Conversion Of Chitin Research Articles

Biocatalytic production of chitosan polymers from shrimp shells, using a recombinant enzyme produced by <i>pichia pastoris</i>

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme

Little information exists for the ability of enterococci to utilize chitin as a carbon source. We show that Enterococcus faecalis V583 can grow on chitin, and we describe two proteins, a family 18 chitinase (ef0361; EfChi18A) and a family 33 CBM (carbohydrate binding module) (ef0362; EfCBM33A) that catalyze chitin conversion in vitro. Various types of enzyme activity assays showed that EfChi18A has functional properties characteristic of an endochitinase. EfCBM33A belongs to a recently discovered family of enzymes that cleave glycosidic bonds via an oxidative mechanism and that act synergistically with classical hydrolytic enzymes (i.e., chitinases). The structure and function of this protein were probed in detail. An ultra-high-resolution crystal structure of EfCBM33A revealed details of a conserved binding surface that is optimized to interact with chitin and contains the catalytic center. Chromatography and mass spectrometry analyses of product formation showed that EfCBM33A cleaves chitin via the oxidative mechanism previously described for CBP21 from Serratia marcescens. Metal-depletion studies showed that EfCBM33A is a copper enzyme. In the presence of an external electron donor, EfCBM33A boosted the activity of EfChi18A, and combining the two enzymes led to rapid and complete conversion of β-chitin to chitobiose. This study provides insight into the structure and function of the CBM33 family of enzymes, which, together with their fungal counterpart called GH61, currently receive considerable attention in the biomass processing field.

Study on Enzymatic Characteristics of Chitin Deacetylase

Chitin deacetylase (CDA) catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues. The results of the fundamental enzymatic properties of chitin deacetylase producing from the high productivity chitin deacetylase strain Z7 show that, the optimum temperature of strain Z7 was 40°C, the optimum pH was 6.5, under the concentration of 1mmol/L, Mg2+, K+, Ca2+and Fe2+had activation effect, of which the strongest activation effect was Mg2+. Pb2+, Cu2+and Mn2+had inhibitory effect, of which the strongest inhibitory effect was Mn2+.

Purification and characterization of a chitinase (sAMC) with antifungal activity from seeds of Astragalus membranaceus

A chitinase (sAMC) was purified from the seeds of Astragalus membranaceus (Fisch.) Bunge, using a combination of 20–60% ammonium sulfate precipitation, regenerated chitin affinity column and Sephadex G-75. Purified chitinase was a monomer with a molecular mass of 35.5kDa on SDS–PAGE. Based on the homology search of amino acid sequences of four internal peptides, sAMC belongs to glycosyl hydrolase (GH) family 19 chitinases which are mostly endochitinases. The optimal pH of sAMC was 5.0, and it was stable over a broad range of pH 4.0–8.0. sAMC exhibited an optimal temperature of 60°C and it was stable up to 60°C. sAMC hydrolyzed colloidal chitin into chitotriose, chitobiose and N-acetyl d-glucosamine suggesting its role in conversion of chitin wastes into useful products. It exhibited antifungal activity against Trichoderma viride, Botrytis cinerea, Fusarium oxysporum and Fusarium solani. This is the first report on chitinases from Astragalus sp.

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.

Development of innovative technologies to decrease the environmental burdens associated with using chitin as a biomass resource: Mechanochemical grinding and enzymatic degradation

The production of N-acetylglucosamine (GlcNAc, chitin monosaccharide) from crab or shrimp shells generally requires numerous steps and the use of deleterious substances. One goal for researchers is the direct production of GlcNAc and NN′-diacetylchitobiose [(GlcNAc) 2, chitin structural dimeric unit] from both chitin and crab shells. The present study reports the development of an intensive ball “converge” mill for the rapid mechanochemical conversion of chitin or crab shells into amorphous, chitinase-sensitive microparticles. Optimal crab shell grinding parameters were determined, and close to 100% direct degradation of chitin from crab shell to GlcNAc was achieved. This is the first report of using a mechanochemical process with enzymatic degradation to decrease the environmental burdens associated with GlcNAc production from chitin.

Characterization of Chitin and Its Hydrolysis to GlcNAc and GlcN

Proton NMR spectra of chitin dissolved in concentrated and deuterated hydrochloric acid (DCl) were found to be a simple and powerful method for identifying chitin from samples of biological origin. During the first hour after dissolving chitin in concentrated DCl (25 degrees C), insignificant de-N-acetylation occurred, meaning that the fraction of acetylated units (FA) of chitin could be determined. FA of demineralized shrimp shell samples treated with 1 M NaOH at 95 degrees C for 1-24 h were determined and were found to decrease linearly with time from 0.96 to 0.91 during the treatment with NaOH. Extrapolation to zero time suggested that chitin from shrimp shells has a FA of 0.96, that is, contains a small but significant fraction of de-N-acetylated units. Proton NMR spectra of chitin ( FA = 0.96) dissolved in concentrated DCl were obtained as a function of time until the samples were almost quantitatively hydrolyzed to the monomer glucosamine (GlcN). The initial phase of the reaction involves mainly depolymerization of the chitin chains, resulting in that almost 90% (molar fraction) of the chitin is converted to the monomer N-acetyl-glucosamine (GlcNAc).Thus, effective conversion of chitin to GlcNAc in concentrated acid is reported for the first time. GlcNAc is then further de-N-acetylated to GlcN. A new theoretical model was developed to simulate the experimental data of the kinetics of hydrolysis of chitin in concentrated acid. The model uses three different rate constants; two for the hydrolysis of the glycosidic linkages following an N-acetylated or a de-N-acetylated sugar unit and one for the de-N-acetylation reaction. The three rate constants were estimated by fitting model data to experimental results. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher as compared to the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. Two chitin samples with different F A values (0.96 and 0.70) were incubated in concentrated DCl until the samples were converted to the maximum yield of GlcNAc and the oligomer composition analyzed, showing that the maximum yield of GlcNAc was much higher when prepared from the chitin with the highest F A value.

Towards new enzymes for biofuels: lessons from chitinase research

Enzymatic conversion of structural polysaccharides in plant biomass is a key issue in the development of second generation ('lignocellulosic') bioethanol. The efficiency of this process depends in part on the ability of enzymes to disrupt crystalline polysaccharides, thus gaining access to single polymer chains. Recently, new insights into how enzymes accomplish this have been obtained from studies on enzymatic conversion of chitin. First, chitinolytic microorganisms were shown to produce non-hydrolytic accessory proteins that increase enzyme efficiency. Second, it was shown that a processive mechanism, which is generally considered favorable because it improves substrate accessibility, might in fact slow down enzymes. These findings suggest new focal points for the development of enzyme technology for depolymerizing recalcitrant polysaccharide biomass. Improving substrate accessibility should be a key issue because this might reduce the need for using processive enzymes, which are intrinsically slow and abundantly present in current commercial enzyme preparations for biomass conversion. Furthermore, carefully selected substrate-disrupting accessory proteins or domains might provide novel tools to improve substrate accessibility and thus contribute to more efficient enzymatic processes.

A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30 degrees C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37 degrees C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Delta and the csr2Delta mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 °C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co 2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.

Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state 13C NMR

Chitin, an important constituent of the exoskeleton of many organisms such as crustacea and insects, and its derivates promote the ordered healing of tissues and are therefore very suitable for use in wound dressings. The degree of substitution (DS) is an important parameter when assessing the conversion of chitin into one of its derivates. The degree of acetylation of chitin and chitosan and the degree of butyrylation of dibutyrylchitin was evaluated. It is found that FT-IR spectroscopy is a relatively easy but indirect way of determining the DS. FT-IR spectroscopy proved to be very useful for comparing the degrees of conversion and -substitution, as well as for differentiating between different chitin types. Absolute DS determinations by FT-IR however are only reliable when a calibration, using a direct technique such as 13C-NMR, is made.

Chitosan from Mucor rouxii: production and physico-chemical characterization

Chitosan is obtained by chemical conversion of chitin, which is a constituent of the exoskeleton of crustacea and insects. An alternative source of chitosan is the cell wall of fungi. Fungal culture media and fermentation condition can be manipulated to provide chitosan of more consistent physico-chemical properties compared to that derived chemically from chitin. Chitosan has been isolated from Mucor rouxii cultured in three different media, viz., molasses salt medium (MSM), potato dextrose broth (PDB) and yeast extract peptone glucose (YPG) medium under submerged condition and their yield has been found to be the almost same, being 0.61 g/l for MSM, 0.51 g/l for PDB and 0.56 g/l for YPG respectively. Their physico-chemical properties such as ash, moisture, protein contents and specific rotation do not show much difference. However, variation has been observed in their polydispersed nature and crystallinity. Chitosan from MSM was less polydispersed and more crystalline compared to those from YPG and PDB.

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a β-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract–peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS–PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10 μg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.

Efficient preparation of β-(1→6)-(GlcNAc) 2 by enzymatic conversion of chitin and chito-oligosaccharides

A chitin-degrading marine bacterium strain, OK2607, was isolated from the sea water of Okinawa, Japan. The bacterium was identified as Alteromonas sp. The extracellular enzyme preparation obtained from the culture media of the bacterium effectively degraded chitin, and the degradation behavior was examined in detail. Unexpectedly, the major product was a β-(1→6)-linked disaccharide of N-acetylglucosamine (GlcNAc), and only small amounts of N,N'-diacetylchitobiose and GlcNAc were detected. High transglycosylation activity of the enzyme preparation was also confirmed with N-acetyl-chito-oligosaccharides giving rise to the same β-(1→6) disaccharide. The results indicate that the enzyme preparation is quite efficient to synthesize the unusual β-(1→6) oligosaccharide starting from the β-(1→6) polysaccharide or the oligosaccharides and would serve as a useful tool to prepare various β-(1→6) oligosaccharides.

Study on ecology of chitin-decomposing microorganisms in donghu lake (in wuhan, china)

对武汉东湖水柱和沉积物中几丁质分解菌的种类组成、数量分布及动态的研究结果表明:几丁质分解菌中.放线菌居于优势,在水柱中约为总数的50-72.3%;在沉积物中约为63-88.5%。经鉴定的102株放线菌中,链霉菌属(Steptomyces)占总数的70.6%;链孢囊菌属(Streptosporangium)、小单孢菌属(Micromonospora)、小多孢菌属(Micropolyspora)分别为8.8%、9.8%和10.8%。细菌中主要是芽孢杆菌属(Bacillus),占鉴定菌株数的42%,其次为沙雷氏菌属(Serratia)、气单胞菌属(Aeromonas)、微球菌属(Micrococcus)和短杆菌属(Brevibacterium)的-些菌株。在数量分布上,沉积物中高于水柱2-3个数量级;东湖Ⅰ站高于Ⅱ站。随着季节更迭,水温上升,其数量亦有相应变化。对菌株分解几丁质的能力和其它生物活性作了比较,并就所得结果进行了讨论。;Chitin is the second most abundant polysaccharide in nature. It has been estimated that 10u tons of chitin are produced annually in the aquatic biosphere in such forms as the exoskeletons of crustanceans, mollusca, coelenterates, in protozoa and in the cell walls of fungi and algae. Therefore the decomposition of chitin is an important component in the recycling of carbon and nitrogen in aquatic ecosystems. In the nature, conversion of chitin to β-1,4 N-acetylglucosamine(NAG) depends on microorganisms. In the present work, the dynamics and distribution of population, the species composition and the decomposing activity of chitin-decomposing microorganism in Donghu Lake (a eutrophic shallow lake in Wuhan, China) were studied. 1. Dynamics and distribution:During March-May, 1991, the numbers of chitin-decomposing microorganisms are higher in the Station I than in the Station I;and much higher in the sediment than in the water column (about 10²-10³). As the temperature increased from 9℃ to 25℃, the chitin-decomposing microorganisms increased with a higher ratio of actinomycetes. (Tab. l, Fig. 1). 2. Spesies composition Actinomycetes and bacteria are included in chitin-decomposing microorganisms. In 155 identified strains of microorganisms there are Streptomyces (72 strains), Streptosporangium (9 strains), Micromonospora (10 strains). Micropolyspora (11 strains), Bacillus (20 strains), (Serreatia (10 strains), Micrococcus (11 strains Aeromonas (11 strains), Brevibacterium (1 strain) and 2 strains unidentified. The Streptomyces and Bacillus are the dominant groups (45.9% and 12.7% respectively). 3. Comparison of decomposing activity of strains; Acording to the value of decomposing clear zone to colony size, the results are shown in Table 2. Worth mentioning is that there are about 55% strains which belong to actinomycetes with higher decomposing activity for chitin, the CZ/CS value over 2.

Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions

The isolation of chitosan from a fungal source offers the potential of a product with controlled physicochemical properties not obtainable by the commercial chemical conversion of crustacean chitin. A variety of culture and processing protocols using Mucor rouxii were studied for their effects on biomass yield and chitosan molecular weight. Weight-averaged molecular weight determined by gel permeation chromotography ranged from 2.0 x 10(5) to approximately 1.4 x 10(6) daltons. The chitosan yield ranged from 5% to 10% of total biomass dry weight and from 30% to 40% of the cell wall. Of the culture parameters studied, length of incubation and medium composition effected biomass production and molecular weight. Modification of the processing protocol, including the type and strength of acid, and cell wall disruption in acid prior to refluxing were used to optimize the efficiency of chitosan extraction.The degree of deacetylation of fungal and commercial chitosans was compared using infrared spectrometry, titration, and first derivative of UV absorbance spectrometry. The chitosan obtained directly from the fungal cell wall had a higher degree of deacetylation than commercial chitosan from the chemical conversion process.

ChemInform Abstract: Conversion of Xylan, Starch, and Chitin into Carboxylic Acids by Treatment with Alkali.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Conversion of xylan, starch, and chitin into carboxylic acids by treatment with alkali

Treatment of xylan, starch, and chitin with m and 3 m sodium hydroxide at 175° and 190° gave mixtures of non-volatile carboxylic acids in yields of 50–70%, 40–60%, and 5–13%, respectively, in which >60 hydroxy monocarboxylic acids and dicarboxylic acids were identified. The main compounds were glycolic, lactic, 2-hydroxybutanoic, 3-deoxypentonic, xyloisosaccharinic, and 3,4,5-trideoxyheptaric acids from xylan; glycolic, lactic, 3,4-dideoxypentonic, glucoisosaccharinic, and 3,4-dideoxyhexaric acids from starch; and glycolic, lactic, 2-hydroxybutanoic, and 3,4-dideoxyhexaric acids from chitin.

N,N-Diacetylchitobiose production from chitin by Vibrio anguillarum strain E-383a

Vibrio anguillarum strain E-383a, isolated from sea water, accumulated a considerable amount of N,N‘-diacetylchitobiose when it was cultivated in a medium containing colloidal chitin. The maximum conversion of chitin to chitobiose was found to be 40·3%. The rate of chitobiose accumulation was accelerated after the cessation of bacterial growth. Small amounts of N-acetylglucosamine and N,N’,N-triacetylchitotriose were also accumulated but no other saccharides were detected. These results may suggest that strain E-383a produces an exo-type chitinase which successively hydrolyses the glycosidic linkages of chitin into biose units. The exclusive accumulation of chitobiose by the bacterial cells may provide a new, selective method for the production of this substance.

Enzymatic potential for decomposition of detrital biopolymers in sediments from Kiel Bay

Abstract Enzymatic activities involved in the solubilization of particulate organic matter (POM) were determined in the surface samples from coastal sediments of Kiel Bay (Baltic Sea). For this purpose dye release assays were developed and standardized to measure the enzymatic conversion of dye-labelled particulate protein, cellulose, chitin, agar, and algal cell walls to soluble fragments. On the average, about half of the total POM-solubilizing activities were proteolytic. These were reduced when oxygen availability or Eh became limiting. Seasonal and spatial distribution patterns showed opposite trends for protein- and polysaccharide-solubilizing enzymes (most clearly for cellulase). Among the polysaccharases investigated, agarase activities were consistently predominant. During periods of intensive advection of macroalgae in winter they reached even the level of proteolytic activities. Intermediate levels of activity were noted for stained cell walls as a mixed natural substrate. Seasonal changes of t...