Production Of Chitin Deacetylase Research Articles

Extraction of chitosan from biologically-derived chitin by bacterial chitin deacetylase: Process optimization and product quality assessment

Commercial chitosan manufacturing process relies on strong chemical treatment on chitin that generates chitosan with undesirable properties and leads to environmental pollution. To overcome the adverse consequences, enzymatic chitosan preparation from chitin was undertaken in the current study. A potent chitin deacetylase (CDA) producing bacterial strain was screened and subsequently identified as Alcaligens faecalis CS4. After optimization 40.69 U/mL of CDA production was achieved. By treating the organically extracted chitin with partially purified CDA chitosan yield of 19.04 % was attained having 71 % solubility, 74.9 % degree of deacetylation, 21.16 % crystallinity index, 246.4 kDa molecular weight and 298 °C highest-decomposition temperature. FTIR and XRD analysis revealed characteristics peaks respectively within 870–3425 cm−1 wavenumber and 10°-20°, for enzymatically and chemically extracted (commercial) chitosan that endorses their structural similarity which validated through electron microscopic study. At 10 mg/mL chitosan concentration 65.49 % DPPH radical scavenging activity endorsed its antioxidant potential. Minimum inhibitory concentration of chitosan was 0.675, 1.75, 0.33 and 0.75 mg/mL for Streptococcus mutans, Enterococcus faecalis, Escherichia coli and Vibrio sp., respectively. Mucoadhesiveness and cholesterol binding properties were also exhibited by extracted chitosan. The present study opens a new vista for eco-friendly extraction of chitosan from chitin that is proficient and sustainable in environmental perspective.

Enhanced Chitin Deacetylase Production Ability of Rhodococcus equi CGMCC14861 by Co-culture Fermentation With Staphylococcus sp. MC7.

Chitin deacetylase (CDA) can hydrolyze the acetamido group of chitin polymers and its deacetylated derivatives to produce chitosan, an industrially important biopolymer. Compared with traditional chemical methods, biocatalysis by CDA is more environment-friendly and easy to control. However, most reported CDA-producing microbial strains show low CDA producing capabilities. Thus, the enhancement of CDA production has always been a challenge. In this study, we report co-culture fermentation to significantly promote the CDA production of Rhodococcus equi CGMCC14861 chitin deacetylase (ReCDA). Due to co-culture fermentation with Staphylococcus sp. MC7, ReCDA yield increased to 21.74 times that of pure culture of R. equi. Additionally, the enhancement was demonstrated to be cell-independent by adding cell-free extracts and the filtrate obtained by 10 kDa ultrafiltration of Staphylococcus sp. MC7. By preliminary characterization, we found extracellular, thermosensitive signal substances produced by Staphylococcus that were less than 10 kDa. We investigated the mechanism of promotion of ReCDA production by transcriptomic analysis. The data showed that 328 genes were upregulated and 1,258 genes were downregulated. The transcription level of the gene encoding ReCDA increased 2.3-fold. These findings provide new insights into the research of co-culture fermentation for the production of CDA and quorum sensing regulation.

An Application of Response Surface Methodology for Optimizing the Production of Chitin Deacetylase Enzyme by Aspergillus Flavus

In this study, the optimum conditions for chitin deacetylase (CDA) production by Aspergillus flavus F1 in solid-state fermentation were investigated via two optimization strategies: classical optimization based on the method of one factor at a time and statistical optimization using response surface methodology. The result of classical optimization showed that corn supplemented with 2% chitin moisturized with mineral salts solution at pH=7 and five days of incubation time were the optimum conditions for increasing CDA production with approximately yield of 219.5 U/g solid substrate. Furthermore, pH, moisture level and inoculum size were systemically evaluated to improve CDA production based on a central composite design using the Design expert 7 software. Based on the enhanced regression model, a maximum predicted CDA enzyme production of 283.8 U/g could be obtained with pH 8, moisture level 1:1 (w/v) and inoculum size 3 ml/10g solid substrate, which contain 1×106 spore/ml. The verification of optimization results and determine accuracy of model showed that the actual response of CDA was 312 U/g, which approximately closed to predicted value 283.89 U/g. The crude extract of CDA was concentrated by sucrose. The results showed that 61% of CDA enzyme was yielded with a purification fold of 1.1 In addition. Then CDA was purified partially by gel filtration chromatography after concentration by sucrose with total enzyme activity and specific activity of 1476 U and 12300 (U/mg protein) respectively. Furthermore, the produced CDA enzyme showed maximum activity in pH ranged from (6-8); in which enzyme activity was 69 U/ ml. however; the enzyme stability has a wide range in acidic and alkaline pH. In addition, the enzyme was maintained its activity at temperatures from 30 to 55C˚. Whereas, the activity was declined in temperature up to 55Cº with a minimum activity (8 U/ml) observed at 80Cº.

Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation.

Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells. A Bacillus licheniformis CDA gene is identified in the genome of the bacterium, cloned, and expressed, yielding enzymatically active recombinant protein. For enzyme production, a growth medium is formulated using carbon and nitrogen sources, which do not compete with the human food chain. The maximum enzyme activity of 320 ± 20 U/mL is achieved under optimized conditions. The CDA productivity is improved by about 23 times in shake flask culture by optimizing operating conditions and medium components. The CDA is purified and the enzyme kinetic values i.e. Km, Vmax and Kcat are reported. Also the effect of cofactors, temperature, and pH on the enzyme activity is reported. Further, economic yield is proposed for production of CDA through this bioprocess.

SSF production, purification and characterization of chitin deacetylase from Aspergillus flavus

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50 °C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn2+ and Zn2+. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70 mM concentration. The Km and kcat values of the enzyme were determined to be 9.45 mg mL−1 and 26.72 s−1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.

Addition of abscisic acid increases the production of chitin deacetylase by Colletotrichum gloeosporioides in submerged culture

The activity of chitin deacetylase from Colletotrichum gloeosporioides was studied by the addition of phytohormones (gibberellic acid, indole acetic acid, and abscisic acid) and amino sugars (glucosamine and N-acetyl glucosamine) in culture media. Abscisic acid exerted a positive and significant effect on enzyme production with 9.5-fold higher activity (1.05Umgprotein−1) than the control (0.11Umgprotein−1). Subsequently, this phytohormone was used in batch culture with higher chitin deacetylase activity being found at acidic pH (3.5) than at neutral pH (7). Furthermore, the highest activity was determined at the acceleration growth phase. The chitin deacetylase production was ascribed to the lag phase within the spore germination process and germ tube elongation instead of during the formation of appressoria, as evidenced by the scanning electronic microscopy results. Therefore, more inoculum and medium containing abscisic acid were added to the fed-batch culture, resulting in a significant increase in chitin deacetylase activity (3.64Umgprotein−1). The addition of abscisic acid led to changes in the acetylation degree of chitin extracted from the cell wall of C. gloeosporioides, with lower degree of acetylation (DA of 75.6%) than that determined with the culture without abscisic acid (DA of 90.6%).

Production of chitin deacetylase by Aspergillus flavus in submerged conditions

ABSTRACTChitosan is a biopolymer obtained by deacetylation of chitin and has been proven to have various applications in industry and biomedicine. Deacetylation of chitin using the enzyme chitin deacetylase (CDA) is favorable in comparison to the hazardous chemical method involving strong alkali and high temperature. A fungal strain producing CDA was isolated from environmental samples collected from coastal regions of South Kerala, India. It was identified as Aspergillus flavus by morphological characteristics and ITS DNA analysis. Nutritional requirement for maximum production of CDA under submerged condition was optimized using statistical methods including Plackett–Burman and response surface methodology central composite design. A 5.98-fold enhancement in CDA production was attained in shake flasks when the fermentation process parameters were used at their optimum levels. The highest CDA activity was 57.69 ± 1.68 U under optimized bioprocess conditions that included 30 g L−1 glucose, 40 g L−1 yeast extract, 15 g L−1 peptone, and 7 g L−1 MgCl2 at initial media pH of 7 and incubation temperature of 32°C after 48 hr of incubation, while the unoptimized basal medium yielded 9.64 ± 2.04 U.

Mustard oil cake as an inexpensive support for production of chitin deacetylase by Penicillium oxalicum SAEM-51 under solid-state fermentation

Abstract Response surface methodology was employed to optimize the cultivation conditions of Penicillium oxalicum SAE M -51 for the enhancement of chitin deacetylase (CDA) production under solid-state fermentation. The enzyme catalyzes deacetylation of N -acetyl glucosamine subunits of chitin resulted in the production of chitosan, widely utilized biopolymer for drug delivery, waste water treatment and as nutraceutics. Among different agro-horticultural substrates evaluated, mustard oil cake had resulted into maximal CDA production. Entrapment of fungal mycelia onto the solid support was studied using scanning electron microscopy. Optimal physico-chemical conditions for maximal CDA production were found to be 4.906 g, 73.62% and 8.578% for substrate amount, moisture content and inoculum size, respectively. The experimental CDA production (1162.03±7.2 U gds −1 ) under optimized condition was observed in close agreement with the values predicted by the quadratic model (1137.85 U gds −1 ). The CDA production by P. oxalicum SAE M -51 was increased significantly by 1.3 fold, as compared to the un-optimized ones (877.56±8.9 U gds −1 ).

Optimization of medium components for production of chitin deacetylase by Bacillus amyloliquefaciens Z7, using response surface methodology

Plackett–Burman design and Box–Behnken response surface methodology (RSM) was employed to optimize the medium components for the chitin deacetylase (CDA) activity from Bacillus amyloliquefaciens Z7. Plackett–Burman design was applied to determine the specific medium components affecting CDA activity and found that starch, chitin and MgSO4 were critical in augmenting CDA activity. These significant parameters were further optimized using Box–Behnken RSM and the optimum concentrations of starch, chitin and MgSO4 were found to be 24.4, 8.8 and 0.19 g/L, respectively. The optimum medium composition was chitin 8.8 g/L, starch 24.4 g/L, yeast extract 10g/L, MgSO4 0.19 g/L, K2HPO4 0.3 g/L and NaCl 5 g/L. Under these optimal conditions, the CDA activity of Bacillus amyloliquefaciens Z7 increased distinctly from 18.75 to 27.48 U/mL (46.6% increase in total yield).

Statistical optimization for production of chitin deacetylase from Rhodococcus erythropolis HG05

A strain producing chitin deacetylase (CDA) was isolated and identified as Rhodococcus erythropolis by morphological characteristics and 16S rDNA analysis, named as R. erythropolis HG05. By Plackett–Burman and central composite design, CDA production from R. erythropolis HG05 was increased from 58.00U/mL to 238.89U/mL. With the crude enzyme from R. erythropolis HG05, the hydrolysate components from colloid chitin were chito-oligosaccharides with polymerization number larger than hexaose.

Optimization of the fermentation conditions of Rhizopus japonicus M193 for the production of chitin deacetylase and chitosan

To improve the production of chitin deacetylase (CDA) for the bioconversion of chitin to chitosan with desirable functionality, the effect of the nutritional requirement on the CDA production from Rhizopus japonicus M193 fermentation was investigated under submerged conditions. Nutritional elements including glucose (g/L), inoculum level (%), and MgSO4·7H2O (g/L), as well as culture time (d) were identified as the most critical factors for the CDA production based on the results from Plackett–Burman design (PBD). Taguchi design with orthogonal array was further employed to optimize R. japonicus M193 fermentation conditions based on the results from PBD, in which 2.5% chitin, 5g/L glucose, 5% inoculum level, 0.6g/L MgSO4·7H2O, and 5d culture time were identified as the optimal fermentation conditions. Under this condition, the maximum CDA production, DDA and MM of produced chitosan were 547.38±12.06U/L, 78.85±1.68%, and 125.63±3.74kDa, respectively. Obtained chitosan displayed similar physicochemical and structural properties to those of commercial chitosan extracted using chemical method based on the results from Fourier transform infrared spectrometer (FT-IR), thermogravimetric analysis (TGA)–differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) assays, while the use of chemical reagents was significantly reduced.

Enhanced production of chitin deacetylase by Penicillium oxalicum SAEM-51 through response surface optimization of fermentation conditions.

Optimization of the fermentation conditions for chitin deacetylase (CDA) production by Penicillium oxalicum SAEM-51 was undertaken in the present study using central composite design (CCD) under submerged condition. CDA is widely employed for bio-catalytic conversion of chitin to chitosan. Chitosan is a biopolymer with immense commercial potential in diverse industrial sectors, viz. pharmaceutics, food, agriculture, water treatment, etc. CDA production was significantly affected by all the variables studied, viz. pH, temperature, inoculum age and size. The optimal conditions that stimulating maximal CDA production were found to be: pH, 7.9; temperature, 28 °C; inoculum age, 90 h, and 11 % inoculum size. Under these optimized conditions, the actual maximal CDA production was 623.57 ± 8.2 Ul−1, which was in good agreement with the values predicted by the quadratic model (648.24 Ul−1), confirming the validity of the model. Optimization of fermentation conditions through CCD had resulted into 1.4-fold enhancement in CDA productivity (Qp = 4.3264 Ul−1 h−1). Results of these experiments indicated that response surface methodology was proved to be a promising method for optimization of CDA production.

Parametric Optimization of Extracellular Chitin Deacetylase Production by Scopulariopsis brevicaulis

Parametric Optimization of Extracellular Chitin Deacetylase Production by Scopulariopsis brevicaulis The culture medium, fermentation conditions and effects of different inorganic salts on producing extracellular chitin deacetylase by Scopulariopsis brevicaulis were optimized.The highest enzymatic activity of deacetylation of chitin was 36 units/mL for optimum culture medium which included 2% (w/v) 3,6-O-carboxymethylchitin, 1% sucrose, 0.2% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.4% peptone, 0.05% MgSO4, 0.05% MnSO4, 0.03% CoCl2 and optimum cultural conditions included temperature, pH, agitation speed, fermenting time and volume and were 29°C, 7.0, 200 rmp min-1, 96 hours and 80 mL in 300 mL Erlenmeyer flask, respectively

Extracellular chitin deacetylase production in solid state fermentation by native soil isolates of Penicillium monoverticillium and Fusarium oxysporum.

Extracellular chitin deacetylase production by native soil isolates of Penicillium monoverticillium CFR 2 and Fusarium oxysporum CFR 8 in solid state fermentation (SSF) using commercial wheat bran (CWB) and shrimp processing by-products (SPP) as solid substrate has been studied. P. monoverticillium produced maximum chitin deacetylase activity of 547.7 ± 45 and 390.2 ± 31 units/g initial dry substrate (U/g IDS) at 96h of incubation in CWB and SPP media, respectively. While, F. oxysporum produced maximum chitin deacetylase activity of 306.4 ± 22U/g IDS at 72h of incubation in CWB medium and 220.1 ± 20U/g IDS at 120h of incubation in SPP medium. Along with chitin deacetylase, P. monoverticillium and F. oxysporum produced other chitin degrading enzymes such as endo-chitinase and β-N-acetylhexosaminidase. P. monoverticillium produced maximum activity (U/g IDS) of endo-chitinase 4.6 ± 0.20 at 120h incubation and β-N-acetylhexosaminidase 82.6 ± 03 at 120h incubation in CWB medium. While, F. oxysporum produced maximum activity (U/g IDS) of endo-chitinase 7.8 ± 0.20 at 144h incubation and β-N-acetylhexosaminidase 38.3 ± 02 at 120h incubation in CWB medium. Production of extracellular chitin deacetylase by P. monoverticillium CFR 2 and F. oxysporum CFR 8 in SSF is being reported for the first time.

Optimization of medium composition for enhanced chitin deacetylase production by mutant Penicillium oxalicum SAE M-51 using response surface methodology under submerged fermentation

Nutritional requirement for maximum production of chitin deacetylase (CDA), an enzyme used for bioconversion of chitin to chitosan, under submerged condition by mutant Penicillium oxalicum SAE M-51 was optimized using response surface methodology (RSM). Among the eight nutritional elements studied, yeast-extract, peptone, (NH 4) 2SO 4 and MgSO 4 were identified as the most critical variables for CDA production by Plackett–Burman design first. Further optimization of these variables was done by using a four factor central composite design (CCD). The model came out to be significant and statistical analysis (analysis of variance, ANOVA) of the results showed that yeast extract had contributed significantly (quadratic effect) to the response and its interaction with magnesium sulfate was strong. Peptone was also found to be another important component in the medium that influence the CDA production. Optimum concentration of the variables for enhanced CDA production determined by CCD were—yeast extract ( X 1) 3.165 g l −1, peptone ( X 2) 4.754 g l −1, (NH 4) 2SO 4 ( X 4) 0.255 g l −1 and MgSO 4 ( X 5) 0.535 g l −1. 2.0 fold enhancement in CDA titres was attained in shake flasks, when the variables were used at their optimum levels. CDA production (414.7 ± 6.32 U l −1) in the optimized medium was in good agreement with the values predicted by the quadratic model (399.08 U l −1), confirming the validity of the model. Employment of optimal growth medium would result into large scale production of microbial deacetylase, which further heighten its potential for biological production of chitosan.

Solid state fermentation production of chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676 using different substrates.

Production of extracellular chitin deacetylase by Colletotrichum lindemuthianum ATCC 56676 under solid substrate fermentation was studied. The suitability of shrimp shell chitin waste (SSCW) and commercial wheat bran (CWB) was evaluated for maximal enzyme production. CWB medium (pH6.4 ± 0.2) supplemented with chitosan favoured maximal chitin deacetylase yield of 460.4 ± 14.7 unit/g initial dry substrate (U/g IDS) at 96h as compared to maximal yield of 392.0 ± 6.4U/g IDS at 192h in SSCW medium (pH8.7 ± 0.2) at 25°C incubation temperature and 60% (w/w) initial moisture content of medium. Along with chitin deacetylase, C. lindemuthianum ATCC 56676 produced maximum endo-chitinase (0.28 ± 0.03U/g IDS at 144h) and β-N-acetylhexosaminidase (0.79 ± 0.009U/g IDS at 192h) in CWB medium and 0.49 ± 0.05U/g IDS of endo-chitinase at 264h and 0.38 ± 0.04U/g IDS of β-N-acetylhexosaminidase at 96h of incubation in SSCW medium. SEM studies indicated the difference in the morphology of mycelia and hyphae of C. lindemuthianum ATCC 56676 when grown on different solid substrates. Production of chitin deacetylase by SSF is being reported for the first time.

Effect of Medium Composition on the Synthesis of Chitinase and Chitin Deacetylase from Thermophilic Paenibacillus sp.Hul

To investigate the effects of nutritional components and initial pH on chitinase and chitin deacetylase production by thermophilic Paenibacillus sp. Hul, and provide basis for fermentation of enzymes and preparation of chito- oligosaccharides. The shake flask culture was employed to research the culture conditions of thermophilic Paenibacillus sp. Hu1 for producing chitinase and chitin deacetylase. The results show that the optimal carbon source was chitin powder with 35g/L; while yeast extract power was the optimal nitrogen source with 10g/L. It was beneficial to synthesize chitinase and chitin deacetylase when the initial pH was 6.0. In addition, the synthesis of chitinase and chitin deacetylase was inhibited by Cu2+, and activated by Ca2+. However, Mg2+ and Zn2+ did not show significant inhibitory or activation effects on the synthesis of the two enzymes. The hydrolysate components were analyzed using chitin as substrate with the crude enzyme, and the result suggested that enzymatic hydrolysates were mainly centralized in chito-oligosaccharides with polymerization number less than hexaose.

Identification of a chitin deacetylase producing bacteria isolated from soil and its fermentation optimization.

Chitin deacetylase (CDA) catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues. In this study, 9 strains of bacteria with chitin deacetylase activity were isolated from canal mud samples from theXiangjiang River in Changsha, Hunan Province. Of these 9 strains, the Z7 strain had the highest chitin deacetylase activity, as determined by enzyme assay screening. Based on its culture, morphological, physiological characteristics and molecular identification, strain Z7 was identified as Bacillus amyloliquefaciens. Yeast extract at a concentration of 1.0% was an ideal nitrogen source for production of CDA (16.78 Eu/ml). Starch at 2% functioned best as the major carbon source for optimum production of CDA (17.84 Eu/ml). Magnesium sulfate at a concentration of 0.04% was an ideal inorganic salt for CDA production (17.45 Eu/ml). Ideal pH and temperature for optimum production of CDA by this strain were pH 6 and 37EsC. Key words: Chitin deacetylase, Chitosan, Bacillus amyloliquefaciens.

Statistical Optimization of Medium Constituents to Improve Chitin Deacetylase Production by Mutant Penicillium oxalicum SAE M-51 under Submerged Fermentation

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Penicillium oxalicum SAE M-51: a mutagenised strain for enhanced production of chitin deacetylase for bioconversion to chitosan

A novel chitin deacetylase (CDA) producing strain Penicillium oxalicum ITCC 6965 was isolated from residual materials of sea food processing industries. Strain following mutagenesis using ethidium bromide (EtBr) and microwave irradiation had resulted into a mutant P. oxalicum SAE(M)-51 having improved levels of chitin deacetylase (210.71 ± 1.65 Ul(-1)) as compared to the wild type strain (108.26 ± 1.98 Ul(-1)). Maximum enzyme production was achieved in submerged fermentation following 144 hours of incubation with notably improved productivity of 1.46 ± 0.82 Ul(-1) h(-1) as compared to the wild type strain (0.75 ± 0.53 Ul(-1)h(-1)). Scanning electron micrographs of mutant and wild type strains had revealed distinct morphological features. Evaluation of kinetic parameters viz. Q(s), Q(p), Y(p/x), Y(p/s), q(p), q(s) had denoted that strain P. oxalicum SAE(M)-51 is a hyper producer of chitin deacetylase. Glucose as compared to chitin or colloidal chitin had resulted in increased levels of enzyme production. However, replacement of glucose with chitinous substrates had prolonged the duration for enzyme production. The mutant strain had two pH optima that is 6.0 and 8.0 and had an optimum temperature of 30 °C for growth and enzyme production.