INTRODUCTION

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We intended to develop a method to recycle organic solid wastes via microbiological treatment at high temperature. As the process may be considered primarily one of waste treatment, it offers a rapid and effective means of converting the substrate in biological solid wastes and yields the additional product of fertilizer. In spite of the validity of the process, the current popularity of incineration in Japan, economic issues, and some social problems are preventing its widespread use. Therefore, it seems to be good strategy to vest the fermented products with further functions profitable for agriculture.

Chitin, a homopolymer of N-acetyl-D-glucosamine (GlcNAc) residues linked by 1-4 bonds, is abundant in nature in the form of integuments of insects and crustaceans and as a component of fungi. Chitin and its derivatives are of interest because they have varied biological functions, e.g., as immunoadjuvants, as flocculants of wastewater sludge, and as agrochemicals. For example, the addition of chitin to soil reduces populations of fungal plant pathogens (13) and plant-parasitic nematodes (14). Such biological activities of chitin oligomers are dependent on chain length and solubility (6).

The enzymatic degradation of chitin appears to occur in two steps, which are similar in procaryotes and eucaryotes. An endochitinase (EC 3.2.1.14) reduces the polymer to oligomers, which are subsequently degraded to monomers by exochitinase (-N-acetylhexosaminidase [EC 3.2.1.52]). We have purified and characterized a thermostable exochitinase from Bacillus stearothermophilus CH-4, isolated from a compost of fermenting organic solid wastes supplemented with some crustaceans from a fishery (23). The purified enzyme hydrolyzed -N-acetyl-D-galactosaminide as effectively as -N-acetyl-D-glucosaminides and was thus designated a -N-acetylhexosaminidase.

Using coloidal chitin as a sole carbon source, we isolated another unique bacterium which grows in hardly any carbon source other than colloidal chitin. The microorganism seemed to belong to the actinomycetes and produced multiple endochitinases in the culture fluid. This paper describes the purification and properties of three endochitinases produced by the thermophilic bacteria. The classification of the microorganism is also presented.

	MATERIALS AND METHODS

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Microorganism and culture. MH-1 was isolated from a compost of fermenting citrus peels and coffee and tea extract residues supplemented with small fish, shrimp, and crabs (3% of the material). MH-1 was cultured at 60°C on agar medium containing 0.5% colloidal chitin, 0.7% (NH₄)₂SO₄, 0.1% K₂HPO₄, 0.1% NaCl, 0.01% MgSO₄ · 7H₂O, 0.05% yeast extract, 1% compost extract, and 1.5% agar (colloidal chitin agar). For the liquid culture, a medium containing 0.5% colloidal chitin, 0.7% (NH₄)₂SO₄, 0.1% K₂HPO₄, 0.1% NaCl, 0.01% MgSO₄ · 7H₂O, 0.05% yeast extract, 1% compost extract, and 0.03% (2,6-O-dimethyl)--cyclodextrin (DMCD) was used (colloidal chitin medium). The microorganism was shaken at 58°C in the colloidal chitin medium for 3 to 4 days. The compost extract was prepared by extracting 50 g of compost (Miroku Co., Oita, Japan) with 100 ml of H₂O at 120°C for 20 min. After centrifugation (8,000 × g; 15 min), the supernatant was used as the 50% compost extract.

Classification. MH-1 was classified mainly according to the methods of Komagata (7) and Bergey's manual (31). For investigating its morphological and physiological properties, seven kinds of International Streptomyces Project media (2), described below, were tested in addition to colloidal chitin medium: yeast extract-malt extract agar, oatmeal agar, inorganic salts starch agar, glycerol-asparagine agar, nitrate broth with 2.0% agar added, glucose-asparagine agar, and peptone-beef extract agar. Czapek sucrose agar was also used.

Purification of three chitinases. All subsequent procedures were performed at temperatures below 4°C, unless otherwise stated.

Enzyme assay. Two types of assay method were used for evaluating endochitinase activity. Routinely, the activity was measured by using p-nitrophenyl-di-N-acetyl--chitobiose [pNP-(GlcNAc)₂] as the substrate. The enzyme was incubated at 60°C in a mixture (0.2 ml) containing 2 mM pNP-(GlcNAc)₂ and 50 mM KPB (pH 7.0) for an appropriate period. The reaction was terminated by adding 1 ml of 0.2 M sodium borate buffer (pH 10.5). The amount of p-nitrophenol released was determined from the absorbance at 400 nm (molar extinction coefficient, 17,700). One unit of enzyme activity was defined as the amount that released 1 µmol of pNP per min at 60°C. The specific activity was expressed as units per milligram of protein. Secondly, the activity was assayed by measuring the change in reducing power (16) of the reaction mixture containing 5 mg of colloidal chitin, 50 mM KPB (pH 6.5), 1 mM CaCl₂, and enzyme. When chitin and chitooligosaccharides were used as substrates, the products of the enzyme reaction were also analyzed by a HPLC (15) equipped with µBondapak CH (Waters) or TSK Gel G2500PW (Toso). Exochitinase activity was assayed with pNP--GlcNAc as the substrate. Chitosanase activity was assayed by measuring the change in the reducing power of the reaction mixture containing chitosan oligomer as a substrate.

Protein measurement. Protein measurement was performed by the method of Lowry et al. (11) with albumin from egg white (Wako Chemicals, Osaka, Japan) as the standard. For column chromatography, the protein concentration was estimated by measuring the absorbance at 280 nm. The molar extinction coefficients of the purified enzymes were calculated by the method of Scopes (24).

Protein analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (9) on a 12.5% polyacrylamide gel. Molecular marker Daiichi III (Daiichi Chemicals Inc., Tokyo, Japan) was the standard protein mixture. The molecular mass of the purified enzyme was estimated by gel filtration on a Superose 12 HR 5/30 column (Pharmacia). Catalase (232 kDa), gamma globulin (160 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) were the standards. The N-terminal amino acid sequence of the purified enzyme was determined by using an Applied Biosystems model 473A gas phase sequencer.

Reagents. DMCD (5) was kindly donated by Y. Suzuki, Teijin Ltd. (Tokyo, Japan). Chitooligosaccharides and their pNP derivatives were obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Colloidal chitin was prepared by the method of Shimahara and Takiguchi (25). Partially acetylated chitosan with a low molecular weight (19,000 to 33,000) was prepared by the method of Kubota and Eguchi (8), and some was kindly donated by N. Kubota, Oita University. All other reagents were of the highest grade available.

	RESULTS

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Morphological and culture properties of MH-1. Strain MH-1 was composed of gram-positive filament cells, often branched, and grew slowly on a colloidal chitin agar, surrounded by a clear zone. It formed a well-developed, white substrate mycelium (diameter, 0.5 mm; length, >20 mm) without any pigment on a colloidal chitin agar but formed a very poor aerial mycelium (Fig. 1). On an aged culture plate, the microorganism seldom formed endospores (0.7 by 1.0 mm). In the colloidal chitin medium, it formed large flocculent mycelia (radii, >1 mm). The bacterium grew aerobically but not anaerobically, and the growth temperature range was 40 to 65°C. The microorganism showed poor growth on the other standard agar media tested. Only colloidal chitin as a carbon source could support its growth. In the colloidal chitin medium, yeast extract and DMCD were essential for the growth of MH-1, and additions of 0.05% and 0.03%, respectively, were most effective. The addition of metals, such as ZnSO₄, FeCl₃, CuSO₄, and (NH₄)₆Mo₇O₂₄, was rather inhibitory. The addition of compost extract enhanced the growth of MH-1, and the greatest growth was observed with a 2.5% addition, although endochitinase activity was highest when the concentration was 0.5%.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   Scanning electron micrograph of MH-1. The microorganism, grown on a chitin agar plate, was treated and observed as described in Materials and Methods.

Chemotaxonomic properties of MH-1. In the cell wall of MH-1, meso-diaminopimelic acid, Glx, Gly, Ala, and glucosamine were found. Mannose, xylose, ribose, arabinose, and rhamnose were detected as whole-cell sugars. The G+C content of the DNA of strain MH-1 was calculated to be 54.6%. A partial 16S rDNA sequence of MH-1 (1,288 bases) was determined (DDBJ accession no., 12934). The similarity rank analysis showed that MH-1 is closely related to Bacillus species, especially to mesothermophilic strains, and Thermoactinomyces vulgaris and Thermoactinomyces candidus are somewhat further removed from strain MH-1 (Fig. 2). Bacillus denitrificans is the nearest neighbor (97.4% homology).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Phylogenetic neighbor-joining tree based on 1,020 nucleotides of 16S rRNA. The numbers in the tree are the percentages of bootstrap replicates in which the cluster was found. Bar (K nuc) = 0.011 substitution per site.

Purification of endochitinases. The results of purification are shown in Table 1. Affinity adsorption to colloidal chitin was quite effective for the purification of the chitinases, and three major proteins were observed by SDS-PAGE after the affinity treatment (Fig. 3). Their molecular masses were 71,000, 62,000, and 53,000 Da, and they were tentatively named chitinase L, M, and S, respectively. The ratio of chitinase L to chitinase M to chitinase S in the preparation after chitin affinity treatment was 5:2:1, estimated by densitometry of the gel. They were effectively separated by subsequent chromatofocusing (Fig. 4). All three proteins showed chitinase activities, although their specific activities decreased slightly. Finally, the chitinases were purified to homogeneity (Fig. 3). When traced by the assay system with colloidal chitin, the results of the purification of the three chitinases were similar to those shown in Table 1 (data not shown).

View larger version (70K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE of the purified chitinases. Final preparations of chitinases L (lane L), M (lane M), and S (lane S) were subjected to SDS-PAGE along with the preparation partially purified by chitin affinity (lane Aff.). Marker proteins (lane Ma) were also applied to the gel electrophoresis.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Chromatofocusing of partially purified chitinases. After chitin affinity treatment, the enzyme preparation (6.59 mg) was applied to a Mono P HR 5/20 column. The chitinase activity of each fraction was assayed, with pNP-(GlcNAc)2 as a substrate. Abs., absorbance.

Physical properties of endochitinases. Gel filtration on Superose 12 gave molecular masses of 68,000, 44,000, and 22,000 Da for chitinases L, M, and S, respectively. Chitinases L, M, and S were determined to have isoelectric points of 4.1, 4.2, and 4.5, respectively (Fig. 4). The N-terminal amino acid sequences were ATPATATYSTDSDWETGFQQKWTIK for chitinase L, EDLVTDPGFESGLSGWT for chitinase M, and VPQWYPAWWPYTWYRVIHRVIHD for chitinase S.

Enzymatic properties of endochitinases. The optimal temperatures for the reaction of chitinases L, M, and S were 75, 65, and 75°C, respectively (Fig. 5). They maintained initial activities after heat treatment (10 min) at 75, 65, and 75°C and were completely inactivated at 90, 80, and 85°C, respectively (Fig. 6).

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Effect of temperature on enzyme activities of chitinases. Reaction mixtures containing 2 mM pNP-(GlcNAc)2, 50 mM potassium phosphate buffer, and chitinase L, chitinase M, or chitinase S were reacted for 10 min at various temperatures.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Effect of temperature on enzyme stabilities of chitinases. After treatment at various temperatures for 10 min (pH 6.5 for chitinase L, pH 5.0 for chitinase M, and pH 5.5 for chitinase S), the enzyme solutions were further reacted with 2 mM pNP-(GlcNAc)2 at 60°C for 10 min.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Time course of hydrolysis of (GlcNAc)6 by three chitinases. The reaction mixture containing 2 mM (GlcNAc)6, potassium phosphate buffer (50 mM; pH 6.0), and purified chitinase L (0.29 mU), chitinase M (0.12 mU), or chitinase S (0.19 mU) was incubated at 60°C for 0 to 40 min, and the products were analyzed by HPLC. Symbols: , GlcNAc; , (GlcNAc)2; , (GlcNAc)3; , (GlcNAc)4; , (GlcNAc)5; , (GlcNAc)6.

	DISCUSSION

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We have isolated a noble thermophilic bacterium strain, MH-1, which has chitinolytic activity and shows characteristic culture properties. In spite of the actinic morphology of strain MH-1, all popular media for actynomycetes tested have failed to support its growth; only colloidal chitin could do so. In addition to yeast extract, the microorganism required DMCD for growth, which might ease an inhibitory effect of some toxic impurity or metabolite in the medium by its host-guest interaction (5). Furthermore, compost extract strongly enhanced its growth.

Although MH-1 morphologically resembles Saccharomonospora, its chemotaxonomical properties, such as the cell wall chemotype, whole-cell sugar pattern, and moles percent G+C of DNA indicated that the strain could not belong to any known genus of actinomycetes. In addition, the partial 16S rDNA sequence showed high similarity to those of Bacillus species, which is consistent with the low moles percent G+C of DNA of strain MH-1. It was reported that the genus Thermoactinomyces (53 to 55 mol% G+C of DNA) was somewhat closer to Bacillus species phylogenetically than to another actinomycete, regardless of their actinic morphology, when their 5S rDNA sequences were compared (18). It is interesting that many Bacillus species neighbors of MH-1 are thermophilic, because it was recently proposed that thermophilic Bacillus species should be in a new genus, Thermobacillus, independent of mesophilic Bacillus species (19). Consequently, we propose that strain MH-1 belongs to the genus Bacillus, in spite of its mycelial morphology. Further investigation, such as DNA-DNA hybridization and analysis of fatty acid composition and menaquinones, would clarify this issue.

Bacillus sp. strain MH-1 produced three endochitinases in its culture fluid. They were distinguishable by their physical properties (molecular masses, pIs, and N-terminal amino acid sequences) and enzymatic properties (thermal and pH stabilities, substrate specificities, and effects of sugars and metals). The results of gel filtration were rather inconsistent with those of SDS-PAGE, possibly because of some interaction between the enzymes and Superose 12, but they might reasonably exist as monomeric peptides. In the case of Streptomyces olivaceoviridis, proteolytic processing is significant for a multiplicity of chitinases (22). On the other hand, Bacillus circulans WL-12 produces several isoforms of chitinases from three independent genes, although proteolytic processing also occurs (1). In the case of MH-1, the production of each chitinase was nearly constant in some experiments, and their content did not change after the culture fluid was incubated at 60°C for several hours (data not shown). This suggests that proteolytic processing of the chitinases in the culture fluid could be negligible for MH-1. The three chitinases have different molecular masses, and their N-terminal amino acid sequences did not show any significant similarity. The N-terminal amino acid sequence of chitinase L showed high homology to those of Streptomyces plicatus CHI (60% homology) (20) and Streptomyces lividans ChiC (56%), and those of chitinase S showed high homology to S. lividans ChiA (60%) (4). Chitinase M did not have any significant sequence showing more than 35% homology to those of other chitinases, including thermostable chitinases of Streptomyces thermoviolaceus OPC-520 (30) and Bacillus licheniformis (partial sequences) (26). These finding indicate that the three chitinases of strain MH-1 might be products of different genes. Also, it is interesting that partial sequences of chitinases L and S showed more similarity to the enzymes from Streptomyces than those from Bacillus, considering the phylogenetic position of MH-1.

All three chitinases showed endo-type hydrolytic activities toward various chitin derivatives. We have reported a thermostable exochitinase of B. stearothermophilus CH-4 which can assimilate colloidal chitin as a carbon source (22). The enzyme is most active toward (GlcNAc)₂. Strain MH-1 did not show exochitinase activity in the culture fluid, and none of the three endochitinases hydrolyzed (GlcNAc)₂. B. licheniformis has been reported to accumulate (GlcNAc)₂ from colloidal chitin, due to a lack of exochitinase activity (27). On the other hand, all three enzymes from MH-1 hydrolyzed pNP-(GlcNAc)₂. This result indicated that the enzymes recognize a structure of (GlcNAc)₂ plus an aglycon, and the aglycon moiety may not necessarily be GlcNAc. Furthermore, chitinase L produced mainly dimeric and trimeric sugars from 66 and 11% acetylated chitosan, respectively, which indicated that the enzyme can recognize the 1-4 bond of GlcNAc-GlcN as well as GlcNAc-GlcNAc. Further investigation will be necessary to clarify the precise enzyme-substrate interactions.

Finally, considering the circumstances under which strain MH-1 was isolated, it would be interesting to know the population of such chitinolytic bacteria, as well as the fate of chitin substances in waste. For analysis of a composting process, it would be useful to demonstrate a characteristic 16S rDNA sequence for such a poorly culturable bacterium.