Family 19 chitinases from Streptomyces thermoviolaceus OPC-520: molecular cloning and characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

The C-terminal module of Chi1 from Aeromonas caviae CB101 has a function in substrate binding and hydrolysis

The chitinase gene chi1 of Aeromonas caviae CB101 encodes an 865-amino-acid protein (with signal peptide) composed of four domains named from the N-terminal as an all-beta-sheet domain ChiN, a triosephosphate isomerase (TIM) catalytic domain, a function-unknown A region, and a putative chitin-binding domain (ChBD) composed of two repeated sequences. The N-terminal 563-amino-acid segment of Chi1 (Chi1DeltaADeltaChBD) shares 74% identity with ChiA of Serratia marcescens. By the homology modeling method, the three-dimensional (3D) structure of Chi1DeltaADeltaChBD was constructed. It fit the structure of ChiA very well. To understand fully the function of the C-terminal module of Chi1 (from 564 to 865 amino acids), two different C-terminal truncates, Chi1DeltaChBD and Chi1DeltaADeltaChBD, were constructed, based on polymerase chain reaction (PCR). Comparison studies of the substrate binding, hydrolysis capacity, and specificity among Chi1 and its two truncates showed that the C-terminal putative ChBD contributed to the insoluble substrate-protein binding and hydrolysis; the A region did not have any function in the insoluble substrate-protein binding, but it did have a role in the chitin hydrolysis: Deletion of the A region caused the enzyme to lose 30-40% of its activity toward amorphous colloidal chitin and soluble chitin, and around 50% toward p-nitrophenyl (pNP)-chitobiose pNP-chitotriose, and its activity toward low-molecular-weight chitooligomers (GlcNAc)3-6 also dropped, as shown by analysis of its digestion processes. This is the first clear demonstration that a domain or segment without a function in insoluble substrate-chitinase binding has a role in the digestion of a broad range of chitin substrates, including low-molecular-weight chitin oligomers. The reaction mode of Chi1 is also described and discussed.     

Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520: Molecular Cloning and Characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70°C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.     

CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.

     

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.     

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.     

Enzymatic deacetylation of chitin by extracellular chitin deacetylase from a newly screened Mortierella sp. DY-52

Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and 28 degrees C with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and 60 degrees C. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of 4-40 degrees C. The enzyme was enhanced in the presence of Co2+ and Ca2+. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers (GlcNAc)2-7.     

G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues

A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.     

Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus

Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was used as the substrate. The K _m value and V _max for the substrate A, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min^?1unit^?1, respectively.     

Electrophoretic Studies of Alkaline Proteinases from Strains of Aspergillus flavus Group

Entevobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Entevobacter sp. G-L To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60,000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10mm EDTA, but was not inhibited by Ca²⁺ (<50 mm) or NaCl (<400 mm). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri,and its biocontrol potential

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag⁺ and Zn²⁺. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC₅₀) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC₅₀ (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera.     

Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium,Laceyella putida

A novel chitinase (LpChiA) was purified to homogeneity from a culture of Laceyella putida JAM FM3001. LpChiA hydrolyzed colloidal chitin optimally at a pH of 4 in an acetate buffer and temperature of 75?ºC. The enzyme was remarkably stable to incubation at 70?ºC up to 1 h at pH 5.2, and its activity half-life was 3 days. The molecular mass of the enzyme was around 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and around 75 kDa by gel filtration, suggesting it is a homodimer. The enzyme activity was enhanced about 60 % when pre-incubated with anionic, cationic, and nonionic surfactants. The gene for LpChiA was cloned by PCR and sequenced. The nucleotide sequence of the gene consisted of 1,683 bp encoding 560 amino acids. The N-terminal and internal amino acid sequences of the purified LpChiA from L. putida suggested that the mature enzyme was composed of 384 amino acids after cleaving its 176 N-terminal amino acids and dimerized to express its activity. The deduced amino acid sequence of the mature enzyme showed the highest similarity to chitinase of Laceyella sacchari with 79 % identity.     

低温几丁质酶基因在乳酸克鲁维酵母中的重组表达及酶学性质表征

【目的】通过构建假交替单胞菌(Pseudoalteromonassp.DL-6)低温几丁质酶(chitinaseA,chi A;chitinase C,chi C)的重组乳酸克鲁维酵母菌株、纯化重组蛋白并对其进行酶学性质表征,为低温几丁质酶潜在工业化生产几丁寡糖奠定理论基础。【方法】人工合成密码子优化的几丁质酶基因,构建重组乳酸克鲁维酵母表达质粒(p KLAC1-chi A、p KLAC1-chi C)并用电脉冲法转化到乳酸克鲁维酵母中,实现低温几丁质酶的可溶表达。利用镍柱亲和层析纯化得到高纯度的重组几丁质酶。【结果】成功构建产低温几丁质酶的重组乳酸克鲁维酵母并纯化获得高纯度的重组几丁质酶。经SDS-PAGE分析在110 k Da与90 k Da附近出现符合预期大小的蛋白条带。铁氰化钾法测得Chi A和Chi C的酶活分别为51.45 U/mg与108.56 U/mg。最适反应温度分别为20°C和30°C,最适p H分别为8.0和9.0。在低于40°C,p H 8.0–12.0时,Chi A和Chi C重组酶较稳定。Chi A和Chi C对胶体几丁质以及粉状底物α-几丁质与β-几丁质具有明显的降解活性,且具有一定协同降解能力。【结论】首次实现假交替单胞菌来源的低温几丁质酶在乳酸克鲁维酵母中的重组表达、纯化、酶学性质及其降解产物分析,为其他低温几丁质酶的研究提供借鉴意义。     

Cloning,sequencing, and expression of the chitinase gene chiA74 from Bacillus thuringiensis

The endochitinase gene chiA74 from Bacillus thuringiensis serovar kenyae strain LBIT-82 was cloned in Escherichia coli DH5 alpha F'. A sequence of 676 amino acids was deduced when the gene was completely sequenced. A molecular mass of 74 kDa was estimated for the preprotein, which includes a putative 4-kDa signal sequence located at the N terminus. The deduced amino acid sequence showed high degree of identity with other chitinases such as ChiB from Bacillus cereus (98%) and ChiA71 from Bacillus thuringiensis serovar pakistani (70%). Additionally, ChiA74 showed a modular structure comprised of three domains: a catalytic domain, a fibronectin-like domain, and a chitin-binding domain. All three domains showed conserved sequences when compared to other bacterial chitinase sequences. A ca. 70-kDa mature protein expressed by the cloned gene was detected in zymograms, comigrating with a chitinase produced by the LBIT-82 wild-type strain. ChiA74 is active within a wide pH range (4 to 9), although a bimodal activity was shown at pH 4.79 and 6.34. The optimal temperature was estimated at 57.2 degrees C when tested at pH 6. The potential use of ChiA74 as a synergistic agent, along with the B. thuringiensis insecticidal Cry proteins, is discussed.     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Chitinases A,B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation

To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)2 almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)6 to (GlcNAc)3, while ChiA exclusively generated (GlcNAc)2 and (GlcNAc)4. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.