High-yield production of a chitinase from Aeromonas veronii B565 as a potential feed supplement for warm-water aquaculture

Chitin, present in crustacean shells, insects, and fungi, is the second most plentiful natural organic fiber after wood. To effectively use chitin in a cost-saving and environmentally friendly way in aquaculture, crustacean shells (e.g., shrimp-shell meal) are supplemented into aquafeed after degradation by chemical methods. Herein, we describe a chitinase from Aeromonas veronii B565, designated ChiB565, which potently degrades shrimp-shell chitin and resists proteolysis. We isolated recombinant ChiB565 of the expected molecular mass in large yield from Pichia pastoris. ChiB565 is optimally active at pH 5.0 and 50 °C and stable between pH 4.5 and 9.0 at 50 °C and below. Compared with the commercial chitinase C-6137, which cannot degrade shrimp-shell chitin, ChiB565 hydrolyzes shrimp-shell chitin in addition to colloidal chitin, powdered chitin, and β-1,3-1,4-glucan. The optimal enzyme concentration and reaction time for in vitro degradation of 0.1 g of powdered shrimp shell are 30 U of ChiB565 and 3 h, respectively. A synergistic protein-release effect occurred when ChiB565 and trypsin were incubated in vitro with shrimp shells. Tilapia were fed an experimental diet containing 5 % (w/w) shrimp bran and 16.2 U/kg ChiB565, which significantly improved growth and feed conversion compared with a control diet lacking ChiB565. Dietary ChiB565 enhanced nitrogen digestibility and downregulated intestinal IL-1β expression. The immunologically relevant protective effects of dietary ChiB565 were also observed for 2 to 3 days following exposure to pathogenic Aeromonas hydrophila.     

Effective production of N-acetyl-β-D-glucosamine bySerratia marcescens using chitinaceous waste

The strain ofSerratia marcescens QM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-β-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of chitin and pretreatment of chitin source were investigated. Also, the effect of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and, ball-milled with a nominal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30°C.     

An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish-processing waste. In this study, shrimp and crab shell powder, prepared by treating shrimp- and crab-processing waste by boiling and crushing, was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus subtilis NPU 001, a strain isolated from soil samples, excreted a chitinase when cultured in a medium containing 2% (w/v) shrimp and crab shell powder as the major carbon source. The chitinase, which was purified by sequential chromatography, had a Mw of 31 kDa and a pI of 5.4. The purified chitinase (2 mg ml⁻¹) inhibited hyphal extension of the fungus Fusarium oxysporum. Compared with other known bacterial chitinases, the unique characteristics of NPU 001 chitinase include antifungal activity against plant-pathogenic fungi and the production of chitotriose as the major enzymatic hydrolysate from colloidal chitin.     

Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

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.     

Chitinase production by a newly isolated thermotolerant Paenibacillus sp. BISR-047

Chitinase is one of the important mycolytic enzymes with industrial significance, and is produced by a number of organisms, including bacteria. In this study, we describe isolation, characterization and media optimization for chitinase production from a newly isolated thermotolerant bacterial strain, BISR-047, isolated from desert soil and later identified as Paenibacillus sp. The production of extracellularly secreted chitinase by this strain was optimized by varying pH, temperature, incubation period, substrate concentrations, carbon and nitrogen source,etc. The maximum chitinase production was achieved at 45 °C with media containing (in g/l) chitin 2.0, yeast extract 1.5, glycerol 1.0, and ammonium sulphate 0.2 % (media pH 7.0). A three-fold increase in the chitinase production (712 IU/ml) was found at the optimized media conditions at 6 days of incubation. The enzyme showed activity at broad pH (3–10) and temperature (35–100 °C) ranges, with optimal activity displayed at pH 5.0 and 55 °C, respectively. The produced enzyme was found to be highly thermostable at higher temperatures, with a half-life of 4 h at 100 °C.     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.     

Production and purification of a hyperthermostable chitinase from Brevibacillus formosus BISR-1 isolated from the Great Indian Desert soils

A strain of Brevibacillus formosus, capable of producing a high level of chitinase, was isolated and characterized for the first time from the Great Indian Desert soils. The production of extracellularly secreted chitinase was analyzed for its biocontrol potential and optimized by varying media pH, temperature, incubation period, substrate concentrations, carbon and nitrogen sources, etc. A twofold increase in chitinase production (798 IU/mL) was achieved in optimized media containing (g l^?1) chitin 2.0, malt extract 1.5, glycerol 1.0, ammonium nitrate 0.3 %, T-20 (0.1 %) and media pH 7.0 at 37 °C. The produced enzyme was purified using a three-step purification procedure involving ultra-filtration, ammonium sulphate precipitation and adsorption chromatography. The estimated molecular weight of the purified enzyme was 37.6 kDa. The enzyme was found thermostable at higher temperatures and showed a t _½ of more than 5 h at 100 °C. Our results show that the chitinase produced by B. formosus BISR-1 is thermostable at higher temperatures.     

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 °C, and activity was stimulated with 25 mM Mn²⁺. In kinetic analysis, Chi18B showed Km values of 9.07?±?0.65 μM and 129.27?±?0.38 μM with the substrates 4-methylumbelliferyl-N-N′-diacetylchitobiose and α-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.     

Improvement of Chitinase Production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> NM101-19 for Antifungal Biocontrol through Physical Mutation

Chitinase is one of the most important enzymes due to its diversity and a variety of potential uses. This study is an attempt to enhance chitinase production for antifungal biocontrol by subjecting Bacillus thuringiensis NM101-19 strain grown on shrimp shell wastes to various doses of gamma irradiation. Six mutants (BM-4, BM-6, BM-8, BM-12, BM-15, and BM-17) obtained at gamma ray doses of 40, 60, 80, 100, 120 and 140 Gy, respectively produced higher levels of chitinolytic activities in comparision to the wild-type strain. The BM-15 mutant strain showed the highest chitinase production (65.41 U/mL) which was 2.60 times more than the wild type (25.11 U/mL). Biocontrol efficacy of the mutants was statistically superior to the wild-type strain against all tested phytopathogens. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) PCR techniques, with five primers for each, were used in order to detect the variation in DNA profile between the mutant and wild-type strains in response to gamma-radiation treatments. RAPD and ISSR analysis indicated the appearance and disappearance of DNA polymorphic bands at different gamma ray doses. The results confirmed that the mutagenesis technique is a potent strategy to enhance the chitinase activity for industrial and agricultural purposes.     

Influence of bioprocess variables on the production of extracellular chitinase under submerged fermentation by Streptomyces pratensis strain KLSL55

Chitinases are the enzymes which are capable of hydrolyzing chitin to its monomer N-acetyl glucosamine (GlcNac). Present study emphasizes on the impact of critical process variables on the production of chitinase from Streptomyces pratensis strain KLSL55. Initially the isolate was noticed to produce 84.67?IU chitinase in basal production medium. At optimization of bioprocess variables, the physical parameters pH of 8.00, 40?°C of incubation temperature, agitation speed of 160?rpm and 1.25?mL of spore suspension were found optimum for improved production of chitinase. Further, formulated production medium with 1.5% colloidal chitin, 1.25% fructose greatly influenced the chitinase production. At all described optimum conditions with formulated production media, a total of 14.30-fold increment was achieved in the chitinase production with final activity of 1210.67?IU when compared to the initial fermentation conditions in basal production medium.     

Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil

A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4–9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR–denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils.     

Preparation of fermentation-processed chitin and its application in chitinase affinity adsorption

A fermentation approach utilizing Paenibacillus sp. to process chitin was developed. The chitin obtained from this process is called fermentation-processed chitin (FPC), and it was further investigated with chitinase affinity adsorption studies together with three other adsorbents, i.e. crab shell chitin, colloid chitin, and enzyme-processed chitin. The results showed that FPC had the highest chitinase adsorption capacity. Under 15 °C and pH 5.0, FPC exhibited an optimal chitinase adsorption capacity of 85.9 U/g, which was 61.9% higher than that of the colloidal chitin. With 0.02 M acetic acid as the eluent, a purification-fold of 10.3 with 97% chitinase recovery was obtained. The results of surface morphology studies indicated that the FPC surface was modified to a fiber-like structure with deep pores. In comparison with the surface morphology of enzyme-processed chitin and colloidal chitin, it is inferred that the enhanced adsorption capacity of FPC for chitinase is attributed to both the effects of chitinase hydrolysis and the bacterial modification.     

Characterization of Chitinase Produced by the Alkaliphilic <Emphasis Type="Italic">Bacillus mannanilyticus</Emphasis> IB-OR17 B1 Strain

The paper reports on the isolation of an extracellular chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain grown in media containing crab shell and bee chitin at a pH of 8–11. The enzyme was 860-fold purified by ultrafiltration and chitin sorption. The molecular weight of the purified chitinase was shown by denaturing electrophoresis to be 56 kDa. The enzyme showed maximum activity at a pH of 7.5–8.0 and 65°C and was stable within a pH range of 3.5–10.5 and temperature range of 75–85°C. With colloidal chitin as substrate, the kinetic characteristics of the chitinase were determined as follows: K_M ~ 1.32 mg/mL and V_max ~ 5.05 μM min^–1. N-acetyl-D-glucosamine and its dimer were the main products of enzymatic chitin cleavage, while the trisaccharide was detected just in minor quantities. The chitinase actively hydrolyzed p-nitrophenyl-GlcNAc₂ according to the exo-mechanism of substrate hydrolysis characteristic of chitobiosidases.     

Efficient decomposition of shrimp shell waste using <Emphasis Type="Italic">Bacillus cereus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis>

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37°C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37°C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.     

Chitinase production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: Their potential in antifungal biocontrol

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na⁺, Mg²⁺, Cu²⁺, and Ca²⁺ caused enhancement of enzyme activities whereas they were markedly inhibited by Zn²⁺, Hg²⁺, and Ag⁺. In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.     

Proficient biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the concomitant production of antifungal chitinase and antioxidant chitosaccharides

This study aimed to optimize the biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the co-production of chitinase and chitosaccharides (CS) under submerged fermentation and evaluation of their bioactivities. Canonical analysis and parametric optimization wrought the peakest production of chitinase (21.48 U/ml) and CS (124 μg/ml) after 66.4 h of fermentation at 37.6 °C. The medium containing 2.64% (w/v) shrimp shell powder, 0.38% (w/v) NaCl, 6.86 × 10⁶ cfu/ml inoculum concentration and an agitation speed of 120 rpm were found best. These optimized parameters were also authenticated by scale up of fermentation in 5 L fermentor and a reproducible results obtained with specific yield of chitinase (Y_P/S^chi) of 958.82 U/g and CS (Y_P/S^CS) 5.5 mg/g. A 59 kD chitinase was purified from culture filtrate by sequential chromatography techniques. The enzyme exhibited high degree of antifungal activity particularly against pathogenic Aspergillus flavus and Fusarium oxysporum by dissolving their cell wall components. The IC₅₀ values for A. flavus and F. oxysporum were 3.7 and 4.5 U/ml of purified chitinase, respectively. Chitosaccharides were extracted from the culture filtrate, quantitatively identified as admixture of N-acetylglucosamine monomer (57.5%) and dimer (39.2%). These chitosaccharides have potential antioxidant activity as detected by in vitro free radical scavenging assay.     

Biodegradation of shrimp processing bio-waste and concomitant production of chitinase enzyme and N-acetyl-D-glucosamine by marine bacteria: production and process optimization

A total of 250 chitinolytic bacteria from 68 different marine samples were screened employing enrichment method that utilized native chitin as the sole carbon source. After thorough screening, five bacteria were selected as potential cultures and identified as; Stenotrophomonas sp. (CFR221?M), Vibrio sp. (CFR173?M), Phyllobacteriaceae sp. (CFR16?M), Bacillus badius (CFR198?M) and Bacillus sp. (CFR188?M). All five strains produced extracellular chitinase and GlcNAc in SSF using shrimp bio-waste. Scanning electron microscopy confirmed the ability of these marine bacteria to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. HPLC analysis of the SSF extract also confirmed presence of 36-65?% GlcNAc as a product of the degradation. The concomitant production of chitinase and GlcNAc by all five strains under SSF using shrimp bio-waste as the solid substrate was optimized by 'one factor at a time' approach. Among the strains, Vibrio sp. CFR173?M produced significantly higher yields of chitinase (4.8 U/g initial dry substrate) and GlcNAc (4.7?μmol/g initial dry substrate) as compared to other cultures tested. A statistically designed experiment was applied to evaluate the interaction of variables in the biodegradation of shrimp bio-waste and concomitant production of chitinase and GlcNAc by Vibrio sp. CFR173?M. Statistical optimization resulted in a twofold increase of chitinase, and a 9.1 fold increase of GlcNAc production. These results indicated the potential of chitinolytic marine bacteria for the reclamation of shrimp bio-waste, as well as the potential for economic production of chitinase and GlcNAc employing SSF using shrimp bio-waste as an ideal substrate.     

Optimization of chitinase production by a new <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> C9 isolate using response surface methodology

The purpose of this article is to use statistical Plackett–Burman and Box–Wilson response surface methodology to optimize the medium components and, thus, improve chitinase production by Streptomyces griseorubens C9. This strain was previously isolated and identified from a semi-arid soil of Laghouat region (Algeria). First, syrup of date, colloidal chitin, yeast extract and K₂HPO₄, KH₂PO₄ were proved to have significant effects on chitinase activity using the Plackett–Burman design. Then, an optimal medium was obtained by a Box–Wilson factorial design of response surface methodology in liquid culture. Maximum chitinase production was predicted in medium containing 2% colloidal chitin, 0.47% syrup of date, 0.25 g/l yeast extract and 1.81 g/l K₂HPO₄, KH₂PO₄ using response surface plots of the STATISTICA software v.12.0.     

Characterization of the chitinase gene in Bacillus thuringiensis Mexican isolates

The chitinase gene was molecularly characterized in five Bacillus thuringiensis Mexican isolates, MR10, MR11, MR21, MR33, and RN52. The proteins derived from these genes were tested for their chitinase activity using fluorogenic chitin derivatives. In order to verify if chitinase genes were functional, they were cloned, and enzymatic activity of recombinant chitinases was also tested. Results indicated that enzymes exhibited endochitinase activity. The highest hydrolytic activity shown against the chitin tetrameric derivative occurred at pH value of 6.5, and the optimum activity temperature was around 60 °C. The recombinant endochitinases showed a molecular mass of ～77 kDa with isoelectric points from 6.5 to 7.0. Analysis of the nucleotide sequences showed highly conserved sequences among all isolates (97–99 %). Gene sequence analysis revealed a putative promoter (?35 TTGAGA and ?10 TTAATA) and a Shine–Dalgarno sequence (5´-AGGAGA-3´) upstream from the open reading frame. The deduced amino acid sequence revealed that the proteins are modular enzymes composed by a family 18 glycosyl hydrolase domain located between amino acids 134 and 549, a fibronectin-binding domain (580 through 656), and a chitin-binding domain (664 through 771). The deduced amino acid sequences of our isolates showed a similarity close to 100 % respect to the sequences reported in the GenBank database.