Chitosanase Activity 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).

Preparation of water-soluble chitosan by hydrolysis with commercial glucoamylase containing chitosanase activity

Water-soluble chitosan (WSC) was prepared by hydrolyzing chitosan with commercial glucoamylase. Factors affecting the enzymatic hydrolysis of chitosan were investigated, and the optimum hydrolysis conditions were as follows: time, 24 h; pH, 4.5; temperature, 55 °C; amount of glucoamylase, 40 mg/500 mL of reaction mixture (26.8 U of chitosanase activity). Under these conditions, the weight-averaged molecular weight (Mw) of chitosan decreased to 1,700 Da. The hydrolysate was neutralized with 1 M NaOH, filtrated, concentrated to 16% (w/v), and precipitated with 5 volumes of ethanol. The product of WSC was obtained by drying the precipitate at 60 °C for 3 h. The content of WSC in the product and the yield of WSC were 95.3% (w/w) and 94.2% (w/w), respectively. The structure of WSC was characterized by Fourier transform infrared spectroscopy (FTIR).

Bioproduction of hydrolytic enzymes using apple pomace waste by A. niger: applications in biocontrol formulations and hydrolysis of chitin/chitosan

Studies were carried out for β-glucosidase production using apple pomace (AP) in solid-state fermentation using 2(4) factorial design and response surface methodology. The influence of four independent variables including initial moisture level and inducers [veratryl alcohol (VA), lactose (LAC) and copper sulfate (CS)] was studied. The experimental design showed that initial moisture level had significant negative effect on the response. Higher β-glucosidase activity of 64.18 IU/gram fermented substrate (gfs) was achieved in solid-state tray fermentation with optimum conditions having initial moisture level 55% (v/w), pH 4.5, 2 mM/kg VA, 2% (w/w) LAC and 1.5 mM/kg CS concentration, respectively,. The non-specific chitinase 70.28 ± 6.34 IU/gfs and chitosanase activities 60.18 ± 6.82 to 64.20 ± 7.12 IU/gfs were observed. The study demonstrated that AP can be potentially used for the β-glucosidase production by Aspergillus niger. Moreover, β-glucosidase can be used for the hydrolysis of chitin/chitosan to depolymerized products and in the formulation of biocontrol agents for enhanced entomotoxicity levels.

Preparation of water soluble chitosan by hydrolysis with commercial α-amylase containing chitosanase activity

Water soluble chitosan (WSC) was prepared by hydrolysing chitosan using α-amylase. The degradation was monitored by WSC recovery and the WSC structure was characterised by Fourier transform infrared spectroscopy. The optimal hydrolysis conditions were found to be at pH 5, 50 °C, 20 mg α-amylase in the reaction solution, with a reaction time of 4 h. Under these conditions, the average molecular weight of chitosan decreased to 730 Da. The resulting products were composed of chitooligosaccharides of DP 3–7. The hydrolysate was neutralised with 1 M NaOH, filtered, concentrated to 16% (w/v), precipitated with five volumes of ethanol to one volume of the concentrated hydrolysate solution, and dried at 60 °C for 3 h to yield a white powder. The WSC content of the product and the yield were 96.2% and 91.2% (w/w), respectively.

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.

Quantitative determination of chitinolytic activity of lysozyme using half-deacetylated chitosan as a substrate

In this paper, we report a novel quantitative assay for chitinolytic activity of lysozyme. Our assay is based on measuring the reducing sugar ends (RSE) released from the water-soluble macromolecular substrate, half-deacetylated chitosan. RSE reacts with 3-methyl-2-benzothiazolinonehydrazone to produce the chromogenic product that has absorption maxima at 615 and 655nm. We found that measurement of low levels of RSE at 655nm yields higher precision and better linearity than that at 615nm. The optimal procedures are: the pre-warmed half-deacetylated chitosan and lysozyme were mixed to make the final solution containing 4mgmL−1 half-deacetylated chitosan at pH 4.0 in sodium acetate buffer, and incubated at 40°C for 1h within steady state time course. The linear range of lysozyme activity was within 94–1006nMmin−1. We demonstrated that our method is simple, rapid, inexpensive and accurate, which other low-sensitive methods cannot be achieved. This method can also be used to quantitatively determine the activity and kinetic parameters of chitinase and chitosanase.

Purification and enzymatic properties of the extracellular and constitutive chitosanase produced by Bacillus subtilis RKY3

BACKGROUND: Purification and enzymatic properties of a chitosanase from Bacillus subtilis RKY3 have been investigated to produce a chitooligosaccharide. The enzyme reported was extracellular and constitutive, which was purified by two sequential steps including ammonium sulfate precipitation and ion exchange chromatography. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified chitosanase revealed one single band corresponding to a molecular weight of around 24 kDa. The highest chitosanase activity was found to be at pH 6.0 and at 60 °C. Although the mercaptide forming agents such as Hg2+ (10 mmol L−1) and p-hydroxymercuribenzoic acid (1 mmol L−1, 10 mmol L−1) significantly or totally inhibited the enzyme activity, its activity was enhanced by the presence of 10 mmol L−1 Mn2+. The enzyme showed activity for hydrolysis of soluble chitosan and glycol chitosan, but colloidal chitin, carboxymethyl cellulose, crystalline cellulose, and soluble starch were not hydrolyzed. The analysis of chitosan hydrolysis by thin-layer chromatography and viscosity variation revealed that the purified enzyme should be endosplitting-type chitosanase. CONCLUSION: The chitosanase produced by Bacillus subtilis RKY3 was a novel chitosanlytic enzyme with relatively low molecular weight, which is a versatile enzyme for chitosan hydrolysis because it could hydrolyze soluble chitosan into a biofunctional oligosaccharide at a high level. Copyright © 2011 Society of Chemical Industry

Depolymerization of chitosan by enzymes from the digestive tract of sea cucumber Stichopus japonicus

A complex of enzymes was isolated in a preparation derived from the digestive tract of sea cucumber, Stichopus japonicus . Hydrolysis of chitosan using this enzyme preparation decreased its molecular weight (Mw), increased its water solubility and produced water-soluble chitosan (WSC). The conditions for hydrolysis were optimized to pH 6.0, temperature 45°C, 16 mg enzyme preparation (22.08 U of chitosanase activity) in a reaction solution (500 ml) containing 5 g chitosan and total reaction time of 3 h. The Mw of hydrolyzed chitosan was 1260 Da, and the WSC content in the resulting product and the yield were 96.7 and 95.4% (w/w), respectively. The structure of WSC was characterized using Fourier transform infrared (FTIR) spectroscopy. Key words : Water-soluble chitosan, complex enzyme preparation, sea cucumber Stichopus japonicus , hydrolysis.

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 < 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 <50 °C, respectively. Tween 40 (2 %, v/v) had stimulatory effect on TKU019 protease activity.

Knock down of chitosanase expression in phytopathogenic fungus Fusarium solani and its effect on pathogenicity

Chitosanases are lytic enzymes involved in the degradation of chitosan, a component of fungal cell walls. The phytopathogenic fungus Fusarium solani produces an extracellular chitosanase, CSN1, the role of which in the physiology and virulence of the fungus remains to be expounded. Here, we studied the expression of the CSN1 gene through gene silencing and examined its effect on fungal pathogenicity. A vector construct encoding a hairpin RNA (hpRNA) of CSN1 was constructed and introduced into the F. solani 0114 strain. The results revealed that majority of the transformants exhibited a significant reduction in chitosanase activity compared with the wild-type strain. Further, transformants with silenced CSN1 exhibited no change in mycelial growth and spore formation. However, pea pod and seedling bioassays indicated that transformants with silenced CSN1 were more virulent compared with the wild-type strain, and in sharp contrast to strains in which overexpression of the CSN1 gene resulted in virulence reduction. Although the mechanism remains unclear, our findings did suggest that F. solani chitosanase has a negative effect on fungal pathogenicity.

Identification, Characterization, and Regulation of a Novel Antifungal Chitosanase Gene ( cho ) in Anabaena spp

Two contrasting cyanobacterial species (Anabaena fertilissima and Anabaena sphaerica) were selected based on differences in antifungal behavior in order to study the mechanism for production of an antifungal enzyme and the genes responsible for this production. In A. fertilissima, chitosanase and antifungal activities were increased significantly under of growth-limiting conditions (8 of light and 16 h of darkness). The lack of such activities in A. sphaerica was associated with high levels of protein that accumulated during the stationary phase (at 28 days) under the same light conditions. The gene putatively responsible for chitosanase and antifungal activities was amplified using specific primers, and sequence analysis of the amplified products (1.086 and 1.101 kb in A. sphaerica and A. fertilissima, respectively) showed that they belong to the glycoside hydrolase 3 (GH3)-like family of Anabaena variabilis ATCC 29413. Pairwise alignment of the corresponding protein sequences identified a putative signal peptide (amino acids 1 to 23) and some amino acid changes in the sequence of A. fertilissima which may be responsible for functioning of the chitosanase and the observed antifungal activity. Hydrolysis of the chitosan oligosaccharide (GlcN)(5) to (GlcN)(2) and (GlcN)(3) confirmed the presence of chitosanase activity in A. fertilissima. Site-directed mutagenesis of the A. fertilissima chitosanase-encoding gene (cho) led to identification of catalytic residues (Glu-121 and Glu-141) important for the antifungal effect of the cho product. The level of expression of cho was monitored by quantitative real-time reverse transcription-PCR, which indicated that transcription of this gene is significantly enhanced under conditions that retard growth, such as a long dark period.

Chitosanase displayed on liposome can increase its activity and stability

The strategy to prepare a novel biocatalyst by the immobilization of chitosanase onto liposome (ICL) was carried out based on the direct interaction of liposomes with cell membrane of Streptomyces griseus cell. The ICL was characterized in relation to the molecular weight of protein, the chitosanase activity, the effect of the surface hydration of various liposomes on hydrolysis activity of immobilized chitosanase and the stability of ICL under various extreme conditions. The SDS-PAGE analysis of the purified ICL sample shows the existence of a protein with approximately 39 kDa that corresponded to the sum of weight of the mature chitosanase and its signal peptide (38.8 kDa). The above protein of ICL also expresses the chitosanase activity that is significantly higher than that of the conventional chitosanase. Furthermore, the surface hydration of liposomes used to prepare ICL that affected the activity of immobilized chitosanase verified the importance of liposome surfaces. Indeed, the stability of ICL assayed by measuring the chitosanase activity is significantly higher than that of conventional chitosanase under various temperatures and pH conditions. These characteristics of ICL show the possible preparation of the biocatalysts that can be prepared by immobilizing enzymes onto liposome vesicles properly.

Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity

BackgroundPlant latex is the cytoplasm of highly specialized cells known as laticifers, and is thought to have a critical role in defense against herbivorous insects. Proteins abundantly accumulated in latex might therefore be involved in the defense system.ResultsWe purified latex abundant protein a and b (LA-a and LA-b) from mulberry (Morus sp.) and analyzed their properties. LA-a and LA-b have molecular masses of approximately 50 and 46 kDa, respectively, and are abundant in the soluble fraction of latex. Western blotting analysis suggested that they share sequence similarity with each other. The sequences of LA-a and LA-b, as determined by Edman degradation, showed chitin-binding domains of plant chitinases at the N termini. These proteins showed small but significant chitinase and chitosanase activities. Lectin RCA120 indicated that, unlike common plant chitinases, LA-a and LA-b are glycosylated. LA-a and LA-b showed insecticidal activities when fed to larvae of the model insect Drosophila melanogaster.ConclusionsOur results suggest that the two LA proteins have a crucial role in defense against herbivorous insects, possibly by hydrolyzing their chitin.

A dye-labelled soluble substrate for the assay of endo-chitosanase activity

Coupling Remazol Brilliant Blue R (RBB) to chitosan that has been subjected to partial enzymatic hydrolysis gave rise to a new dyed derivative of chitosan which is highly soluble in slightly acidic buffers or microbiological media. The RBB:chitosan ratio has been carefully optimized, as well as the reaction conditions, to obtain a quasi-linear, sensitive assay for chitosanase. The substrate (sRBB-C) is useful for microtitre plate-based screening and for assay of chitosanase activity in the presence of high backgrounds of reducing sugars. Due to its solubility, sRBB-C is particularly suitable for the detection of chitosanase-positive organisms on agar media.

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.

키틴함유 DFMs 급여 한우송아지 분변내 키틴 및 키토산분해효소 생산 미생물 선발 및 동정

본 연구는 키틴으로부터 면역증강물질인 N-acetyl-Dglucosamine을 축적시킴으로써 동물의 면역력을 향상시켜 폐사율을 줄이고자 키틴이 함유된 DFM을 급여한 생후 3~7개월령된 한우 송아지의 분변에서 키틴분해 효소능을 가진 미생물을 분리 동정하기 위해서 수행하였다. 송아지 분변 1g당 키틴이 함유된 배지상에서 <TEX>$10^5$</TEX> 이상의 집락이 형성된 10균주 중 키틴을 분해하는 2균주와 키토산을 분해하는 2균주를 선발하였으며 이들은 모두 용혈성을 갖지 않는 것으로 나타났다. 키틴을 분해하는 균주는 선발한 4균주 모두 그람 음성균으로서 HANDI 110과 HANDI 309는 키틴을 분해하는 균주로 선발되었으며 HANWOO와 HANYOO는 키토산을 분해하는 균주로 선발되었다. 분리된 미생물은 형태학적, 물리화학적 및 유전학적 분류를 통해 HANDI 110는 Escherichia fergusonii으로, HANDI 309는 Acinetobacter parvus으로, HANWOO는 Comamonas koreensis으로 HANYOO는 Chryseobacterium indologenes으로 각각 동정하였다. This study was carried out to screen and identify the chitinase and chitosanase producing microorganisms from the feces of calves medicated DFM sincluding chitin in order to do the immune fortification of Korean Native calves. Ten isolates were grown in the medium containing chitin and chitosan that had more than <TEX>$10^5$</TEX> cfu/g in feces. Among these 10 strains, 2 strains (HANDI 110 and HANDI 309) had the chitinase activities and 2 strains (HANWOO and HANYOO) had the chitosanase activities in calves' feces. They showed no reaction in hemolysis tests by utilizing chitin and chitosan. The results from morphological, physicochemical and genetical identification indicated the HANDI 110 as a strain of Escherichia fergusonii, HANDI 309 was identified as a strain of Acinetobacter parvus, HANWOO was identified as a strain of Comamonas koreensis, and HANYOO as a strain of Chryseobacterium indologenes.

Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and beta-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and beta-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose.

Enhancement of the activity and pH-performance of chitosanase from Bacillus cereus strains by DNA shuffling

DNA shuffling was carried out with two chitosanase genes belonging to glycoside hydrolase family eight from Bacillus cereus KNUC51 and B. cereus KNUC55. The shuffled products, YM18 and YM20, which showed higher activity than the parents at 40 degrees C, were selected for further studies. The 50 kDa chitosanases were purified using affinity chromatography with glutathione-Sepharose 4B. In general, the specific activity of YM18 is enhanced 250% and that of YM20 is 350% compared to the parents. YM20 exhibits a shift of the optimal pH level from 5.5 to 6.5. DNA sequence analysis revealed that YM18 and YM20 contained 2 amino acid substitutions (I13T and A87V for YM18; K66R and N352S for YM20). We presumed that these amino acid substitutions increase the specific activity and change the property of the two variants.

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