Colloidal Chitin Research Articles

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Entomopathogenic efficacy of the chitinolytic bacteria: Aeromonas hydrophila isolated from Siwa Oasis, Egypt

Thirty-four bacterial isolates were isolated from soil samples collected from the North Western Coast and a water sample collected from brackish water at Siwa Oasis, Matrouh Governorate, Egypt. Only six isolates showed chitinase activity when screened on colloidal chitin agar medium. The highest chitinolytic activity was achieved by a bacterial isolate labeled as A.S. This isolate was identified as Aeromonas hydrophila based on analysis of 16S rRNA gene sequence and morphological, physiological, and biochemical characteristics. Optimization of the cultural conditions for maximum chitinase production by A. hydrophila revealed that the highest level of chitinase was recorded when the bacterium was grown in malt nitrogen-based medium containing 1% colloidal chitin at pH7 for 48 h incubation at 30 °C. Crude chitinase from isolate A. hydrophila was evaluated against first instar larvae of the greater wax moth; Galleria mellonella L. (Lepidoptera: Pyralidae) at different concentrations of 0, 185, 205, 235, 265, 295 U/mg protein. It increased larval and pupal mortality rates in a concentration-dependent manner. The tested crude chitinase significantly induced a decrease in adults’ emergence rate and their fecundity.

Characterization of Stackebrandtia nassauensis GH 20 Beta-Hexosaminidase, a Versatile Biocatalyst for Chitobiose Degradation.

An unstudied β-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The β-N-acetylhexosaminidase was able to hydrolyze β1,2-linked, β1,3-linked, β1,4-linked, and β1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the β-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.

BIOLOGICAL CONTROL OF SOIL -BORNE FUNGAL PATHOGENS

Seventeen bacterial isolates have been successfully isolated and purified from soil rhizosphere samples collected from different agricultural areas Buhayra, Sharqia and Alqilyubia of Egypt, and screened for production of chitinase enzymes had the highest chitinolytic activities amongst those investigated. Isolate S3-C and S1-C were Identified by biochemical, physiological tests and 16s rRNA gene primer as Bacillus cereus S3C, Gene Bank NCBI accession MK185696 and Bacillus cereus S1C accession MK185697. The production of chitinase by B.cereus S3C and Bacillus cereus S1C was optimized using colloidal chitin medium amended with 1% colloidal chitin at 30°C after five days of incubation. B.cereus S3C had potential for cell wall lysis of many phytopathogenic fungi tested such as Fusarium oxysporum and Rhizoctonia solani Potato phytopathogenic fungi by in vitro antagonistic test. The addition of B. cereus S3C chitinase was more effective than that of B.cereus S1C in increasing the resistance of Potato plants infected with various Soil- borne phytopathogenic fungi.

ESTIMATION OF ANTIBACTERIAL EFFECT ON VIBRIO CHOLERAE (ON THE EXAMPLE OF THE SUBSTANCE ACETYL-N- CYSTEINE–L)

We studied the effect of the substance acetyl-N-cysteine-L on cholera Vibrio. Its minimum inhibitory concentration for the strains of Vibrio cholerae El Tor O1 and O139 serogroup was 1–2.5 mg/ml in solid nutrient media - Martin's agar (pH 7.6) and LB (pH 7.2), while in Bhascaran synthetic medium, using glucose (0.1per cent) as the sole carbon source, N-acetyl-β-D-glucosamine (0.05 per cent) or colloidal chitin (0.027 per cent) it was reduced to 50–250 μg/ml. The ability of the substance acetyl-N-cysteine-L substance to suppress the activity of the purified enzyme N-acetyl-β-D-glucosaminidase (chitobiasis) was found. Antibacterial effect detected of the substance acetyl-N-cysteine-L against the strains of Vibrio cholerae El Tor O1 and O139 serogroups with different epidemic significance (presence / absence of ctxAB and tcpA genes) indicates the advisability of considering the issue on the possibility of including this substance in composition of solution components used in the rehydration therapy of diarrheal diseases cases.

Identification and characterization of a marine-derived chitinolytic fungus, Acremonium sp. YS2-2.

Chitin is the most abundant biopolymer in marine environments. To facilitate its utilization, our laboratory screened marine-derived fungal strains for chitinolytic activity. One chitinolytic strain isolated from seawater, designated YS2-2, was identified as Acremonium species based on morphological and phylogenetic analyses. Acremonium species are cosmopolitan fungi commonly isolated from both terrestrial and marine environments, but their chitinolytic activity is largely unknown. The extracellular crude enzyme of YS2-2 exhibited optimum chitinolytic activity at pH 6.0-7.6, 23-45°C, and 1.5% (w/v) NaCl. Degenerate PCR revealed the partial cDNA sequence of a putative chitinase gene, chiA, in YS2-2. The expression of chiA was dramatically induced in response to 1% (w/v) colloidal chitin compared to levels under starvation, chitin powder, and glucose conditions. Moreover, the chiA transcript levels were positively correlated with chitinolytic activities under various colloidal chitin concentrations, suggesting that ChiA mediates chitinolytic activity in this strain. Our results provide a basis for additional studies of marinederived chitinolytic fungi aimed at improving industrial applications.

The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly

There is no structural information about any chitinase synthesized by Bacillus thuringiensis, the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74ΔspE211N and ChiA74ΔspD207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc3. ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc2).

Biochemical characterization of a β-N-acetylhexosaminidase from Streptomyces alfalfae and its application in the production of N-acetyl-d-glucosamine

N-Acetyl-d-glucosamine (GlcNAc) is a valuable monosaccharide widely used in the medical, agricultural, biofuel, and food industries. Its efficient and environment-friendly production depends on the binary system of β-N-acetylhexosaminidase (HEX) and chitinase. In the present study, a HEX of glycoside hydrolasefamily 20 was identified in Streptomyces alfalfae ACCC40021, and was overexpressed in Escherichia coli. The purified recombinant SaHEX showed maximal activities at 60°C and pH 5.5, and retained stable up to 45°C. The enzyme not only exhibited broad substrate specificity including p-nitrophenyl β-N-acetylglucosaminide, p-nitrophenyl β-N-acetylgalactosaminide, chitooligosaccharides and colloidal chitin, but also had higher specific activities (up to 1149.7±72.6 U/mg) towards natural and synthetic substrates. When combined with a commercial chitinase, it achieved a conversion rate of 93.7% from 1% of colloidal chitin to GlcNAc in 6h, with the product purity of >98%. These excellent properties make SaHEX a potential enzyme candidate for the chitin conversion for various industrial purposes.

Efficient enzymatic hydrolysis of ionic liquid pretreated chitin and its dissolution mechanism

Colloidal chitin, the substrate of chitinase with an open hydrated gel-like structure, can be obtained by treatment using either traditional hydrochloric acid (HCl) or ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). IL-pretreated chitin provided an efficient production of N-acetylglucosamine (175.62 mg g chitin) and N,N’-diacetylchitobiose (341.70 mg g chitin) with a conversion of 61.49% at 48 h catalyzed by chitinase from Streptomyces albolongus ATCC 27414. A short time second homogenization treatment after IL pretreatment can increase the conversion to 76.11%. A comprehensive characterization and comparison of chitin with different pretreatments suggested that enzymatic performances were correlated with the structural changes (size of the grains and porosity), high decrease in crystallinity, and high enzyme adsorption. The NMR spectroscopy studies of N-acetylglucosamine solvation in [Emim][OAc] clearly suggest that hydrogen bonding is formed between the hydroxyls of N-acetylglucosamine and both the anions and cations of the IL.

Molecular engineering of chitinase from Bacillus sp. DAU101 for enzymatic production of chitooligosaccharides

Enzymatic production of chitooligosaccharides has high value in medicine and other fields. However, low chitinase activity and yield of chitooligosaccharides limit the production and application. Herein, we used a series of molecular biology strategies to increase the expression of chitinase in Bacillus subtilis WB600. Upon addition of the signal peptide NprB, Chisb was successfully secreted to the outside of the cell and extracellular expression level reached 35.54 U/mL. Furthermore, optimizing Ribosome Binding Sites (RBSs) with spacer sequences, and combining molecular docking technology with site-directed mutagenesis, the expression level and the specific activity of Chisb was further increased to 51.67 U/mL and 249.62 U/mg, respectively. When colloidal chitin was used as the substrate, the chitooligosaccharides detected by ion chromatography were (GlcNAc)1-5, and the total yield of chitooligosaccharides was 14.4%. Our results indicate that strategies for increasing Chisb expression contribute to the study and application of chitinase and the production of chitooligosaccharides.

High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc

β-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60°C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55°C for one hour and at 4°C for 12h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.

Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides.

Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from Saccharomyces cerevisiae, ScCDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. ScCDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that ScCDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. ScCDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore ScCDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 °C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 °C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences.

Purification and characterization of exo-β-1,4-glucosaminidase produced by chitosan-degrading fungus, Penicillium sp. IB-37-2A.

Chitosan-degrading fungal strain, Penicillium sp. IB-37-2A, produced mainly extracellular chitosanolytic enzymes under submerged agitating cultivation in presence of soluble chitosan or colloidal chitin as main carbon source. Significant N-acetyl-β-D-glucosaminidase activity (8-18 × 103 U·ml-1) was also detected in culture filtrate of the fungal strain. Alone major exo-chitosanase from culture filtrate of Penicillium sp. IB-37-2A was purified in 46-fold using ultrafiltration, affinity sorption on colloidal chitosan and hydrophobic chromatography on Phenyl-Sepharose CL 4B and characterized. Molecular weight of the exo-β-1.4-glucosaminidase is 41kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55°C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55°C. Activity of the enzyme is strongly inhibited by 1mM Hg2+ and Ag+, in less degree-10mM Cu2+, Zn2+, Ni+ and Fe2+, slightly activated-with 1mM Mg2+, 10mM Ca2+, tween-80 (10mM) and Triton X-100 (1mM). Viscosimetric assay confirmed reported earlier exo-splitting manner of the enzyme activity. Soluble chitosan (deacetylation degree (DD) 80-85%) is most rapidly hydrolyzed by the enzyme (Vmax = 7.635µM × min-1 × mg-1, KM ~ 0.83mg/ml). Purified exo-chitosanase also degraded laminarin, β-glucan, colloidal chitin and showed significant chitobiohydrolase activity (V ~ 50µM × ml-1 × min-1 for pNP-GlcNAc2) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo-β-1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.

The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity.

Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.

Isolation and identification of marine strains of Stenotrophomona maltophilia with high chitinolytic activity.

Chitin is the second most abundant organic compound in nature and represents a rich carbon and nitrogen source that is primarily transformed by bacterial communities. Bacteria capable of gradually hydrolyzing chitin into N-acetylglucosamine monomers can have applications in the transformation of residues from shrimp and other crustaceans. The objective of the present study was to isolate, characterize and identify microorganisms with high chitinolytic activity. These microorganisms were isolated and characterized based on macro- and microscopic morphological traits. Strains were selected on colloidal chitin agar medium primarily based on a hydrolysis halo larger than 2 mm and a growing phase no longer than 6 days. Secondary selection consisted of semi-quantitative evaluation of chitinolytic activity with a drop dilution assay. From the above, ten strains were selected. Then, strain-specific activity was evaluated. The B4 strain showed the highest specific activity, which was 6,677.07 U/mg protein. Molecular identification indicated that the isolated strains belong to the species Stenotrophomonas maltophilia.

Isolation, screening and identification of haloarchaea with chitinolytic activity from hypersaline lakes of Iran

Halophiles produce stable enzymes under extreme conditions. The scant information about chitinolytic haloarchaea led us to conduct the present study in order to isolate and screen native halophilic archaea with chitinolytic activity and to optimize the enzyme production conditions. Among 500 haloarchaeal strains isolated from water samples from different hypersaline lakes of Iran, five strains showed chitinolytic activity. Based on biochemical, morphological and molecular analyses, we established that all five potent strains belonged to the genus Natrinema. Besides, observing chitinase function in culture media, through an additional molecular test the presence of the chitinase gene in chitinase-producing strains was also confirmed by PCR amplification. Compared with other potent strains, Natrinema sp. strain BS5 showed significant chitinase production. The production of chitinase in strain BS5 accompanied growth, started at the logarithmic phase and increased to its maximum level at the beginning of the stationary phase. Maximum chitinase production was obtained at 37?C, pH 7.5, 3 M NaCl and 1% colloidal chitin. The strain BS5 showed 38%, 30%, 24% and 28% decreases in enzyme production at 40?C, pH 8, 3.5 M NaCl and 0.5% substrate, respectively. This strain was able to produce the enzyme in NaCl 4 M and in the absence of MgCl2 and MgSO4. This study revealed the strong potential of the genus Natrinema to produce chitinase at high salt concentrations without Mg2+ requirement.

Phân lập và tuyển chọn những chủng xạ khuẩn triển vọng đối kháng với tuyến trùng Pratylenchus sp. trong điều kiện phòng thí nghiệm

The study on isolation and selection of antagonistic Actinomyces against nematode Pratylenchus sp. was conducted in laboratory condition. Sixty-four strains of Actinomyces were isolated from vegetable farm soil collected from Tra Vinh and Can Tho. All Actinomyces strains were screened on hydrolytic ability of chitin and protein which are components of nematode cell wall, results showed that six strains i.e. 4A1, BM15, 8.11.1, 9.3.1, 5A6 and SM8 exhibited high chitinase and protease enzymes. Especially, three strains i.e 4A1, BM15, 8.11.1 expressed hydrolytic ability of chitin and protein higher than other strains. Investigation the effect of culture extract of strains 4A1, BM15, 8.11.1 and mix 4A1+BM15+8.11.1 in liquid medium ISP2 contained colloidal chitin on the survival of nematode in soil pot, results found that all treatments expressed effect in killing nematode Pratylenchus sp. with number of alive nematodes were significant lower than control, especially the treatment mixture of 3 strains (4A1+BM15+8.11.1) showed highest efficacy in killing nematode at 48 and 72 hours of testing.

Biocontrol of Root-Knot Nematode Meloidogyne incognita by Chitinolytic Trichoderma spp.

The inhibitory potential of chitinolytic Trichoderma spp. was assayed against Meloidogyne incognita in vitro and in vivo. Sixteen Trichoderma isolates were assayed for chitinase activity using basal solid medium supplemented with colloidal chitin. Isolates recorded high chitinolytic activity were identified morphologically as Trichoderma asperelloides, T. hamatum, T. harzianum and T. viride. The four species were quantitatively assayed for total and specific chitinase activity. T. asperelloides showed maximum chitinase activity (1.736 U/mL). In vitro studies revealed that egg hatching and second juveniles vitality of root-knot nematodes were significantly inhibited by chitinase-containing growth culture of the four selected chitinolytic Trichoderma species. However, the reduction effect was directly correlated with the concentration of fungal growth suspension. Under greenhouse conditions, three sequentially treatments of soil with homogenized culture suspension were studied. Trichoderma species significantly reduced the final nematode population by 97.81% and root galling by 92.81%. Moreover, all tested Trichoderma species obviously promoted tomato growth compared to untreated control. Significant positive correlation between the biocontrol efficacy of Trichoderma species against root-knot nematodes and their chitinolytic activity was confirmed. The most potential chitinolytic species, T. asperelloides, also recorded the highest biocontrol activity against M. incognita.

Purification and biochemical characterization of a novel acido-halotolerant and thermostable endochitinase from Melghiribacillus thermohalophilus strain Nari2AT

An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI–TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-β-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.