Hydrolysis Of Chitin Research Articles

Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin

The enzymatic bioconversion of chitin is of considerable interest for the natural production of bioactive compounds such as chitooligosaccharides and N-acetyl-d-glucosamine (GlcNAc). Key enzymes are involved in the natural processing of chitin, hydrolyzing this abundant biopolymer to yield chitooligosaccharides with substantial value to the medicinal and biotechnological fields. In this study, chitinase C (ScChiC) from the soil bacterium and chitin decomposer Streptomyces coelicolor A3(2) was expressed, purified and characterized. We also optimized a Streptomyces lividans system generating ScChiC expression yields nearly 500-fold higher than the previously reported heterologous expression in Escherichia coli. The purified enzyme was found to be stable below 55°C for a broad range of pH values (pH 3.5–9) and exhibited high activity against chitin and chitooligosaccharides to form chitobiose (C2) as main product. Crab shell chitin hydrolysis profiles also revealed that ScChiC catalyzes the bioconversion of chitopolysaccharides through an endo-nonprocessive mode of action. When combining ScChiC with an N-acetylhexosaminidase from S. coelicolor A3(2) (ScHEX) in an assay using crude extracts and crystalline chitin as substrate, GlcNAc was generated as final product with a yield over 90% after 8h incubation. This chitin hydrolysis yield represents one of the most efficient enzyme bioconversion of chitopolysaccharides to GlcNAc characterized to date, making the S. coelicolor ScChiC–ScHEX pair a potentially suitable contender for the viable industrial production of this important bioactive compound.

Crystal structures of chitin binding domains of chitinase from Thermococcus kodakarensis KOD1

Chitinase from T.kodakarensis (TkChiA) catalyzes the hydrolysis of chitin. The enzyme consists of two catalytic and three binding domains (ChBD1, ChBD2 and ChBD3). ChBD2 and ChBD3 can bind to not only chitin but also cellulose. In both domains, the intervals of the side chains of the three tryptophan residues, which are located on the molecular surface, correspond to twice the length of the lattice of the chitin. A binding model with crystalline chitin implies that the tryptophan residues and a glutamate residue interact with the hexose ring by CH-π interactions and the amide group by a hydrogen bond, respectively.

Effect of pH and Temperature on Bacillus sp. R2 Chitinase Activity and Stability

The purified chitinase of Bacillus sp. R2 showed a molecular weight of 41.68 KDa, an optimal pH and temperature of 7.5 and 40°C respectively. The enzyme was stable between pH 7 to 8 but it lost 57% and 62% of its initial activity at the pH 6 and 9 respectively. The purified chitinase also exhibited complete heat stability for 1h at 40°C and retained above 50% and 30% of its original activity after 30min heating at 50°C and 60°C respectively. The chitinase activation energy for chitin hydrolysis was 12.93 Kcal/mol. To the best of our knowledge, this is the first study about marine chitinase characterization from novel gram variable Bacillus isolated from the Red sea.

Role of Vibrio cholerae exochitinase ChiA2 in horizontal gene transfer.

Vibrio cholerae exochitinase ChiA2 plays a key role in acquisition of nutrients by chitin hydrolysis in the natural environment as well as in pathogenesis in the intestinal milieu. In this study we demonstrate the importance of ChiA2 in horizontal gene transfer in the natural environment. We found that the expression of ChiA2 and TfoX, the central regulator of V. cholerae horizontal gene transfer, varied with changes in environmental conditions. The activity of ChiA2 was also dependent on these conditions. In 3 different environmental conditions tested here, we observed that the supporting environmental condition for maximum expression and activity of ChiA2 was 20 °C, pH 5.5, and 100 mmol/L salinity in the presence of chitin. The same condition also induced TfoX expression and was favorable for horizontal gene transfer in V. cholerae. High-performance liquid chromatography analysis showed that ChiA2 released a significant amount of (GlcNAc)2 from chitin hydrolysis under the favorable condition. We hypothesized that under the favorable environmental condition, ChiA2 was upregulated and maximally active to produce a significant amount of (GlcNAc)2 from chitin. The same environmental condition also induced tfoX expression, followed by its translational activation by the (GlcNAc)2 produced, leading to efficient horizontal gene transfer.

Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.

STUDY OF THE ADSORPTION OF METHYLENE BLUE DYE IN CHITOSAN MICROSPHERES

Chitosan is a polymer that can be used as adsorbent material. In this work, chitosan was synthesized via alkaline hydrolysis of chitin. The chemical structure was characterized by IR spectroscopy and the degree of deacetylation (% DG) of the acetamido groups present in the polymer matrix by neutralization titration. The most suitable adsorption model for analysis of experimental data was Langmuir. The maximum adsorption capacity of the microspheres (Ns) of chitosan was 3.36 x 10-6 mol g-1 adsorbent. The rate of adsorption is governed by the kinetics of the pseudo-second order, where the saturation of adsorption sites occurred after two hours of contact between the adsorbent and the adsorbate. By analyzing data across the isotherm model Dubinin-Radushkevich (DR) it was found that this process adsorption is physical in nature.

MYCOLYTIC ACTIVITY OF PHOSPHATE MOBILIZING SOILBA CTERIA OF GENUS BACILLUS COHN

The aim of this research was study of the chitinolytic activity of phosphateobilizing soil bacteria of genus Bacillus and their antagonistic properties towards microscopic fungi. Methods. In this research 16 strains of phosphate-mobilizing soil bacteria of genus Bacillus and saprophytic and plant pathogenic strains of micrtomycetes were investigated. The antagonistic activity of bacilli against micromycetes was determined by the method of blocks and holes. Bacilli on agar medium and also their supernatants were used. Inhibition of fungal growth under co-cultivation with bacilli under static conditions in the liquid culture medium was determined gravimetrically by the difference of dry weight mycelium in control and in experiment. Bacilli chitinolytic activity was determined photocolorimetrically by the formation of reducing sugars by hydrolysis of colloidal chitin using 3,5-dinitrosalicylic acid. Student test for 5% significance level was used for statistical processing of the obtained data. Results. The presence of chitinolytic activity in all tested bacilli in colloidal chitin medium was established. B. megaterium 16, B. pumilus 7, B. cereus v mycoides 10 strains showed high chitinolytic activity, they were also characterized by high antagonistic activity against plant pathogenic fungi (Fusarium oxysporum 54 and Trichothecium roseum 658). Bacilli were capable to using autoclaved mycelium as the sole of carbon and nitrogen source. Cultivation of bacteria and microscopic fungi under static conditions showed growth inhibition and changes in morphological features – blistering and areas of lysis of mycelium. Conclusions. It was established that phosphate-mobilizing strains of soil bacteria of the genus Bacillus showed chitinolytic activity and growth inhibition of plant pathogens and caused their morphological changes in co-culture. The direct relationship between the antagonistic and chitinolytic activity of bacilli was demonstrated. The bacilli ability to use autoclaved fungal mycelium as a sole source of carbon and nitrogen was showed on the example of B. cereus v. mycoides 10.

EVALUATION OF CHITINOLYTIC GUT MICROBIOTA IN SOME CARPS AND OPTIMIZATION OF CULTURE CONDITIONS FOR CHITINASE PRODUCTION BY THE SELECTED BACTERIA

Present study was aimed at isolation of autochthonous chitinase-producing bacteria from the gastrointestinal tracts of 3 Indian Major Carps (Labeo rohita, Catla catla, Cirrhinus mrigala) and 3 exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio). Altogether, 119 bacteria were isolated from both the proximal and distal intestine and screened for chitinolytic activity. On the basis of chitin hydrolysis zone, 63 isolates were primarily selected for chitinase production, from which 34 potent strains were further studied for quantitative enzyme assay. Amongst them, the strains HMH1 and CMF2 exhibited potent chitinolytic activity and were identified as Bacillus pumilus (KF454036) and Bacillus flexus (KF454035), respectively by 16S rRNA partial gene sequence analysis. Optimization of various fermentation parameters (e.g., temperature, pH, inoculums size, surfactant, colloidal chitin concentration, incubation time, carbon sources, organic and inorganic nitrogen sources) were carried out in chitinase production medium. Incubation for 72 h at 350C and initial pH 7.5 revealed optimum chitinase productions by B. pumilus HMH1 in the media supplemented with colloidal chitin 0.1% (w/v), maltose 2% (w/v), ammonium sulphate 1.0% (w/v) and Tween-80 0.2% (v/v). However, B. flexus CMF2 required 48 h incubation at 35°C and initial pH 8.0 with colloidal chitin 0.15% (w/v), sucrose 1% (w/v), yeast extract 2.0% (w/v) and Tween-20 0.2% (v/v) supplementation for optimum yield. The results indicate that there is ample scope for further research to appraise fish gut microorganisms for chitinase production or as probiotics to improve feed efficiency in fish.

Thermodynamic Relationships with Processivity in Serratia marcescens Family 18 Chitinases.

The enzymatic degradation of recalcitrant polysaccharides is accomplished by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive which means that they remain attached to the substrate in between subsequent hydrolytic reactions. Chitinases are GHs that catalyze the hydrolysis of chitin (β-1,4-linked N-acetylglucosamine). Previously, a relationship between active site topology and processivity has been suggested while recent computational efforts have suggested a link between the degree of processivity and ligand binding free energy. We have investigated these relationships by employing computational (molecular dynamics (MD)) and experimental (isothermal titration calorimetry (ITC)) approaches to gain insight into the thermodynamics of substrate binding to Serratia marcescens chitinases ChiA, ChiB, and ChiC. We show that increased processive ability indeed corresponds to more favorable binding free energy and that this likely is a general feature of GHs. Moreover, ligand binding in ChiB is entropically driven; in ChiC it is enthalpically driven, and the enthalpic and entropic contributions to ligand binding in ChiA are equal. Furthermore, water is shown to be especially important in ChiA-binding. This work provides new insight into oligosaccharide binding, getting us one step closer to understand how GHs efficiently degrade recalcitrant polysaccharides.

A simple procedure for preparing chitin oligomers through acetone precipitation after hydrolysis in concentrated hydrochloric acid

Chitin oligomers are of interest because of their numerous biologically relevant properties. To prepare chitin oligomers containing 4–6 GlcNAc units [(GlcNAc)4–6], α- and β-chitin were hydrolyzed with concentrated hydrochloric acid at 40°C. The reactant was mixed with acetone to recover the acetone-insoluble material, and (GlcNAc)4–6 was efficiently recovered after subsequent water extraction. Composition analysis using gel permeation chromatography and MALDI-TOF mass spectrometry indicated that (GlcNAc)4–6 could be isolated from the acetone-insoluble material with recoveries of approximately 17% and 21% from the starting α-chitin and β-chitin, respectively. The acetone precipitation method is highly useful for recovering chitin oligomers from the acid hydrolysate of chitin. The changes in the molecular size and higher-order structure of chitin during the course of hydrolysis were also analyzed, and a model that explains the process of oligomer accumulation is proposed.

Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution

The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

A Diverse Range of Bacterial and Eukaryotic Chitinases Hydrolyzes the LacNAc (Galβ1–4GlcNAc) and LacdiNAc (GalNAcβ1–4GlcNAc) Motifs Found on Vertebrate and Insect Cells

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

STUDY OF COLLOIDAL CHITIN HYDROLYSIS TO PRODUCE THE N ACETYL GLUCOSAMINE FROM SHRIMP SHELL WASTE USING HYDROCHLORIC ACID AND NITRIC ACID

Shrimp processing industries produces shrimp waste by 30-75% is wasted without being processed causing environmental pollution. The purpose of this study examines efforts to produce monomers of chitin hydrolysis of colloidal chitin compound derived from shrimp shells are chemically (hydrochloric acid and nitric acid) to produce N acetyl glucosamine bioactive compounds that have health benefits. The method used in this study is the optimization of chemical hydrolysis conditions using a solution of HCl and HNO3. On determining the optimum time to sample variation colloidal chitin 0,5%, 1% and 2%, the sample is heated with time variation 1,3,6, 9,12, and 24 hours at a temperature of 60 oC and the concentration of acid was 4 M. In the determination of the optimum temperature, each sample was heated at a temperature variation of 40, 60, and 80 oC at the optimum time (9 hours ) and acid concentration of 4 M. In the determination of the optimum concentration of acid, each sample was heated at a temperature and the optimum reaction time. Production results showed the highest compound N acetyl glucosamine as 623.3 ppm, using hydrochloric acid hydrolysis reaches optimum temperature 40° C, the optimum reaction time 9 hours, the optimum concentration of 4 M hydrochloric acid, the concentration of 2% colloidal chitin. Production of N acetyl glucosamine supreme as 625.83 ppm, using nitric acid hydrolysis reaches optimum temperature 60° C, the optimum reaction time 9 hours, the optimum concentration of 4M nitric acid, and the concentration of 1% colloidal chitin. Therefore we can conclude the results of 1% colloidal chitin hydrolysis using nitric acid more efficiently produce N acetyl glucosamine than hydrochloric acid, due to the temperature, reaction time, and acid concentration are not the same ability to hydrolyze colloidal chitin. Differences in the results of hydrolysis reached 11,59 ppm. Keywords : Chitin, N acetyl glucosamine, acid hydrolysis, optimum reaction

1H NMR and 1H–13C HSQC surface characterization of chitosan–chitin sheath-core nanowhiskers

Surface deacetylation of chitin nanowhiskers (CtNWs) to chitosan–sheath/chitin-core nanowhiskers (CsNWs) was successfully monitored by liquid-state high-resolution NMR of colloidal suspensions of these never-dried nanowhiskers. CtNWs were derived from acid hydrolysis (3N HCl, 30mL/g, 90min, 104°C) of chitin at 65% yield and 86% CrI. Deacetylation (50% NaOH, 48h, 50°C) of CtNWs generated CsNWs with unchanged nanowhisker morphology and overall length and width dimensions, but a reduced CrI of 54%. Successful step-wise exchanging the aqueous media with acetone, then D2O prevented agglomeration of nanowhiskers and enabled NMR detection of individual nanowhiskers. The crystalline structure of CtNWs and CsNWs provided different chemical environments for the glucosamine hydrogen atom H2, splitting the NMR signals into 2 peaks (δ 3.0 and δ 3.35ppm) which differed from that reported for soluble chitosan (δ 3.2ppm). Besides, 1H–13C HSQC was only possible for CsNWs indicating the NMR phenomenon observed to represent that of the surfaces where the outer layers were highly mobile and less crystalline. The degree of acetylation at the surfaces was determined from 1H NMR data to be 56% and 9% for CtNWs and CsNWs, respectively.

Production and optimization of extracellular fungal chitinase produced byMetarhizium anisopliae(M.) Sorokin through Submerged and Solid State Fermentation

Chitinase, one of the major metabolite produced by many organisms for the degradation of chitin. Chitinases also play a role in the utilization of waste from crustacean and other insects resulting in the hydrolysis of chitin to its monomer N-acetyl-D-glucosamine. The production of Chitinase can be enhanced by changing the constituents in the medium. In present study chitin production from Metarhizium anisopliae isolated from the dead insect showed production with different concentration of chitin in submerged media and solid state fermentation using fish waste.

Chitin oligomers and monomers production by coupling γ radiation and enzymatic hydrolysis

The main purpose of this study was the chitin enzymatic hydrolysis enhancement by a pretreatment, consisting of chitin exposure to gamma radiation at several doses, ranging from 15kGy to 210kGy. The solid chitin molecular weight reduction achieved by Co-60 gamma rays treatment reached a 90% rate at the t used, with a dose of 210kGy. The partial depolymerization of chitin was realized by irradiation allowing an enzyme affinity increase toward this substrate. The monomers and oligomers obtained from their radiated chitin production rate were almost 100%. It was also demonstrated that the NAG production by enzymatic hydrolysis of irradiated chitin at 210Gy follows Michaelis–Menton kinetics.

Preparation, characterization, and antimicrobial activity of chitin nanofibrils reinforced carrageenan nanocomposite films.

Present study illustrates the preparation of chitin nanofibrils (CNF) reinforced carrageenan nanocomposite films by the solution-casting technique. CNF was prepared by acid hydrolysis of chitin, followed by high speed homogenization and sonication. FTIR result demonstrated that the chemical structure of chitin had not changed after acid hydrolysis. However, the crystalinity of CNF was found to be higher than chitin. The crystallite size of chitin and CNF was 4.73 and 6.27 nm, respectively. The char content at 600 °C of chitin (19.2%) was lower than the CNF (25%). The carrageenan/CNF composite films were smooth and flexible and the CNF was dispersed uniformly in the carrageenan polymer matrix. The tensile strength and modulus of carrageenan film were increased significantly (p<0.05) after CNF reinforcement with up to 5 wt%, however, elongation at break, water vapor permeability, and transparency decreased slightly. Carrageenan/CNF nanocomposite films showed strong antibacterial activity against a Gram-positive food-borne pathogen, Listeria monocytogenes.

Microbial responses to chitin and chitosan in oxic and anoxic agricultural soil slurries

Abstract. Microbial degradation of chitin in soil substantially contributes to carbon cycling in terrestrial ecosystems. Chitin is globally the second most abundant biopolymer after cellulose and can be deacetylated to chitosan or can be hydrolyzed to N,N′-diacetylchitobiose and oligomers of N-acetylglucosamine by aerobic and anaerobic microorganisms. Which pathway of chitin hydrolysis is preferred by soil microbial communities is unknown. Supplementation of chitin stimulated microbial activity under oxic and anoxic conditions in agricultural soil slurries, whereas chitosan had no effect. Thus, the soil microbial community likely was more adapted to chitin as a substrate. In addition, this finding suggested that direct hydrolysis of chitin was preferred to the pathway that starts with deacetylation. Chitin was apparently degraded by aerobic respiration, ammonification, and nitrification to carbon dioxide and nitrate under oxic conditions. When oxygen was absent, fermentation products (acetate, butyrate, propionate, hydrogen, and carbon dioxide) and ammonia were detected, suggesting that butyric and propionic acid fermentation, along with ammonification, were likely responsible for anaerobic chitin degradation. In total, 42 different chiA genotypes were detected of which twenty were novel at an amino acid sequence dissimilarity of less than 50%. Various chiA genotypes responded to chitin supplementation and affiliated with a novel deep-branching bacterial chiA genotype (anoxic conditions), genotypes of Beta- and Gammaproteobacteria (oxic and anoxic conditions), and Planctomycetes (oxic conditions). Thus, this study provides evidence that detected chitinolytic bacteria were catabolically diverse and occupied different ecological niches with regard to oxygen availability enabling chitin degradation under various redox conditions on community level.

Chitinase expression in Listeria monocytogenes is positively regulated by the Agr system.

The food-borne pathogen Listeria monocytogenes encodes two chitinases, ChiA and ChiB, which allow the bacterium to hydrolyze chitin, the second most abundant polysaccharide in nature. Intriguingly, despite the absence of chitin in human and mammalian hosts, both of the chitinases have been deemed important for infection, through a mechanism that, at least in the case of ChiA, involves modulation of host immune responses. In this study, we show that the expression of the two chitinases is subject to regulation by the listerial agr system, a homologue of the agr quorum-sensing system of Staphylococcus aureus, that has so far been implicated in virulence and biofilm formation. We demonstrate that in addition to these roles, the listerial agr system is required for efficient chitin hydrolysis, as deletion of agrD, encoding the putative precursor of the agr autoinducer, dramatically decreased chitinolytic activity on agar plates. Agr was specifically induced in response to chitin addition in stationary phase and agrD was found to regulate the amount of chiA, but not chiB, transcripts. Although the transcript levels of chiB did not depend on agrD, the extracellular protein levels of both chitinases were reduced in the ΔagrD mutant. The regulatory effect of agr on chiA is potentially mediated through the small RNA LhrA, which we show here to be negatively regulated by agr. LhrA is in turn known to repress chiA translation by binding to the chiA transcript and interfering with ribosome recruitment. Our results highlight a previously unrecognized role of the agr system and suggest that autoinducer-based regulation of chitinolytic systems may be more commonplace than previously thought.

Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues

The optimization studies for production of chitinase were carried out by response surface methodology (RSM) based on statistics experimental design using three substrates, which were wheat, rice and red gram bran. 24 full factorial central composite design was applied to evaluate optimal combinations of variables. These variables were chitin concentration, initial moisture content, inoculum level, and incubation time. The results of second order polynomial showed that all four variables had significant effect on chitinase production. Maximum chitinase activity was recorded for wheat bran (2443.23 U g−1) than rice (1216.65 U g−1) and red gram bran (961.32 U g−1). An overall 3-fold increase in chitinase activity was achieved using optimized strategies of RSM. Growth of the fungus on all bran particles have been visualized by scanning electron microscopy. These results indicated the potential of Penicillium ochrochloron for economical production of chitinase using agricultural residues. TLC and HPLC analysis of colloidal chitin hydrolysate with partially purified chitinases revealed that the major reaction product was monomeric GlcNAc indicating the potential of these enzymes for efficient production of GlcNAc.