Chitosanase Production Research Articles

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by Trichoderma spp.

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40-50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4IU/gds, followed by T. viride (~1.2IU/gds) and T. harzianum (1.06IU/gds).

SELECTION OF STRAIS PRODUCING FUNGAL CHITOSANASE AND KINETICS

In this work it was performed a selection of fungal strains able to grow in a specific medium containing chitosan (Chitosan Detector Agar) as the sole carbon source at 0.2%, and a kinetic study was performed to evaluate the best conditions for production of chitosanase from the best producing strain. The selection was made from five strains, previously isolated from soil. In the evaluation of kinetics was accompanied by cell concentration, the protein concentration (mg/mL), the enzyme activity (U/mL) for two volumes of fermentation broth (0.05 and 0.10 mL). The results showed a significant increase in all parameters studied in the first 12 hours of culture. Comparing the activity values for the two volumes of fermentation broth was observed that the volume of 0.05 mL broth showed enzymatic activity always greater than the volume of 0.1 mL

Uncoupling chitosanase production from chitosan

There is a growing interest in chitosanases as enzymatic tools to hydrolyze chitosan into bioactive forms: low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS). However chitosanases are still expensive and methods of large-scale production of these enzymes are not yet established. The article reviews the approaches used for chitosanase production in various bacterial hosts, pointing out the difficulties resulting from the necessity to include chitosan into the medium composition. A mutated Streptomyces host allows for the efficient production of several chitosanases originating from actinobacteria in the absence of chitosan as inducer.

Statistical optimization of chitosanase production by Aspergillus sp. QD-2 in submerged fermentation

Cultivation of chitosanase production by Aspergillus sp. QD-2 under submerged fermentation was optimized. Factors including (NH4)2SO4, inoculum size and initial pH were identified by two-level Plackett-Burman design (PBD) as significant for chitosanase production. The path of steepest ascent was undertaken to determine the optimal region of three significant factors. To determine the optimal values of the significant variables, Box-Behnken design (BBD) and response surface analysis were employed. The quadratic regression model of producing chitosanase showed that the optimal cultivation conditions ((NH4)2SO4 5.164 g/l, inoculum size 8.204% (v/v) and initial pH 4.074) resulted in the improvement of chitosanase activity (85.816 U/ml) as compared to the initial level (26.515 U/ml) after 72 h of fermentation. The optimal pH and temperature of the crude chitosanase were determined to be 5.6 and 55°C, respectively.

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

BackgroundChitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans.ResultsTwo S. lividans strains were used for induction experiments: the wild type strain and its mutant (ΔcsnR), harbouring an in-frame deletion of the csnR gene, encoding a negative transcriptional regulator. Comparison of chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous chitosanase gene csnN106. Using the ΔcsnR host and a mutated csnN106 gene with a modified transcription operator, substantial levels of chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed.ConclusionsThis new chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.

Biodegradation of shellfish wastes and production of chitosanases by a squid pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024

Two chitosanases (CHSA1 and CHSA2) were purified from the culture supernatant of Acinetobacter calcoaceticus TKU024 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHSA1 and CHSA2 determined by SDS-PAGE were approximately 27 and 66kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSA1 and CHSA2 were (pH 6, 50°C, pH 4-10, <90°C) and (pH 7, 60°C, pH 6-11, <70°C), respectively. CHSA1 and CHSA2 had broad pH and thermal stability. CHSA1 and CHSA2 were both inhibited by EDTA and were inhibited completely by 5mM Mn(2+). CHSA1 and CHSA2 degraded chitosan with DD ranging from 60 to 98%, and also degraded some chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU024 culture supernatant (1.5% SPP) incubated for 5days has the most reducing sugars (0.63mg/ml). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.

Optimization of Chitosanase Production by Trichoderma koningii sp. Under Solid-State Fermentation

This study aimed at the optimization of the production of chitosanase in solid culture. Trichoderma koningii sp., an entomopathogenic fungus, was used to produce chitosanase under solid-state fermentation using a mixture of wheat bran and chitosan. The incubation period; addition of moistening water and culture medium composition were optimized. The protocol to extract the enzyme was also optimized. The optimal conditions for chitosanase production by T. koningii were obtained using a mixture of 3.0 g of wheat bran and 1.5 g of chitosan, with the addition of 2.5 mL of moistening water (pH 5.5) and of 2.5 mL of saline solution (pH 5.5) containing NaNO3 (1.0 g/L), (NH4)2HPO4 (1.0 g/L), MgSO4.7H2O (1.0 g/L), and NaCl (1.0 g/L). Optimal enzyme extraction was carried out adding 20 mL of sodium acetate buffer (200 mM, pH 5.5) at 30 °C under orbital agitation at 150 rpm for 6 min. The optimized production yielded 4.84 IU/gds.

Chitosanase production by Paenibacillus ehimensis and its application for chitosan hydrolysis

The chitosanase production by Paenibacillus ehimensis was studied in submerged cultures and the chitosan hydrolysis was evaluated by using these enzymes without purification. The bacterium produced inducibles enzymes after 12 h of growth in a culture medium containing 0.2% (w/v) of soluble chitosan as carbon source. The enzyme production was strongly repressed by the presence of glucose. The production started as soon as the available sugars finished in the culture medium. The maximum level of chitosanase activity was 500 U.L-1 at 36°C after 36 h incubation. The crude enzyme was optimally active at pH 6.0 and 55°C and in these conditions, the enzyme presented good stability (6 days). The enzyme without purification was used to hydrolyze the chitosan which resulted chitooligosaccharides between 20 and 30 min of reaction.

Purification and Characterization of a Chitosanase and a Protease by Conversion of Shrimp Shell Wastes Fermented by Serratia Marcescens Subsp. Sakuensis TKU019

AbstractA chitosanase and a protease were purified from the culture supernatant of Serratia marcescens subsp. sakuensis TKU019 with shrimp shell as the sole carbon/nitrogen source. The culture condition suitable for production of chitosanase was found to be shaken at 37 °C for 3 days in 100 mL of medium containing 0.5 % shrimp shell powder, 0.1 % K2HPO4 and 0.05 % MgSO4•7 H2O at pH 7. The TKU019 chitosanase was suppressed by the simultaneously existing protease, which also showed the maximum activity at the third day of incubation. The molecular masses of the chitosanase and protease determined by SDS‐PAGE were approximately 36 kDa and 58 kDa, respectively. Cu2+, Mn2+ and Zn2+ inhibited the chitosanase activity, and all of tested divalent metals inhibited in the protease activity. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase was pH 7, 60 °C, pH 4–7, and &lt; 60 °C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the protease was pH 10, 50 °C, pH 5–10, and &lt;50 °C, respectively. Tween 40 (2 %, v/v) had stimulatory effect on TKU019 protease activity.

Oxidative/heat stress enhanced production of chitosanase from Streptomyces griseus cells through its interaction with liposome

The effective production and secretion of chitosanase from S. griseus cells, in the presence of hydrogen peroxide, were studied by the treatment of the cells with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes under heat condition. The variation of the conformation and activity of the chitosanase caused by the interaction of liposomes with target chitosanase under stress condition was systematically investigated by using circular dichroism (CD) spectra and dielectric dispersion analysis (DDA). The effect of the oxidative stress (hydrogen peroxide) on the lipid and protein peroxide of cell membrane of S. griseus pretreated with and without liposomes under heat stress at 41 degrees C was further carried out. The possible utilization of membrane-membrane interaction between liposomes and cell membrane induced by the heat treatment was further investigated to enhance the production of chitosanase from S. griseus under oxidative stress condition.

Bioprospecting for genes involved in the production of chitosanases and microcystin-like compounds in Anabaena strains

Bioinformatic tools guided PCR amplification assays were employed for analyzing two Anabaena strains A. laxa and A. iyengarii which exhibited chitosanase activity, allelopathic and fungicidal activity. Sequencing of a 297 bp fragment obtained by amplification with primers directed towards mcy A gene (involved in the production of microcystins), revealed significant similarity with the condensation domain, while amplification with specific primers towards N-methyltransferase (NMT) domain showed 59% similarity with a homologous domain in a toxic strain of Microcystis aeruginosa. An amplified product of 172 bp obtained using specific primers derived from the coding region of chitinase (chi IS) gene in Streptomyces sp., showed 100% similarity with hydrogenbyrinic acid a, c-diamide cobaltochelatase gene in Anabaena, and significant similarity with chi IS gene of Streptomyces sp. under less stringent conditions. The 663 bp sequence obtained by employing specific primers for chitosanase (choA) derived from Mitsuaria chitosanitabida 3001 strain, showed 100% similarity with glycoside hydrolase family three domain like protein(s). This study is a first time report on the presence of homologues of chitosanase in cyanobacteria which can play a role in allelopathic activity exhibited by these oxygenic photosynthetic prokaryotes.

Conversion and degradation of shellfish wastes by Bacillus cereus TKU018 fermentation for the production of chitosanases and bioactive materials

Two chitosanases (CHSB1 and CHSB2) were purified from the culture supernatant of Bacillus cereus TKU018 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of CHSB1 and CHSB2 determined by SDS–PAGE were approximately 44 kDa and 22 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHSB1 and CHSB2 were (pH 5, 60 °C; pH 5–7, <40 °C) and (pH 7, 50 °C; pH 4–7, <50 °C), respectively. CHSB1 and CHSB2 were both inhibited by EDTA and CHSB1 was inhibited completely by 5 mM Zn 2+. CHSB1 and CHSB2 degraded chitosan with DD ranging from 60% to 95%, but did not degrade chitin. The most susceptible substrate was 60% deacetylated chitosan. Furthermore, TKU018 culture supernatant (1.5% SPP) incubated for 3–4 days has 75% relative antioxidant activity (DPPH scavenging ability). With this method, we have shown that shellfish wastes may have a great potential for the production of bioactive materials.

Optimization of chitosanase production from Bacillus sp. RKY3 using statistical experimental designs

AbstractBACKGROUND: The culture medium and fermentation conditions for the production of constitutive chitosanase from a newly isolated Bacillus sp. RKY3 were optimized statistically.RESULTS: The variables significantly influencing both chitosanase production and cell growth were screened through the Plackett–Burman design, by which maltose, beef extract, MgSO4, and incubation time were identified as the most significant variables. The optimum values of the selected variables and their mutual interactions were determined through the steepest ascent method and Box–Behnken experimental design. The results demonstrated that 62.30 U mL−1 chitosanase activity was predicted with optimum conditions of maltose (30.18 g L−1), beef extract (15.25 g L−1), MgSO4 (0.26 g L−1), and incubation time (50.02 h). The predicted response was verified by the validation experiments, and the optimum conditions resulted in a maximum chitosanase activity of 63.53 ± 1.22 U mL−1.CONCLUSION: The optimization of fermentation variables resulted in an approximately 11.3‐fold increase in chitosanase activity compared with that observed under unoptimized conditions (from 5.63 U mL−1 to 63.53 U mL−1). Copyright © 2009 Society of Chemical Industry

Degradation of chitin and production of bioactive materials by bioconversion of squid pens

Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K 2HPO 4, and 0.05% MgSO 4. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications.

Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment

We have developed a culture system for efficient production of chitosanase by Bacillus sp. TKU004. TKU004 was cultivated by using squid pen powder as the sole carbon/nitrogen source. The effects of autoclave treatments of the medium on the production of chitosanase were investigated. Autoclave treatment of squid pen powder for 45 min remarkably promoted enzyme productivity. When the culture medium containing an initial squid pen powder concentration of 3% was autoclaved for 45 min, the chitosanase activity was optimal and reached 0.14–0.16 U/mL. In addition, extracellular surfactant-stable chitosanase was purified from the TKU004 culture supernatant. The antioxidant activity of TKU004 culture supernatant was determined through the scavenging ability of DPPH, with 70% per mL. With this method, we have shown that marine wastes can be utilized efficiently through prolonged autoclave treatments to generate a high value-added product, and have revealed its hidden potential in the production of functional foods.

Overexpression of the Chitosanase Gene in Fusarium solani via Agrobacterium tumefaciens-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (approximately 2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.

Enhanced Release of Chitosanase from Streptomyces griseus through Direct Interaction of Liposome with Cell Membrane under Heat Stress

A direct interaction of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes with membrane of Streptomyces griseus cell under the heat stress at 41 degrees C increased the chitosanase production and release to 2.2 times higher than that at 37 degrees C without the POPC liposomes. Amount of chitosanase released across the lipid mimicking cell membrane (LMCM) liposome under a heat at 41 degrees C in the presence of POPC liposomes was 17% of initially-entrapped chitosanase while it was only 1% in the absence of POPC liposomes, even under a heat stress at 41 degrees C, clearly showing the importance of the direct interaction between membrane and membrane.

Strain improvement and optimization of the media composition of chitosanase-producing fungus Aspergillus sp. CJ 22-326

A mutant Aspergillus sp. CJ 22-326-14 with higher production of chitosanase was selected by serial mutation procedure, and the optimization of medium components was performed using one-factor-at-a-time combined with orthogonal array design method. The results showed that chitosan, wheat bran and (NH4)2SO4 were the most important components by one-factor-at-a-time experiments. And the optimal concentrations were obtained by orthogonal array design method as (g/L): chitosan 20, wheat bran 10, (NH4) 2SO4 2, KH2PO4 2 and MgSO4·7H2O 0.5. After the strain improvement and optimization of the media, the chitosanase activity from Aspergillus sp. CJ 22-326-14 reached to 3.92 U/mL in 250 mL flasks, which was nearly 4-fold increase of chitosanase production at the parent lowest level.

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.

Enhancement of Gongronella sp. JG Chitosanase Production by Ion Beam Implantation

Gongronella sp. JG was a fungal strain which expressed extracellularchitosanase of about 800 U/L during its growth in production medium.To improve its enzyme production, low energy N+ implantation wasemployed to mutate spores of JG. The implantation condition wasoptimized and the parameters of 15 keV and 60 × 2.6× 1013 ions/cm2 were selected for further breedingexperiments. A mutant designated as SG was obtained. It showedincreased chitosanase production (1800 U/L) and shortenedcultivation period (from 72 h to 60 h). Five-generation cultivationof SG indicated that its chitosanase production was stable at about1800 U/L.