Crude Chitinase Research Articles

Sustainable quick quantitative screening for the physiology of chitinases production by Bacillus thuriengiensis gallariae and detection of Aedes aegypti biopesticide activity

Background Chitin is considered the world’s first and most abundant amino-carbon substrate. Objective The proposed method aims to speed up the detection and measuring of microbial chitinase production. Materials and methods Solid-state fermentation, plates, viscosity, and spectrophotometric methods were used to detect microbial chitinase activity. Analysis of variance was used for statistical analysis. Results and conclusion The trailed Bacillus thuriengiensis gallariae strain was differentiated from other tested strains of Bacillus thuriengiensis in terms of mosquito resistance, besides being superior to other studied Bacillus sphaericus. As anticipated, results from the clear zone plates method were not clear compared with the viscosity method where the strain exhibited the best. Worthy, the same result was reached in the color methods. The superlative incubation period for the physiological properties of chitinase production from Bacillus thuriengiensis gallariae was attained after 5 days. Results indicated that the best size of chitin particles was 1 mm, the best percentage of humidity of the growth was 67%, the best inoculum volume was 10 ml, and the best carbon source for the production of crude chitinases was glucose. The optimal concentration for the production of chitinases was 20% of the weight of the growth medium, yet 5% of the weight of the growth medium was the optimal percentage of glucose as a carbon source for the production of other protein metabolites. The proposed colorimetric method seemed to be an effective and rapid method to survey the aptitude of huge quantities of microorganisms to produce chitinases.

Chitinase Producing Gut-Associated Bacteria Affected the Survivability of the Insect Spodoptera frugiperda.

Fall armyworm (Spodoptera frugiperda) is a highly destructive maize pest that significantly threatens agricultural productivity. Existing control methods, such as chemical insecticides and entomopathogens, lack effectiveness, necessitating alternative approaches. Gut-associated bacteria were isolated from the gut samples of fall armyworm and screened based on their chitinase and protease-producing ability before characterization through 16S rRNA gene sequence analysis. The efficient chitinase-producing Bacillus licheniformis FGE4 and Enterobacter cloacae FGE18 were chosen to test the biocontrol efficacy. As their respective cell suspensions and extracted crude chitinase enzyme, these two isolates were applied topically on the larvae, supplemented with their feed, and analyzed for their quantitative food use efficiency and survivability. Twenty-one high chitinase and protease-producing bacterial isolates were chosen. Five genera were identified by 16S rRNA gene sequencing: Enterobacter, Enterococcus, Bacillus, Pantoea, and Kocuria. In the biocontrol efficacy test, the consumption index and relative growth rate were lowered in larvae treated with Enterobacter cloacae FGE18 by topical application and feed supplementation. Similarly, topical treatment of Bacillus licheniformis FGE4 to larvae decreased consumption index, relative growth rate, conversion efficiency of ingested food, and digested food values. The presence of gut bacteria with high chitinase activity negatively affects insect health. Utilizing gut-derived bacterial isolates with specific insecticidal traits offers a promising avenue to control fall armyworms. This research suggests a potential strategy for future pest management.

Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm–3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

Nematode egg parasitic fungus, Purpureocillium lilacinum: efficacy of indigenous strains for the management of Meloidogyne incognita in chickpea

BackgroundNematode egg parasitic fungus, Purpureocillium lilacinum is the most effective biocontrol agent and has been widely used commercially in many countries for the management of root-knot nematode, Meloidogyne incognita. Availability of indigenous potential strains specific to an agro ecosystem is very crucial for their successful commercial exploitation for suppression of nematode population. Hence, an attempt was made to isolate, characterize, evaluate and identify potential indigenous strains of P. lilacinum from pulse ecosystem for root-knot nematode management in chickpea.ResultsThe fungal colony was initially white and when spore was formed it turned into pink colour in 72 to 96 h. Hyphae was hyaline and septate, conidiophore was blunt, and phialides were with wide base and long neck bearing round to oval conidia in chains. Molecular identification of the species, P. lilacinum was carried out based on ITS1-5.8S-ITS2 region of the genomic DNA. In vitro bioassay of cultural filtrates on juvenile mortality revealed that maximum percentage of mortality was observed in IIPR-Pl-11 (88.36%). Spectrophotometric assay on chitinolytic activity showed that the strain IIPR-Pl-11 produced significantly high chitinolytic activity, chitinase enzyme and total protein content (0.139, 51.1 and173.75 µg/ml at 5days); (0.245, 90.1 and 272.67 µg/ml at 10 days) and (0.273, 100.4 and 306.25 µg/ml), respectively, at 15 days of culturing in colloidal chitin-enriched medium C. 2D gel electrophoresis of the crude chitinase suspension showed the presence of chitinase (32, 46 kDa size) in the sample from chitinase-induced medium C. In vitro bioassay of the cultural filtrates of the fungus grown in chitin-enriched medium C on inhibition on egg hatching revealed that the highest percent inhibition on egg hatching showed by IIPR-Pl-8 strain at 5 days of inoculation (42.6%) and IIPR-Pl-11 at 10 and 20 days of inoculation (62.80 and 93.50%), respectively. In vivo pot experiment revealed that among all strains, IIPR-Pl-11 was efficient in promoting plant growth very effectively by reducing gall number (41.3 per plant), egg mass (28.3 per plant) and soil population (284.3 per 200cc of soil) compared to control.ConclusionP. lilacinum strain IIPR-Pl-11 was the highest potential strain from pulse rhizosphere for the management of root-knot nematode, M. incognita in chickpea.

PH and temperatures optimation activity of crude chitinase of oil palm endophytic bacteria Bacillus cereus which have antagonistic properties on pathogenic fungi Ganoderma boninense

Endophytic bacteria is promised solution to suppress basal stem rot disease caused by fungus Ganoderma boninense. The antifungal activities of selected isolate endophytic bacteria from oil palm plantation in South Kalimantan was studied. Bacillus cereus was tested for its antifungal activities of crude chitinase and secondary metabolites against the growth of Ganoderma boninense mycelium with dual cultured method. The results were showed that chitinase had the largest inhibition zone (18.5%) against growth inhibition of fungi Ganoderma boninense’s mycelium. A series of optimation assays of chitinase activity were conducted at pH 3 to 8 with 0.5 interval, and temperature at 30 to 70°C, with 5°C interval. Specific chitinase activities was measured using the colorimetric method. The highest specific chitinase activity significantly at 1.66393±0.04807 mU/μg (95% confidence level) at pH 5.5 and temperature 45°C.

Bioproduction of Chitin Hydrolysate Containing N-Acetylglucosamine by Serratia marcescens PT6 Crude Chitinase and Its Effects on Bacterial Growth Inhibition in Various Temperature

N-acetylglucosamine (GlcNAc), a chitin monomer, can be used as a natural preservative to ensure food quality and safety. Combining natural preservatives with low storage temperature offers physical hurdles to bacterial growth in food. This study aimed to produce chitin hydrolysate containing GlcNAc using Serratia marcescens PT6 crude chitinase and investigate its effect on bacterial growth rate as a function of temperature. Crude chitinase from partial purification was used to hydrolyze 1.3% colloidal chitin. The optimal enzymatic conditions were pH 6 and 45˚C for 120 min, at an enzyme:substrate ratio of 1:1, yielding a 65.6 µg/mL GlcNAc. Inhibitory activity of hydrolysate containing 2.5-7.5 ppm GlcNAc on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus was measured at 4, 15, and 30oC in nutrient broth. Bacterial growth was measured using of optical density for each combination of GlcNAc concentration and temperature. Growth curves fitted by the Baranyi and Roberts model were developed using DMFit software. The growth rate was converted to the square root and then modeled as a function of temperature using the Ratkowsky square root model. Incubation temperature exerted a pronounced effect on the inhibition of all bacterial species (P<0.0001), with the greatest effect observed for E. coli at 30°C (P<0.0001), and the least effect for V. parahaemolyticus (P=0.0878). The inhibitory effect of GlcNAc in chitin hydrolysate was only significant for E. coli (P<0.0001) and S. aureus (P=0.0041). This study revealed that the effect of temperature in growth inhibition was more significant than GlcNAc addition. However, a reduction in bacterial growth with the addition of GlcNAc at 30°C was observed, which may be effective for food encountered thermal abuse conditions. Further investigation of the effect of GlcNAc on bacteria structure and metabolism is required to elucidate the mechanism of GlcNAc as a food preservative.

Bioprospecting of Chitinolytic Diazotroph Rhizospheric Bacterial Isolated From Mucuna bracteata as Biocontrol Against Ganoderma boninense

Ganoderma boninense is an infectious phytopathogenic fungus of basal stem rot in oil palm with the need of significant biocontrol strategy or alternative. Bioprospecting of chitinolytic diazotroph bacteria from rhizosphere of Mucuna bracteata as Legume Crop Cover (LCC) in oil palm plantations is potential to investigated. The aims of this study were to obtain total population of diazotroph bacteria and chitinolytic diazotroph bacteria, to evaluate its antagonistic properties followed by identification of the isolate based on 16S rRNA gene encoding. Diazotroph bacteria were isolated from M. bracteata rhizosphere originating from three oil palm plantations (PTPN III, PTPN IV unit Adolina Medan, and Perkebunan rakyat Simalingkar), followed with chitinolytic assay, morphological characterization, antagonistic assay against G. boninense, lytic assay of G. boninense mycelium by using crude chitinase and molecular identification of potential isolate. Total population of diazotroph bacteria in M. bracteata rhizosphere ranged between 2.80–3.83 × 106 CFU/g. Screening of chitinolytic diazotrophs using colloidal chitin medium obtained 23 bacterial isolates and 14 of them were known as antagonists to G. boninense. Five isolates with the highest anti Ganoderma were DK17 (71,15%), DK10 (69,70%), DK07 (59,63%), DK21 (53,48%), and DK19 (52,0%). The highe1st lytic activity of crude chitinase to G. boninense mycelium was produced by DK17. Molecular identification of five potential isolates revealed different identities, e.g. Enterobacter aerogenes (DK07), Mycobacterium senegalense (DK10), Bulkhoderia cepacia (DK17), Pseudomonas stutzeri (DK19), and Bulkhoderia cepacia (DK21). Based on these results, chitinolytic diazotroph bacteria isolated from M. bracteata rhizosphere were able to control G. boninense

Antifungal Chitinase Production by Bacillus paramycoides B26 using Squid Pen Powder as a Carbon Source

This study aimed to optimize the medium compositions and cultural conditions for improved chitinase production by a potential strain of Bacillus isolated from the marine environment and determine the antifungal activity of its chitinase against plant pathogenic fungi. Five potential isolates were cultured for chitinase production by submerged fermentation using colloidal chitin in a liquid medium. In this study, chitinase activity was determined by measuring reducing sugars, which were determined by the 3,5-dinitrosalicylic acid (DNS) assay. The most potential isolate, B26, showed similarity to Bacillus paramycoides based on the 16S rRNA gene sequence. The maximum chitinase production was achieved at 6.52±0.02 U/mL after 72 h of incubation in a medium containing 2% squid pen powder, supplemented with 0.5% sodium nitrate and 2% NaCl, with an initial pH of 7. It was observed that the optimization of cultural conditions resulted in 2.83 times higher chitinase production than an unoptimized medium. The antifungal activity of crude chitinase against phytopathogenic fungi was evaluated by a well-diffusion method. The chitinase of B. paramycoides B26 effectively inhibited the growth of Fusarium solani TISTR 3436 (83.4%) and Penicillium chrysogenum TISTR 3554 (80.12%).

Chitinolytic and Antagonistic Activity of Streptomyces Isolated from Fadama Soil against Phytopathogenic Fungi.

Chitinases which degrade chitin have attracted attention as biological antifungal agents. The purpose of this study is to isolate Streptomyces from Fadama soil and assess its chitinolytic and antagonist potential against phytopathogenic fungi for application as biocontrol agent. Streptomyces were isolated from Fadama soil. The selected isolate CT02 exhibited chitinolytic characteristics. Chitinase production was performed under different temperatures, pH and varying incubation period. The highest chitinase production by CT02 isolate was observed after five days of cultivation. The highest chitinase activity was observed at 35°C and pH 7. The crude extracellular enzyme exhibited a specific activity of 4.20 U/μg whereas partially purified extracellular enzyme exhibited a specific activity of 6.19 U/μg with purification fold of 1.47. The selected isolate CT02 and its extracellular crude chitinase showed in vitro antifungal antagonist potential by inhibiting the growth of Aspergillus niger and Aspergillus oryzae. This indicates that Streptomyces derived chitinases are potential biocontrol agents against phytopathogenic fungi.

Characterization of Novel Chitin-degrading laceyella spp. Strains from New Athos Cave (Abkhazia) Producing Thermostable Chitinases

Nine chitin-degrading isolates of moderately thermophilic bacteria with temperature optimum for growth and chitin destruction at 50–55°C were isolated from soils of the New Athos Cave (Abkhazia). Four most active bacterial cultures with chitinase production yields 0.11–0.28 U mL–1 (at 48°C) were screened from seven isolates forming aerial mycelium. Selected isolates were identified as Laceyella sp. species based on their cultural, morphological, physiological, and biochemical characteristics, as well as on phylogenetic analysis of their 16S rRNA gene sequences; these formed a cluster with L. sacchari, L. sediminis, and L. tengchongensis on the phylogenetic tree with sequence identity of 96.43–97.21%. Occurrence of isolated thermoactinomycetes in the cave was confined to tour circuit zones and the area inundated with flood waters. Laceyella spp. strains NAC-16-5 A-4 and NAC-16-5 A-5 degraded 85–100% of colloidal chitin (5 g L–1) after 96 h of submerged cultivation (48°C). Inducible synthesis of chitinolytic enzymes was observed in all selected strains and reached its maximum in the presence of crab shell chitin. The enzymatic complex of Laceyella sp. NAC-16-5 A-4 was active in pH range 4–10.5 with two optima at pH 5.5 and 7.0. While crude chitinases of all studied strains exhibited maximal activity at 60–70°C with similar temperature optima (65°C), the enzyme complexes from strains NAC-16-5 A-2 and NAC-16-5 A-4 were most stable at 70°C.

Production of N-acetylglucosamine from shrimp shells’ chitin using intracellular chitinase from Mucor circinelloides

Chitin is a natural biopolymer found in shrimp shells and can be processed into Nacetylglucosamine which is extensively used as a dietary supplement to treat osteoarthritis, back pain and knee pain. This research was conducted to determine the optimum pH, temperature, substrate concentration and incubation period to produce Nacetylglucosamine using crude and semi pure intracellular chitinase extracted from Mucor circinelloides. Chitinase activity was measured to determine optimum pH and temperature by using various pHs (3, 4, 5, 6, 7, 8 and 9) and temperatures (30oC, 40oC, 50oC, 60oC, 70oC and 80oC). Different substrate concentrations (0.5%, 1.0%, 1.5% and 2.0%) and incubation periods (2, 4, 6 and 24 hrs) were used to determine the optimum condition to produce N-acetylglucosamine. Results showed that crude intracellular chitinase had an optimum pH of 5 with chitinase activity of 4.16±0.07 U/mL and optimum temperature of 60oC with chitinase activity of 4.22±0.07 U/mL. The optimum substrate concentration obtained was 0.5% and the optimum incubation period obtained was 6 hrs with about 961.67±9.13 ppm N-acetylglucosamine produced. Semi pure intracellular chitinase had an optimum pH of 4 with chitinase activity of 4.75±0.09 U/mL and optimum temperature of 50oC with chitinase activity of 5.03±0.08 U/mL. The optimum substrate concentration obtained was 1.5% and the optimum incubation period obtained was 4 hrs with about 1150.56±12.55 ppm N-acetylglucosamine produced.

Termiticidal activity of chitinase enzyme of Bacillus licheniformis, a symbiont isolated from the gut of Globitermes sulphureus worker

Abstract Chitinase is a digestive enzyme that breaks down glycosidic bonds in chitin. Chitin is a biopolymer that can be found in the exoskeleton of insects, fungi, yeast, and algae, and in the internal structures of other vertebrates. Bacillus licheniformis strain USMW10IK was isolated from the guts of Globitermes sulphureus workers. The production of chitinase was highest during the stationary phase (72–96 h of incubation) with the enzyme activity was 0.0057 U/mL – 0.0058 U/mL. The crude chitinase extracted after 72 h was used as biotermiticide against termite G. sulphureus. A pathogenic bioassay was done in four methods; no choice, treated sand medium, topical (Petri dish lined with filter paper), and topical (Petri dish lined with sterile sand). Termite mortality was observed for 24–48 h as the crude chitinase stability has decreased by 22.8% after 48 h. As a result, topical (Petri dish lined with filter paper) has caused significant termite mortality after 24 h and topical (Petri dish line with sterile sand) gave significant termite mortality after 48 h while no choice and sand treated medium gave no significant mortality. These findings suggest that chitinase is effective to be used as a biotermiticide when it directly contacts with the termite skin.

A Cold-Adapted Chitinase-Producing Bacterium from Antarctica and Its Potential in Biocontrol of Plant Pathogenic Fungi.

To obtain chitinase-producing microorganisms with high chitinolytic activity at low temperature, samples collected from Fildes Peninsula in Antarctica were used as sources for bioprospecting of chitinolytic microorganisms. A cold-adapted strain, designated as GWSMS-1, was isolated from marine sediment and further characterized as Pseudomonas. To improve the chitinase production, one-factor-at-a-time and orthogonal test approaches were adopted to optimize the medium components and culture conditions. The results showed that the highest chitinolytic activity (6.36 times higher than that before optimization) was obtained with 95.41 U L−1 with 15 g L−1 of glucose, 1 g L−1 of peptone, 15 g L−1 of colloid chitin and 0.25 g L−1 of magnesium ions contained in the medium, cultivated under pH 7.0 and a temperature of 20 °C. To better understand the application potential of this strain, the enzymatic properties and the antifungal activity of the crude chitinase secreted by the strain were further investigated. The crude enzyme showed the maximum catalytic activity at 35 °C and pH 4.5, and it also exhibited excellent low-temperature activity, which still displayed more than 50% of its maximal activity at 0 °C. Furthermore, the crude chitinase showed significant inhibition of fungi Verticillium dahlia CICC 2534 and Fusarium oxysporum f. sp. cucumerinum CICC 2532, which can cause cotton wilt and cucumber blight, respectively, suggesting that strain GWSMS-1 could be a competitive candidate for biological control in agriculture, especially at low temperature.

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.

Biosynthesis of biologically active chitinase utilizing some Egyptian chitinaceous wastes and the properties of the synthesized enzyme

Background and objective Chitin-degrading enzymes have an utmost practical importance in many fields such as medicine, agriculture, and industry. These enzymes are used as effective antibacterial, antifungal, antihypertensive, and antioxidant agents and also as excellent food quality enhancers. The objective of the present article was to formulate the production medium and to pinpoint the proper growth conditions for the chosen microorganism producing highly active chitinase enzymes. The general properties of the crude enzyme preparation were determined to define the proper conditions for enzyme action. Under the specified conditions, the capability of the enzyme preparation for antimicrobial and antioxidant activities were decided. Materials and methods Eighteen recommended microbial strains were screened for biologically active chitinolytic enzymes productivity. Chitinase enzyme was determined, and also the important properties of the crude chitinase were duly pinpointed. Finally, biological activities of the crude enzyme were studied. Results and conclusion and conclusion Among all the 18 organisms, Streptomyces halstedii H2 was the most potent producer of an influential chitinase enzyme. The maximum chitinase activity of 49.5 U/ml was obtained from medium contains glucose 6 g/l, ammonium nitrate (0.9 g/l), and urea (0.64 g/l) at 30°C and pH 9.0. The important properties of the streptomycetal chitinase were duly pinpointed as follows: optimum enzyme and substrate concentrations were 1.6 mg/ml and 1.4% (w/v), respectively, and optimum reaction pH and temperature were 7.2 and 45°C, respectively. The crude preparation was stable for 60 min at pH 7.2 and 30°C and retained 92.6% of the original activity. Under the specified conditions, at varying concentrations, the enzyme preparation exhibited considerable 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity accompanied with low antimicrobial activity, pointing out the partial purification necessity of the crude enzyme preparation.

Isolation of Chitinolytic Bacteria From Two Lizard Digestive Tract and Characterization of Their Crude Chitinase

Isolation of Chitinolytic Bacteria From Two Lizard Digestive Tract and Characterization of Their Crude Chitinase

Mode of Parasitism of the Antagonist Trichoderma Viride towards the Phytopathogen Alternaria Solani

In a previous work, the optimized crude chitinase of Trichoderma viride exhibited strong inhibitory activity against food spoilage moulds (phytopathogens) Alternaria solani, Botrytis cinerea, Penicillium oxalicum and P. italicum, with the first species being the most affected. Currently, on testing the antagonistic activity of T. viride against the previous phytopathogens, the antagonist exhibited a strong activity particularly towards A. solani. The antagonist displayed more than one mechanism of parasitism on A. solani including penetration of the pathogen hyphae, abnormal pathogen hyphae swelling, lack of conidia formation, conidia malformation as well as degradation of the pathogen cell wall chitin through secretion of chitinase enzyme which is a major cause of antagonism. Fortunately, the present chitinase secreted by Trichoderma viride is found to be a constitutive enzyme. The previously optimized enzyme showed also a powerful insecticidal activity against home insects cockroach, spider and ant where the exoskeleton was softened and hydrolyzed, followed by rapid killing of the insects within 12 minutes.

Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates.

N-Acetyl-D-glucosamine (GlcNAc) is a monosaccharide with great application potential in the food, cosmetic, pharmaceutical, and biomaterial areas. GlcNAc is currently produced by chemical hydrolysis of chitin, but the current processes are environmentally unfriendly, have low yield and high cost. This study demonstrates the potential to produce GlcNAc from α-chitin using chitinases of ten marine-derived Aeromonas isolates as a sustainable alternative to the current chemical process. The isolates were characterized as Aeromonas caviae by multilocus sequence analysis (MLSA) using six housekeeping genes (gltA, groL, gyrB, metG, ppsA, and recA), not presented the virulence genes verified (alt, act, ast, ahh1, aer, aerA, hlyA, ascV and ascFG), but showed hemolytic activity on blood agar. GlcNAc was produced at 37°C, pH 5.0, 2% (w/v) colloidal chitin and crude chitinase extracts (0.5UmL-1) by all the isolates with yields from 14 to 85% at 6h, 17-89% at 12h and 19-93% after 24h. The highest yield of GlcNAc was observed by A. caviae CH129 (93%). This study demonstrates one of the most efficient chitin enzymatic hydrolysis procedures and A. caviae isolates with great potential for chitinases expression and GlcNAc production.

Intergeneric fusant development using chitinase preparation of Rhizopus stolonifer NCIM 880.

Fungal chitinase have tremendous applications in biotech industries, with this approach we focused on extracellular chitinase from Rhizopus stolonifer NCIM 880 for the formation of fungal protoplasts. The maximum chitinase production reached after 24 h at 2.5% colloidal chitin concentration in presence of starch as an inducer. Chitinase was extracted efficiently at 65% cold acetone concentration and then purified by using DEAE-Cellulose column chromatography. Purified chitinase having molecular weight 22 kDa with single polypeptide chain was optimally active at pH 5.0 and temperature 30 °C. The purified chitinase revealed kinetic properties like Km 1.66 mg/ml and Vmax 769 mM/min. Crude chitinase extract efficiently formed protoplasts from A. niger, A. oryzae, T. viride and F. moniliforme. The formed protoplasts of A. niger and T. viride showed 70 and 66% regeneration frequency respectively. Further, intergeneric fusants were developed successfully and identified at molecular level using RNA profiling. Thus, this study could be useful for strain improvement of various fungi for biotechnological applications.

Optimization, Production and Antifungal Activity of Chitinase Produced by Trichoderma harezianum

Twelve of Trichoderma spp. Isolates were obtained from 30 soils samples collected from differentcrops fields in Baghdad city. Among them, isolate designated as TLT4 which produced highestchitinase activity in primary and secondary screenings was identified as Trichoderma harezianum.The high level of chitinase production was observed in chitinase production media (CPM)amended with 0.4% colloidal chitin with pH6, incubation temperature 30°C, incubation period 6days, peptone as nitrogen source and inoculum size 1× 105spore/ml using submerged cultures.The antifungal activity of crude T. harezianumchitinase was investigated against fourdermatophytes ncluding: Trichophytonment agrophytes, T. rubrum, Microsporumcanis, M.gypsum. The crude chitinase was found to inhibit the growth of all dermatophytes tested withvarying degrees. Clotrimazole drug was used to compare.