insoluble inclusion body. A fraction of soluble recombinant GST-chitosanase was obtained when expression during the incubation of the recombinant strain was performed at 20°C. The production of soluble recombinant GST-chitosanase was dependent on induction with IPTG. The maximum yield of soluble material (about 20 mg/liter) was achieved upon induction with 0.2 mM IPTG. Computer analysis of the deduced amino acid sequence revealed that the C-terminal region of the enzyme had a high similarity with that of B. subtilis, but not with those of other groups. Streptomyces sp. strain N174 had two essential residues, Glu-22 and Asp-40, localized within the conserved N-terminal region for catalytic activity (7). In the case of glycosyl hydrolases, most catalytic amino acids are aspartate or glutamate residues conserved in regions sharing amino acid sequence similarities. N-terminal segments of all bacterial chitosanases had conserved Glu-22 and Asp-40, which were thought to be putative catalytic residues, like those in Streptomyces sp. strain N174 chitosanase.

The only significant difference between the chitosanases from Bacillus sp. strain CK4 and B. subtilis is thermostability. The two enzymes have a relatively low homology sequence in positions 86 to 110 of the Bacillus sp. strain CK4 enzyme. This portion might have a role in the thermostability of chitosanase. This is consistent with the idea that a considerable increase in the thermal resistance of proteins can be acquired by the addition of only a few intramolecular bonds such as hydrogen, ionic, and hydrophobic bonds (5). Compared with the amino acid sequences of other, thermolabile bacterial chitosanases, there were several conserved residues and/or regions in the primary structure of the thermostable chitosanase. It has been reported that the conserved residues and/or regions were important in the catalytic activity of the enzyme. Studies on the thermostability and heat inactivation of alanine dehydrogenases from B. subtilis and Thermus thermophilus (32) have also indicated that the factors related to the thermoresistance of T. thermophilus have not affected the catalytic ability of the enzyme. This suggests that the structures related to catalytic activity could be almost identical, although the thermostability was distinctly different. Therefore, the structural differences between the two enzymes would be subtle. Moreover, the B. subtilis enzyme contains only one cysteine residue, at position 76. This indicates that there is probably no intramolecular disulfide bond in the B. subtilis chitosanase. It is noteworthy that the more thermostable enzyme from Bacillus sp. strain CK4 contains 2 additional cysteine residues, at positions 49 and 211, besides the 1 residue located at position 76 that is equivalent to the cysteine in the B. subtilis enzyme. This contrasts with the lack of cysteine in the T. thermophilus enzyme, which also has a very high thermostability. Considering that free cysteine occurring in the exterior of a protein is a potential source of thermal instability, the Cys-211 of the Bacillus sp. strain CK4 enzyme may occur in the interior of the protein and form a disulfide bond with either Cys-49 or Cys-72, exerting a positive effect on thermostability (19, 30, 33).

The catalytic role of the Asp-66 residue (Fig. 4) is identified by some recently obtained data (H. G. Yoon, H. Y. Kim, Y. H. Lim, H. K. Kim, D. H. Shin, B. S. Hong, and H. Y. Cho, unpublished data). In the site-directed mutagenesis experiment, Asp-66 was proposed as a catalytic residue, corroborating the conclusion drawn from the present work. On the other hand, glutamate residues (Glu-50 and Glu-62) are not found to play an important role in catalysis, seemingly essential for the Streptomyces sp. strain N174 chitosanase. Furthermore, it was found that Cys-211, which may occur in the interior of the protein, exerts a positive effect on the thermostability of the enzyme.

The optimum temperature for recombinant chitosanase activity is in the range of the optimal growth temperature of Bacillus sp. strain CK4 (55°C), and the purified chitosanase shows high thermostability in this temperature range compared to other bacterial chitosanases. The purified enzyme belongs to the enzyme group that is able to hydrolyze only chitosan. The previously reported chitosanases classified into the group hydrolyzing only chitosan also can catalyze colloidal chitin and can partially catalyze O-hydroxyethylated chitosan as well. However, this new enzyme is distinct from other enzymes in the substrate specificity of colloidal chitin degradation. The substrate specificity of this enzyme is very high compared with that of other enzymes in the group. Among the hydrolysis products of colloidal chitosan, (GlcN)₄ was detected as the major product, with high levels of the trimer, pentamer, and hexamer, but no monomer (Fig. 7). This suggests that the mode of action of the enzyme is of the endo type. Endo-type chitosanases from several microorganisms have been reported, and their degrading patterns on chitosan are similar. Although the amounts of oligomers were variable in each case, these enzymes were previously reported to hydrolyze chitosan into oligomers of 1 to 6 units by an endo-type catalytic action. However, the thermostable enzyme described here produces functional oligomers, trimers through hexamers, with a high rate of about 80% of total yield at temperatures under 55°C for 12 h. The reaction pattern of this chitosanase, with its thermostability, makes the enzyme a good candidate for biotechnological applications in the industrial production of functional chitooligosaccharides.