Characterization Of Chitinase Research Articles

Computational identification and characterization of chitinase 1 and chitinase 2 from neotropical isolates of Beauveria bassiana.

The fungus Beauveria bassiana is widely used for agronomical applications, mainly in biological control. B. bassiana uses chitinase enzymes to degrade chitin, a major chemical component found in insect exoskeletons and fungal cell walls. However, until recently, genomic information on neotropical isolates, as well as their metabolic and biotechnological potential, has been limited. Eight complete B. bassiana genomes of Neotropical origin and three references were studied to identify chitinase genes and its corresponding proteins, which were curated and characterized using manual curation and computational tools. We conducted a computational study to highlight functional differences and similarities for chitinase proteins in these Neotropical isolates. Eleven chitinase 1 genes were identified, categorized as chitinase 1.1 and chitinase 1.2. Five chitinase 2 genes were identified but presented a higher sequence conservation across all sequences. Interestingly, physicochemical parameters were more similar between chitinase 1.1 and chitinase 2 than between chitinase 1.1 and 1.2. Chitinases 1 and 2 demonstrated variations, especially within chitinase 1, which presented a potential paralog. These differences were observed in their physical parameters. Additionally, CHIT2 completely lacks a signal peptide. This implies that CHIT1 might be associated with infection processes, while CHIT2 could be involved in morphogenesis and cellular growth. Therefore, our work highlights the importance of computational studies on local isolates, providing valuable resources for further experimental validation. Intrinsic changes within local species can significantly impact our understanding of complex pathogen-host interactions and offer practical applications, such as biological control.

Production, purification and characterization of chitinase from Micromonospora sp. AR17

N‐acetylglucosamine (NAG) is the monomer product of chitin, which has been widely used as a bioactive com‐ pound in applications such as anti‐tumor, anti‐microbial, and antioxidant activities. In production, biological processes using enzymes are preferable to chemicals due to environmental issues. This study aims to determine the activity, purity level, and molecular weight of purified chitinase from Micromonospora sp. AR17 determines the concentration of NAG produced by purified chitinase that has been characterized. Chitinase was produced by fermentation in colloidal chitin broth at 40 °C, pH 7, for 7 days, while chitinase activity was checked every 24 h. The optimal fermentation time was used to produce chitinase for a further purification step. Enzyme purification was carried out by ultrafiltration, ammonium sulfate precipitation, ion exchange chromatography (Q Sepharose Fast Flow), and gel filtration (Sephacryl S‐300). The purified enzyme was then char‐ acterized for optimum time, pH, and temperature to produce NAG. The results suggested that the fourth day was the optimal time for chitinase production, with chitinase activity of 0.0040 U/mL and a NAG concentration of 7.62 µg/mL. The purifica‐ tion step successfully increased the purity by 6.82 times with chitinase‐specific activity at 1.4648 U/mg. Production of NAG with purified chitinase produced a NAG concentration of 32.472 µg/mL with an incubation time of 30 min at 40 °C and pH 7.

Characterization of chitinases from the GH18 gene family in the myxomycete Physarum polycephalum

BackgroundPhysarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. MethodsLow stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. ResultsCatalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. ConclusionsPhysarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. General significanceMyxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field.

Characterization of chitinase from Exiguobacterium antarcticum and its bioconversion of crayfish shell into chitin oligosaccharides

Crayfish have become a heavily consumed food and its chitin-rich shell is of great value in terms of waste conversion. This study found a novel chitinase (EaChi40) from a marine bacterium Exiguobacterium antarcticum. The gene was cloned and expressed as a soluble protein of 40 kDa, having optimal activity at pH 6.0 and 30 °C. EaChi40 showed good stability and high specific activity, and kinetic studies found Km and Vmax were 0.86 mg/mL and 13.66 μmol/min/mg. For conversion crayfish shell into oligosaccharides, ball milling and ultrasound-assisted hydrogen peroxide decolorization were applied to pretreat crayfish shell to facilitate its hydrolysis. After the enzymatic conversion, the hydrolysis products of chitobiose and N-acetyl-D-glucosamine were 9.09 mg/mL and 9.21 mg/mL, respectively. EaChi40 efficiently degraded crayfish with a high hydrolysis rate of 76.1%. It is expected to be a good candidate for the production of chitin oligosaccharides in the food and biological fields.

Analysis and Characterization of Chitinase in Bacillus salmalaya Strain 139SI

Introduction: Chitinases are enzymes that hydrolyze the internal β-1,4 glycosidic linkages of chitin, a significant structural component of arthropod exoskeletons and fungus cell walls. Objective: Main objective of current study was to determine the protein and to check the antimicrobial, antifungal activity of chitinase in Bacillus salmalaya strain 139SI. Methodology: Purified and estimation enzymes were quantified by the method of Lowry method. Antimicrobial action of chitinase was done using standard Disc Diffusion method while the hyphal extension inhibition assay was used to test the antifungal activity of pure chitinase strains 139SI, 140SI, and 141SI. Results: In this study, Bacillus salmalaya 139SIexhibited strong hemolytic activity and their protein concentration was measured as 56.43 mg/mL. In addition, strain 139SI had strong antifungal activity against phytopathogenic fungus including Fusarium sp., R. solani and Phytophthora sp. Strain 139SI had the capability in degrading the peptidoglycan component of cell walls of gram-negative bacteria such as Escherichia coli but not against the gram-positive, Staphylococcus aureus. Chitinase activity was observed when 200μl crude extract of 139SI able to degrade 0.09 g chitin of shrimp shell by breaking down shrimp shell structure and bonds of chitin effectively as early as 2 days or up to 7 days. Conclusion: Hence, based on the results, B. salmalaya 139SI has potential to be a novel biofunctional chitinase that could use as a biological agent in degrading the chitin component of fungal cell walls and shells waste of many kind of insect and crustaceans for solving future problem in the agricultural sector and fishery industry.

Purification and characterization of chitinase from Paenibacillus sp. .

The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on SephadexG-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50°C with colloidal chitin as a substrate. The enzyme is stable below 45°C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca2+ , Fe2+ , Fe3+ , and Ni2+ . It is inhibited by SDS, H2 O2 , ascorbic acid, Cu2+ , Mg2+ , Ba2+ , Sn2+ , Cr3+ , and K+ . With colloidal chitin as substrate, the Km and the Vmax of the chitinase are 4.28 mg/mL and 14.29 μg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethylchitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.

Characterization of chitinase from Streptomyces luridiscabiei U05 and its antagonist potential against fungal plant pathogens

AbstractStreptomyces luridiscabiei U05 was isolated from wheat rhizosphere. It produced chitinase, which showed in vitro antifungal properties. The crude enzyme inhibited the growth of Alternaria alternata, Fusarium oxysporum, F. solani, Botrytis cinerea, F. culmorum and Penicillium verrucosum. The chitinase enzyme of the molecular weight of 45 kDa was purified using affinity chromatography of chitin. Streptomyces luridiscabiei U05 produced different chitinolytic enzymes. The highest enzyme activity was observed with the use of 4‐MU‐(GlcNAc), which points to the presence of an β‐N‐acetylhexosaminidase. The optimum activity was obtained at 35–40°C and pH 7–8. The enzyme showed thermostability at 35–40°C during 240 min of preincubation and lost its activity at 50°C and 60°C in 60 min. The chitinase activity from S. luridiscabei U05 was strongly inhibited by Hg2+ and Pb2+ ions, and sodium dodecyl sulphate (SDS). The Ca2+, Cu2+ and Mg2+ ions stimulated the activity of the enzyme.

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

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

Characterization of Chitinase Produced by the Alkaliphilic Bacillus mannanilyticus 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: KM ~ 1.32 mg/mL and Vmax ~ 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.

Purification and characterization of chitinase from the integument of the Mediterranean flour moth, Ephestia kuehniella (Lepidoptera: Pyralidae) and its antibacterial role

AbstractChitinases are found in species from all kingdoms, including Kingdom Animalia. We purified chitinase, poly [1,4-(N-acetyl-β-d-glucosaminide)] glycanohydrolase, by cation exchange chromatography on SP-Sepharose from the integument of a lepidopteran, the Mediterranean flour moth, Ephestia kuehniella Zeller, and further characterized it. Ephestia kuehniella chitinase has similar properties to other insect chitinases, with respect to the molecular mass. Its molecular mass was 79.7 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for activity were 6 and 40 °C, respectively. The purified enzyme exhibited stability at a pH range from 4.5 to 8.0 and showed high sensitivity to Co2+, Mn2+, Ca2+, Hg2+ and Ag+. The Km and Vmax values of E. kuehniella chitinase were, respectively, 3.98 mg/mL and 7.11 μmol/min/mg for colloidal chitin. In comparison to the values determined for the substrate affinity, the enzyme had a 2.36-fold higher affinity for Carboxymethyl–Chitin–Remazol Brilliant Violet (CM–Chitin–RBV) as a soluble substrate. We examined the antibacterial effect of E. kuehniella chitinase on the growth of both Gram-positive and Gram-negative bacteria. The enzyme inhibited the growth of Bacillus subtilis, Bacillus thuringiensis and Escherichia coli, where the average diameter of inhibition zone was 20, 23 and 19 mm, respectively. The antimicrobial effect and chitinase activity strengthen the hypothesis that the enzyme could be used in biological control against plant pests.

Characterization of Chitinase from Bacillus thuringiensis Isolated from the Dynastid Beetle, Allomyrina dichotoma L.

Chitin is a linear polysaccharide that consists of β-1,4-linked N-acetyl-glucosamine (GlcNAc) and it forms the carbohydrate backbone of crustacean and insect exoskeletons. Chitinase has the ability to degrade these chitins to its product chitin oligosaccharides which are used in potential applications of biomedicine, nutrition and bio-technology. Bacillus thuringiensis is one of the well-known bacteria producing chitinase. In present study, we identified and purified the chitinase from B. thuringiensis PRCII. Chitinase activity assay using crap shell chitin and T. molitor chitin indicates that three chitinases approximately 37, 42, and 71 kDa were detected from B. thuringiensis PRCII. In addition, we cloned and expressed the 71 kDa chitinase from B. thuringiensis PRCII. These chitinases exhibited two chitinase protein activity 71 and 50 kDa in both native and rBtchitinase using crap shell and T. molitor chitin. On the basis of the in silco analysis of Btchitinase, Btchitinase lacks chitin binding domain. However, it exhibited the active chitinase activity, suggesting that chitin binding domain is not necessary for chitin hydrolysis.

Production and Characterization of Chitinases from Thermophilic Bacteria Isolated from Prataan Hot Spring, East Java

Thermophilic bacteria producing chitinase were collected from Prataan hot spring, East Java, Indonesia and screened. The isolated bacterium was analyzed using 16S rRNA gene sequencing analysis and identified as Paenibacillus sp. The molecular identification was confirmed through morphological and physiological analyses. The production of chitinase was conducted at various incubation times, temperatures, pH and concentrations of colloidal chitin. The optimum condition of the isolate to produce the highest chitinase was 0.9% (w/v) of colloidal chitin (pH 7.0) at 48 °C for 24 hours. The obtained chitinases were optimally active at 55 °C and pH 6.0-7.0. The chitinases were gradually purified by ammonium sulfate precipitation, Sephadex G-100 gel filtration, followed by DEAE"“cellulose ion exchange chromatography (IEC). The purification method gave a purification factor of 9.43 and a yield of 2.68%. Two protein fractions were obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 68 and 82 kDa.

Production and Characterization of Chitinases from Thermophilic Bacteria Isolated from Prataan Hot Spring, East Java

Production and Characterization of Chitinases from Thermophilic Bacteria Isolated from Prataan Hot Spring, East Java

Isolation and characterization of chitinase from soil fungi, Paecilomyces sp.

Chitinolytic fungal strains were isolated from soil in Thailand. They were screened as chitinase producers by testing their shrimp shell digestion ability on potato dextrose agar plates. The chitinase activity was tested with colloidal chitin in culture medium C and basal medium. There was greater activity in culture medium C than in the basal medium. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from the culture filtrate of medium C showed three protein bands at about 40 kDa, 46 kDa and 56 kDa. The chitinase gene was sequenced from genomic DNA. The obtained sequence consisted of 713 bp upstream, a 1499 bp open reading frame that was interrupted by three introns and 1698 bp downstream sequences. The intron lengths were 63 bp, 57 bp and 110 bp, respectively. The sequence was found to be the most similar to the chitinase gene of Paecilomyces lilacinus (EF183511). Pairwise alignment of the 1499 bp and P. lilacinus resulted in 72.5% DNA sequence identity, while alignment of the 1269 bp coding sequence and P. lilacinus resulted in 78.5% cDNA sequence identity and 83.5% amino acid sequence identity. The protein structure contained two conserved domains of the putative substrate binding site (S-I-G-G) and catalytic domain (D-G-I-D-L-D-W-E), suggesting that this fungal chitinase belonged to the glycosyl hydrolases family 18 chitinase (GH18). Phylogenetic analysis of the chitinase gene from the nematopathogenic fungi suggested that this chitinase sequence was class V chitinase.

Characterization of Extraceluller Chitinase Produced by <i>Bacillus licheniformis</i> JP2 from Penen Hot Springs, North Sumatera

The research was about characterization of extraceluller chitinase produced by Bacillus licheniformis JP2 from Penen Hot Springs, North Sumatera. Precipitation of crude chitinase was performed with different levels of ammonium sulphate. Chitinase activity was determined colorimetrically by detecting the amount of N-acetylglucosamine (GlcNAc) released from colloidal chitin substrate with N-acetylglucosamine. Characterization of chitinase activity was measured at different pH, different temperature, Km and Vmax values. The optimum activity of chitinase was produced 4 days after incubation with chitinase activity of 0.0063 U/ml. Optimum chitinase activity was higher after 50% ammonium sulphate precipitation with activity of 0.0087 U/ml followed by decreasing in activity supernatant with activity of 0.0012 U/ml. Chitinase activity increased at pH 6 and at 60°C by 0.05066 U/ml with specific activity 3.8113 U/mg after dialysis. Km and Vmax values were 0.321 mg/ml and 71.429 µg, respectively.

Purification and characterization of chitinases from ridgetail white prawn Exopalaemon carinicauda.

In this paper, we purified two native chitinases from the hepatopancreas of the ridgetail white prawn Exopalaemon carinicauda by using ion-exchange resin chromatography (IEC) and gel filtration. These two chitinases, named EcChi1 and EcChi2, were identified by chitinolytic activity assay and LC-ESI-MS/MS. Their apparent molecular weights were 44 kDa and 65 kDa as determined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of EcChi1 and EcChi2 was 1305.97 U·mg−1 and 28.69 U·mg−1. The optimal temperature and pH of EcChi1 were 37 °C and pH 4.0, respectively. Co2+, Fe3+, Zn2+, Cd2+, and Cu2+ had an obvious promoting effect upon chitinase activity of EcChi1. For colloidal chitin, the Km and Vmax values of EcChi1 were 2.09 mg·mL−1 and 31.15 U·mL−1·h−1.

Cloning and characterization of chitinases from interior spruce and lodgepole pine

Chitinases have been implicated in the defence of conifers against insects and pathogens. cDNA for six chitinases were cloned from interior spruce (Picea glauca x engelmannii) and four from lodgepole pine (Pinus contorta). The cloned interior spruce chitinases were annotated class I PgeChia1-1 and PgeChia1-2, class II PgeChia2-1, class IV PgeChia4-1, and class VII PgeChia7-1 and PgeChia7-2; lodgepole pine chitinases were annotated class I PcChia1-1, class IV PcChia4-1, and class VII PcChia7-1 and PcChia7-2. Chitinases were expressed in Escherichia coli with maltose-binding-protein tags and soluble proteins purified. Functional characterization demonstrated chitinolytic activity for the three class I chitinases PgeChia1-1, PgeChia1-2 and PcChia1-1. Transcript analysis established strong induction of most of the tested chitinases, including all three class I chitinases, in interior spruce and lodgepole pine in response to inoculation with bark beetle associated fungi (Leptographium abietinum and Grosmannia clavigera) and in interior spruce in response to weevil (Pissodes strobi) feeding. Evidence of chitinolytic activity and inducibility by fungal and insect attack support the involvement of these chitinases in conifer defense.

Purification and characterization of thermostable chitinase from a novel S. maltophilia strain

Aims: The presents study examines the purification and characterization of a chitinase from S. maltophilia SJ602 strain isolated from a soil sample collected from Jamia Hamdard, New Delhi. Methodology and Results: The purification steps included chitin affinity using colloidal chitin as the affinity matrix and column chromatography using Sephadex G-100. The chitinase was purified to 66 fold having a yield of 17%. The molecular weight of the chitinase was found to be around 29 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima of the purified chitinase were found to be at pH 5.5 and 60 °C, respectively. Conclusion, Significance and Impact of the study: Besides showing a significant yield, the enzyme has a high thermal stability which has its applicability in the recycling of chitin waste.

Isolation and Characterization of Chitinase from Tomato Infected by Fusarium oxysporum f. sp. lycopersici

AbstractChitinases are important component of plant defence in response to attack by pathogens. To identify specific chitinase, we constructed a cDNA library using total RNA from a genotype‐resistant tomato inoculated with conidia of isolates race 2 of Fusarium oxysporum f.sp. lycopersici (Fol). One chitinase (SolChi) clone was isolated and sequence analysis shows that the cDNA clone SolChi encodes an acidic isoform of class III chitinase. Southern blotting indicated that SolChi was present only once in the tomato genome. Real‐time quantitative RT‐PCR assay show that the expression of this gene is induced upon infection with Fol, and the accumulation of transcripts for this R protein was rapid in the resistant genotype during the first 24 h. A putative role for chitinase in tomato is defence against fungal pathogens.

Purification and characterization of chitinase from Gliocladium catenulatum strain HL-1-1

Chitinase was isolated and purified from Gliocladium catenulatum through ammonium sulfate sedimentation, sephadex G-25 gel filtration, deionization, ultra filtration condensation, negative ion exchange separation, and non-denaturing gel electrophoresis. A 51 kDa chitinase was purified from the fungus through sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum temperature and pH for chitinase activity were 60°C and pH 6.0, respectively. The enzyme solution was stable from 20 to 40°C and pH 4 to 5. Co2+ and Ca2+ were conducive to the enzyme activity, whereas the Fe3+, Cu2+, and Ag+ evidently inhibited the enzyme activity. Fe2+, Na+, K+, Zn2+, and Mn2+ showed less inhibitory effects on the enzyme activity. The Km of the chitinase was 2.832 mg ml-1. The chitanase were found to inhibit the hyphal growth, conidial germination and sclerotial germination of various plant pathogenic fungi. Key words: Gliocladium catenulatum, chitinases, purification, characteristics.