Effective production of chitinase and chitosanase byStreptomyces griseus HUT 6037 using colloidal chitin and various degrees of deacetylation of chitosan

The advantages of the organismStreptomyces griseus HUT 6037 is that the chitinase and chitosanase using chitinaceouse substrate are capable of hydrolyzing both amorphous and crystalline chitin and chitosan. We attempted to investigate the optimization of induction protocol for high-level production and secretion of chitosanase and the influence of chitin and partially deacetylated chitosan sources (75–99% deactylation). The maximum specific activity of chitinase has been found at 5 days cultivation with the 48 hours induction time using colloidal chitin as a carbon source. To investigate characteristic of chitosan activity according to substrate, we used chitosan with various degree of deacetylation as a carbon source and found that this strain accumulates chitosanase in the culture medium using chitosanaceous substrates rather than chitinaceous substrates. The highest chitosanase activity was also presented on 4 days with 99% deacetylated chitosan. The partially 53% deacetylated chitosan can secrete both chitinase and chitosanase which was defined as a soluble chitosan. The specific activities of chitinase and chitosanase were 0.89 at 3 days and 1.33 U/mg protein at 5 days, respectively. It indicate that chitosanase obtained fromS. griseus HUT 6037 can hydrolyze GlcNAc-GlcN and GlcN-GlcN linkages by exo-splitting manner. This activity increased with increasing degree of deacetylation of chitosan. It is the first attempt to investigate the effects of chitosanase on various degrees of deacetylations of chitosan byS. griseus HUT 6037. The highest specific activity of chitosanase was obtained with 99% deacetylated chitosan.     

Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Production of chitin and chitosan from the exoskeleton of adult two‐spotted field crickets (Gryllus bimaculatus)

Chitin and chitosan were extracted from all specimens of Type I and II two‐spotted field crickets (Gryllus bimaculatus) following chemical treatment with an acid and alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve demineralization and deproteinization, respectively. For chitosan extraction, 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis, respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis, respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and 7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %, 90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi Poise), respectively. The molecular weight of chitosan from adult exoskeletons of G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G. bimaculatus could be used as a source of chitin and chitosan for use as functional additives in industrial animal feeds.     

Effects of Spray Drying on Physicochemical Properties of Chitosan Acid Salts

The effects of spray-drying process and acidic solvent system on physicochemical properties of chitosan salts were investigated. Chitosan used in spray dryings was obtained by deacetylation of chitin from lobster (Panulirus argus) origin. The chitosan acid salts were prepared in a laboratory-scale spray drier, and organic acetic acid, lactic acid, and citric acid were used as solvents in the process. The physicochemical properties of chitosan salts were investigated by means of solid-state CP-MAS ¹³C nuclear magnetic resonance (NMR), X-ray powder diffraction (XRPD), differential scanning calorimetry, and Fourier transform infrared spectrometry (FTIR) and near-infrared spectroscopy. The morphology of spray-dried chitosan acid salts showed tendency toward higher sphericity when higher temperatures in a spray-drying process were applied. Analysis by XRPD indicated that all chitosan acid salts studied were amorphous solids. Solid-state ¹³C NMR spectra revealed the evidence of the partial conversion of chitosan acetate to chitin and also conversion to acetyl amide form which appears to be dependent on the spray-drying process. The FTIR spectra suggested that the organic acids applied in spray drying may interact with chitosan at the position of amino groups to form chitosan salts. With all three chitosan acid salts, the FTIR bands at 1,597 and 1,615 cm⁻¹ were diminished suggesting that –NH groups are protonated. The FTIR spectra of all chitosan acid salts exhibited ammonium and carboxylate bands at 1,630 and 1,556 cm⁻¹, respectively. In conclusion, spray drying is a potential method of preparing acid salts from chitosan obtained by deacetylation of chitin from lobster (P. argus) origin.     

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

Reduction of the algicidal properties of copper and mercury ions by chitin and chitosan

Chitin, obtained from the shell of the tail portions of the prawn Nephrops norvegicus (L), and chitosan prepared by deacetylation of the chitin, were added to growing cultures of Chlorella containing various quantities of toxic metals. It was found that while the presence of copper and mercury ions inhibited the growth of Chlorella, the presence of chitin or chitosan reduced the toxic effects of these metals when they were present in concentrations up to 10 mg dm^-3.     

Effect of chitin sources on production of chitinase and chitosanase byStreptomyces griseus HUT 6037

The advantage of usingStreptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystal-line chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75–99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 days of cultivation with 99% deacetylated chitosan. Soluble chitosan (53% deacetylated chitosan) was found to induce chitinase as well as chitosanase. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)₃, (GlcN)₄ and (GlcN)₅ were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)₃ was homogeneous and those of (GlcN)₄ and (GlcN)₅ were heterogeneous.     

Microwave technique for efficient deacetylation of chitin nanowhiskers to a chitosan nanoscaffold

A chitosan nanoscaffold in the form of a colloidal solution was obtained from the deacetylation of chitin whiskers under alkaline conditions by using a microwave technique in only 1/7 of the treatment time of the conventional method. Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (¹H NMR) techniques confirm the degree of deacetylation to be above 90% within 3 h. The wide-angle X-ray diffraction (WAXD) pattern clearly shows that the highly crystalline chitin whiskers are changed to amorphous chitosan. SEM micrographs show the aggregation of branched nanofibers, whereas the TEM micrographs reveal the scaffold morphology.     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.     

Chitin and chitosan biopolymer production from the Iranian medicinal fungus Ganoderma lucidum: Optimization and characterization

Abstract

Chitin and chitosan with unique properties and numerous applications can be produced from fungus. The production of chitin and chitosan from the mycelia of an Iranian Ganoderma lucidum was studied to improve cell growth and chitin productivity. Inoculum size and initial pH as two effective variables on the growth of G. lucidum and chitin production were optimized using response surface method (RSM) by central composite design (CCD). The results verified the significant effect of these two variables on the cell growth and chitin production. In optimum conditions, including pH?=?5.7 and inoculum size of 7.4%, the cell dry weight was 5.91?g/L and the amount of chitin production was 1.08?g/L with the productivity of 0.083?g/(L day). The produced chitin and chitosan were characterized using XRD and FTIR. Moreover, the antibacterial activity of the produced chitosan was investigated and compared with the commercial chitosan. The results showed that the produced chitin and chitosan had suitable quality and the Iranian G. lucidum would be a great source for safe and high-quality chitin and chitosan production.     

Extraction of chitin and chitosan from larval exuvium and whole body of edible mealworm,Tenebrio molitor

The purpose of this study was to investigate the production of chitin and chitosan from both the exuvium and whole body of mealworm (Tenebrio molitor) larvae. Chitin from the exuvium and whole body of T. molitor larvae was chemically extracted with acid and alkali solutions to achieve demineralization (DM) and deproteinization (DP), respectively. The average DM (%) and DP (%) on a dry weight (DW) basis was 32.56 and 73.16% from larval exuvium, and 41.68 and 91.53% from whole body, respectively. To obtain chitosan, chitin particles from the exuvium and whole body of T. molitor larva were heated at various temperatures in different concentrations of NaOH. Average chitin yields were 18.01% and 4.92% of DW from the exuvium and whole body, respectively. The relative average yield of chitosan from whole body was 3.65% of DW. On average, over 90% of chitosan derived from whole body was deacetylated. The viscosity of chitosan from whole body was ranged from 48.0 cP to 54.0 cP. The chitin content of dry and wet byproducts from whole body were 17.32% and 16.94% respectively, compared to dry weight. The chitosan contents of byproducts on a DW basis were 14.48% in dry and 13.07% in wet byproduct. These results indicate that the exuvium and whole body of T. molitor larva may serve as a source of chitin and chitosan for use in domestic animal feed.     

New chitin,chitosan, and O-carboxymethyl chitosan sources from resting eggs of Daphnia longispina (Crustacea); with physicochemical characterization,and antimicrobial and antioxidant activities

The paper describes the isolation and characterization of chitin and chitosan from Daphnia longispina resting eggs harvested from a reservoir. Resting eggs are fertilized eggs that are encased in chitinous shells called ‘ephippia’ and which ensure the survival of the Daphnia population in adverse conditions. The chitin-content of D. longispina resting eggs was found to be 23 ～ 25% and the chitosan (having a 70 ～ 75% deacetylation degree) yield of the chitin was 76 ～ 77%. This high chitin-content indicates that D. longispina resting eggs can be exploited as a chitin source. The structure and thermal properties of chitin, extracted from D. longispina resting eggs, were characterized by employing Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction and scanning electron microscopy. The crystallinity of the chitin was found to be very low (48%). Physicochemicallycharacterized chitosan and the produced O-carboxymethyl chitosan were tested for their antimicrobial and antioxidant activity. It has been observed that chitosan displays antimicrobial activity against all pathogenic bacteria, whereas O-carboxymethyl chitosan only exhibits inhibition activity against L. garvieae, L. Monocytogenes ATCC 7644, Y. enterocolitica NCTC 11175 and S. aureus ATCC 25923. In a free radical scavenging activity assay, the IC₅₀ values of chitosan, O-carboxymethyl chitosan and butylated hydroxytoluene were found to be 23.01, 56.43 and 0.05, respectively. The ferric-reducing power of O-carboxymethyl chitosan (EC₅₀ = 8.30) indicated higher activity than chitosan (EC₅₀ = 10.12).     

Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

Extraction and characterization of chitin and chitosan from local sources

Chitin has been extracted from six different local sources in Egypt. The obtained chitin was converted into the more useful soluble chitosan by steeping into solutions of NaOH of various concentrations and for extended periods of time, then the alkali chitin was heated in an autoclave which dramatically reduced the time of deacetylation. Chitin from squid pens did not require steeping in sodium hydroxide solution and showed much higher reactivity towards deacetylation in the autoclave that even after 15 min of heating a degree of deacetylation of 90% was achieved. The obtained chitin and chitosan were characterized by spectral analysis, X-ray diffraction and thermo gravimetric analysis.     

Effects of molecular weight and deacetylation degree of chitin/chitosan on wound healing

We studied the effects of chitin/chitosan on wound healing with reference to chemical properties using a linear incisional wound model in rats. Wound break strength of the chitosan group (D-glucosamine (GlcN), chito-oligosaccharide (COS), chitosan) was higher than the chitin group (N-acetyl-D-glucosamine (GlcNAc), chiti-oligosaccharide (NACOS), chitin). Collagenase activity was also higher in the chitosan group than the chitin group. There was no significant change between the concentration of the sample and the break strength and collagenase activity in all samples. In histological findings, collagen fibers run perpendicular against the incisional line in the oligosaccharide group (NACOS, COS), and many activated fibroblasts were observed around the wound in the chitosan group. As for the deacetylation degree, the higher the deacetylation degree becomes, the more the stronger the break strength becomes. Also, activated fibroblasts appeared more in the higher deacetylation degree.     

Characterization and Quantification of Chitosan Extracted from Nacre of the Abalone Haliotis tuberculata and the Oyster Pinctada maxima

This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan. Received February 16, 2000; accepted July 4, 2000.     

Chitin deacetylase product inhibition

Chitin deacetylase is the only known enzyme catalyzing the hydrolysis of the acetamino linkage in the N-acetylglucosamine units of chitin and chitosan. This reaction can play an important role in enzymatic production of chitosan from chitin, or in enzymatic modification of chitosan, which has applications in medicine, pharmacy or plant protection. It was previously shown that acetic acid, a product of the deacetylation process, may act as an inhibitor of chitin deacetylase. Here we show the mechanism of inhibition of chitin deacetylase isolated from Absidia orchidis vel coerulea by acetic acid released during the deacetylation process. The process follows competitive inhibition with respect to acetic acid with an inhibition constant of K(i) = 0.286 mmol/L. These results will help to find the optimal system to carry out the enzymatic deacetylation process for industrial applications.     

Purification and Mode of Action of Chitosanolytic Enzyme from Enterobacter sp. G-1

A chitosanolytic enzyme was purified from Enterobacter sp. G-1 by fractionation of 30% saturation with ammonium sulfate, isoelectric focusing, and Sephadex G-100 gel chromatography. The purified enzyme. showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the molecular mass was estimated to be 50 kDa. The enzyme degraded N-acetyl-chitooligosaccharides, glycol chitin, colloidal chitin, and colloidal chitosan (about 80% deacetylated), but did not degrade chitooligosaccharides, colloidal chitosan (100% deacetylated), or Micrococcus lysodeikticus cell walls. It hydrolyzed GlcNAc_4–6 and colloidal chitin to GlcNAc₂, finally. The main cleavage site with GlcNAc_3–6 was the second linkage from the non-reducing end, based on the pattern of pNp-GlcNAc_2–5. Colloidal chitosan was hydrolyzed to GlcNAc₂ and to similar partially N-acetylated chitooligosaccharides.     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.