Hydrolysis Of Chitin Research Articles

Effects of Nanochitin on the Enhancement of the Grain Yield and Quality of Winter Wheat

This study investigated the effects of different rates of nanochitin in soil on the grain yield and quality of winter wheat. Nanochitin obtained by acidic hydrolysis of shrimp chitin was a rod-like whisker possessing a hydrodynamic diameter of 143 nm and ζ potential of 55.7 mV. Two varieties of winter wheat, multi-spike wheat (MSW) and large spike wheat (LSW), were treated with the nanochitin suspension in outside pot experiments. The results showed that 0.006 g kg-1 of nanochitin in soil could significantly enhance the yield by 23.0% for MSW and 33.4% for LSW, with significant increases of net photosynthesis rate, stomatal conductance, intercellular CO2 concentrations, and transpiration rate in flag leaf at the grain filling stage. Grain protein, iron, and zinc contents in wheat treated with nanochitin were also increased by 5.0, 10.3, and 22.1% for MSW and 33.4, 32.0, and 27.0% for LSW, respectively. This indicated that utilization of nanochitin has a great potential in future agriculture sustainability and crop production.

Characterization of maize chitinase-A, a tough allergenic molecule.

Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70°C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.

Intensifying chitin hydrolysis by adjunct treatments – an overview

AbstractChitin is, after cellulose, the most abundant organic natural polysaccharide on Earth, being synthesized as a dominant component in the exoskeletons of crustaceans, among other sources. In the processing of seafood for human consumption, between 40 and 50% of the total raw material mass is wasted, causing a significant problem for the environment owing to its slow degradation. Efforts to find uses for chitin derivatives, particularly their oligomers, have intensified since these chemicals are highly functional and offer a wide range of applications, especially as antimicrobial agent. As a consequence, some adjunct treatments, either chemical or physical in nature, have been employed to assist acid and enzymatic hydrolysis. This work provides a detailed review of the methods employed to intensify the formation of chitin oligomers, particularly focusing on the adjunct treatments used (microwave, ultrasonication, steam explosion and gamma irradiation), and an evaluation of the yield and characteristics of the oligomers formed. Adjunct treatments are more suitable for enzymatic hydrolysis since these treatments modify the chitin structure, and enhance the hydrolysis rate and yield of the oligomers, under milder reaction conditions. For future research, it would be worth trying pre‐treatments like the application of high‐pressure to chitin in order to lower its crystallinity. © 2017 Society of Chemical Industry

Investigation of Ionization Pattern of the Adjacent Acidic Residues in the DXDXE Motif of GH-18 Chitinases Using Theoretical pKa Calculations.

GH-18 chitinases are chitinolytic enzymes, primarily responsible for the recycling of insoluble chitin biomaterials. These enzymes contain three invariant acidic active-site residues within a DXDXE motif, which play a synergistic role in the catalytic cycle of chitin degradation. We employed a pKa calculation approach to approximate the protonation states of residues D1, D2, and E in the DXDXE motif of 75 GH-18 chitinases. Theoretical pH-activity profiles of these enzymes were subsequently constructed and compared with the experimentally determined pH-activity profiles. Theoretical pKa data indicate that in the majority of chitinases the D1 side-chain is in the "up" and the E side-chain in the "down" position, while the position of the D2 side-chain is versatile and depends on the state of the enzyme. The pKa values in 75 GH-18 chitinases were predicted to be <0 for D1, 8-13 for D2, and 6-9 for E, indicating that the D1-D2 pair holds exactly one net negative charge. On the other hand, the catalytic acid E is protonated over the active pH-range, agreeing with the pH-activity curves reported previously for most chitinases. The results obtained from this study help to elucidate the mechanistic details of the concerted participation of D1, D2, and E in the catalytic cycle of chitin hydrolysis by GH-18 chitinases.

High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors

Lipo-chitinoligosaccharides (LCOs) are key molecules for the establishment of plant-microorganisms symbiosis. Interactions of leguminous crops with nitrogen-fixing rhizobial bacteria involve Nod factors, while Myc-LCOs improve the association of most plants with arbuscular mycorrhizal fungi. Both Nod factors and Myc-LCOs are composed of a chitinoligosaccharide fatty acylated at the non-reducing end accompanied with various substituting groups. One straightforward way to access LCOs is starting from chitin hydrolysate, an abundant polysaccharide found in crustacean shells, followed by regioselective enzymatic cleavage of an acetyl group from the non-reducing end of chitin tetra- or pentaose, and subsequent chemical introduction of N-acyl group. In the present work, we describe the in vitro synthesis of LCO precursors on preparative scale. To this end, Sinorhizobium meliloti chitin deacetylase NodB was produced in high yield in E. coli as a thioredoxin fusion protein. The recombinant enzyme was expressed in soluble and catalytically active form and used as an efficient biocatalyst for N-deacetylation of chitin tetra- and pentaose.

The effect of room temperature ionic liquids on the selective biocatalytic hydrolysis of chitin via sequential or simultaneous strategies

N-Acetylglucosamine and N,N′-diacetylchitobiose are efficiently and selectively produced from chitin biomass by using RTILs and chitinases.

Chitinase from Pseudomonas fluorescens and its insecticidal activity against Helopeltis theivora.

The tea mosquito bug (TMB), Helopeltis spp. (Hemiptera: Miridae) is an insidious pest that poses a significant economical threat to tea plantations. Pseudomonas cultures are being used extensively for pest management which, however, resulting in a low mortality rate of insects and which has prompted us to search for a new microbial metabolite for TMB control. A chitinase purified from P. fluorescens and partially characterized by our group showed insecticidal activity against TMB. The mode of action behind chitinase toxicity is the enzymatic hydrolysis of chitin, which is a common constituent of the insect exoskeleton and gut lining of the peritrophic membrane. A chitinase-secreting strain MP-13 was characterized based on 16S rRNA sequencing and validated as Pseudomonas fluorescens. In the present study, purified chitinase (0.048 units/ml) enzyme from P. fluorescens MP-13 revealed 100% TMB mortality under in-vitro conditions. The results of this study can be utilized for future crop improvement programs and integrated pest management strategies.

An excellent anaerobic respiration mode for chitin degradation by Shewanella oneidensis MR-1 in microbial fuel cells

Shewanella oneidensis MR-1, a model electroactive bacterium, can oxidize a variety of fermentative organics and utilize an electrode as an electron acceptor for anaerobic respiration. Chitin as the main component of food wastes (shrimp and crab shells) is hardly degraded for bioenergy generation. This study demonstrated that S. oneidensis MR-1 could degrade chitin in microbial fuel cell (MFC). The metabolites during chitin degradation in MFC were succinate, lactate, acetate, formate, and ethanol. Their concentrations produced in MFC were higher than those in the fermentation system, as well as additional electricity could be recovered in MFC. Furthermore, the degradation of GlcNAc (the intermediate of chitin hydrolysis) by S. oneidensis MR-1 was faster than chitin in MFC and fermentation systems. Moreover, the mechanism of enhanced chitin degradation in MFC was speculated. This work might provide a new insight for biomass treatment and energy recovery by S. oneidensis MR-1 in MFC.

Chitin degradation and electricity generation by Aeromonas hydrophila in microbial fuel cells

Chitin is one of the most abundant biopolymers in nature and the main composition of shrimp and crab shells (usually as food wastes). Thus it is essential to investigate the potential of degrading chitin for energy recovery. This study investigated the anaerobic degradation of chitin by Aeromonas hydrophila, a chitinolytic and popular electroactive bacterium, in both fermentation and microbial fuel cell (MFC) systems. The primary chitin metabolites produced in MFC were succinate, lactate, acetate, formate, and ethanol. The total metabolite concentration from chitin degradation increased seven-fold in MFC compared to the fermentation system, as well as additional electricity generation. Moreover, A. hydrophila degraded GlcNAc (the intermediate of chitin hydrolysis) significantly faster (0.97 and 0.94 mM C/d/mM-GlcNAc) than chitin (0.13 and 0.03 mM C/d/mM-GlcNAc) in MFC and fermentation systems, indicating that extracellular hydrolysis of chitin was the rate-limiting step and this step could be accelerated in MFC. Furthermore, more chemicals produced by the addition of exogenous mediators in MFC. This study proves that the chitin could be degraded effectively by an electroactive bacterium in MFC, and our results suggest that this bioelectrochemical system might be useful for the degradation of recalcitrant biomass to recover energy.

Chitosan oligosaccharide: Biological activities and potential therapeutic applications.

Chitosan oligosaccharide (COS) is an oligomer of β-(1➔4)-linked d-glucosamine. COS can be prepared from the deacetylation and hydrolysis of chitin, which is commonly found in the exoskeletons of arthropods and insects and the cell walls of fungi. COS is water soluble, non-cytotoxic, readily absorbed through the intestine and mainly excreted in the urine. Of particular importance, COS and its derivatives have been demonstrated to possess several biological activities including anti-inflammation, immunostimulation, anti-tumor, anti-obesity, anti-hypertension, anti-Alzheimer's disease, tissue regeneration promotion, drug and DNA delivery enhancement, anti-microbial, anti-oxidation and calcium-absorption enhancement. The mechanisms of actions of COS have been found to involve the modulation of several important pathways including the suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) and the activation of AMP-activated protein kinase (AMPK). This review summarizes the current knowledge of the preparation methods, pharmacokinetic profiles, biological activities, potential therapeutic applications and safety profiles of COS and its derivatives. In addition, future research directions are discussed.

Giant mimivirus R707 encodes a glycogenin paralogue polymerizing glucose through α- and β-glycosidic linkages.

Acanthamoeba polyphaga mimivirus is a giant virus encoding 1262 genes among which many were previously thought to be exclusive to cellular life. For example, mimivirus genes encode enzymes involved in the biosynthesis of nucleotide sugars and putative glycosyltransferases. We identified in mimivirus a glycogenin-1 homologous gene encoded by the open reading frame R707. The R707 protein was found to be active as a polymerizing glucosyltransferase enzyme. Like glycogenin-1, R707 activity was divalent-metal-ion-dependent and relied on an intact DXD motif. In contrast with glycogenin-1, R707 was, however, not self-glucosylating. Interestingly, the product of R707 catalysis featured α1-6, β1-6 and α1-4 glycosidic linkages. Mimivirus R707 is the first reported glycosyltransferase able to catalyse the formation of both α and β linkages. Mimivirus-encoded glycans play a role in the infection of host amoebae. Co-infection of Acanthamoeba with mimivirus and amylose and chitin hydrolysate reduced the number of infected amoebae, thus supporting the importance of polysaccharide chains in the uptake of mimivirus by amoebae. The identification of a glycosyltransferase capable of forming α and β linkages underlines the peculiarity of mimivirus and enforces the concept of a host-independent glycosylation machinery in mimivirus.

Hydrolysis of chitin and chitosan in low temperature electron-beam plasma

Abstract Hydrolysis of natural chitin and chitosans was performed in the electron beam plasma (EBP) of oxygen, by means of specially designed electron beam plasmachemical reactor (EBPR). Low molecular water-soluble chitin oligosaccharides with weight-average molecular mass 800–2000 Da and polydispersion index 1.5–2.5 were produced due to action of active oxygen species formed in the EBP. By optimizing the treatment conditions the 95% yield of chitin oligosaccharides was obtained after 2 min whereas the conventional chemical hydrolysis usually takes several days. The studies of the antimicrobial activity of low molecular products formed due to EBP-stimulated degradation showed that they inhibit the multiplication of various mycelial and yeast-like fungi. The technique involved is likely to be promising for the production of bioactive low molecular chitin oligosaccharides and the EBP-stimulated hydrolysis appears to be competitive with technologies conventionally used in the industry.

The role of microorgansisms in maintaining the chitin balance in the Barents Sea

In this study, we investigated chitin hydrolysis by the bacteria inhabiting the ground of the Barents Sea. Four microbial cultures isolated from the ground were described as the genera of Rhodococcus sp., Bacillus sp., Pseudomonas sp., and Acinetobacter sp. Protein complexes with endochitinase and exochitinase activities were purified from the culture liquid. These microorganisms can participate in chitin degradation in sea water. The average molecular weight of the protein fraction with the chitinolytic activity constituted 92–135 kDa. The ratio of the endo-/exochitinase activities of the enzymatic systems was increased in the order Pseudomonas sp. < Bacillus sp. < Acinetobacter sp. < Rhodococcus sp.

Bio-inspired production of chitosan/chitin films from liquid crystalline suspensions

Inspired by chitin based hierarchical structures observed in arthropods exoskeleton, this work reports the capturing of chitin nanowhiskers’ chiral nematic order into a chitosan matrix. For this purpose, highly crystalline chitin nanowhiskers (CTNW) with spindle-like morphology and average aspect ratio of 24.9 were produced by acid hydrolysis of chitin. CTNW were uniformly dispersed at different concentrations in aqueous suspensions. The suspensions liquid crystalline phase domain was determined by rheological measurements and polarized optical microscopy (POM). Chitosan (CS) was added to the CTNW isotropic, biphasic and anisotropic suspensions and the solvent was evaporated to allow films formation. The Films’ morphologies as well as the mechanical properties were explored. A correlation between experimental results and a theoretical model, for layered matrix’ structures with fibers acting as a reinforcement agent, was established. The results evidence the existence of two different layered structures, one formed by chitosan layers induced by the presence of chitin and another formed by chitin nanowhiskers layers. By playing on the ratio chitin/chitosan one layered structure or the other can be obtained allowing the tunning of materials’ mechanical properties.

A Method to Determine Lethal and Sublethal Concentrations of Recombinant Bioinsecticides Derived from Bacillus thuringiensis Against Larvae of Spodoptera litura

The increased awareness of environmental pollution have led to the growing interests of the application of biological agents to control insects such as lepidopteran family .Thus , this study aimed to produce genetically improved bioinsecticide agents to be used to control Spodoptera litura which would be useful in reducing environmental pollution ,preventing resistance to bioinsecticides and for safe food production .Four Bacillus thuringensis strains were used as recipients ' while ' Seratia marcescens was used as a doner strain in the conjugation to induce new recombinants in Bacillus thuringiensis over expressed chitinase.Four matings were conducted in this study between Serratia marcescence and the four Bacillus thuringiensis strains which having opposite genetic markers.Two efficient transconjugants based on chitin hydrolysis on agar medium were selected from each conjugation to be evaluated for toxicites against the larvae of cotton leafworm in relation to their parents .The efficient LC50 was appeared by Tr4 ( 1.93 x105 ppm ) followed by Tr6 and Tr7 ( 2x105 ppm). However , more than 995% toxicity index was recorded by Tr4,Tr6 and Tr7. Higher toxicity index observed by these transconjugants may be the related to the overexpression of chitinase genes.Therefore , the lethal concentration values obtained was necessary to kill 50% of larval population from Spodoptera litura in a certain period .The virulence of Bt strains showed that a lower concentration from Tr4 was required to kill 100% of larval population .It must be pointed out that higher toxicity index obtained by transconjugants reflected the interaction between crystal genes from Bt and chitinase genes from Seratia marcescens which enhanced the possibility of larval mortality as a genetic helpful tool in integrated pest management in cotton fields.

X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1) Reveals Features of Its Chitin Binding Domain.

Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBDCHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBDCHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBDCHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBDCHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain.

Preparation of chitooligosaccharides from fungal waste mycelium by recombinant chitinase

This study aimed to develop an enzymatic method for conversion of chitin from fungal waste mycelia to chitooligosaccharides. The recombinant chitinase LlChi18A from Lactococcus lactis was over-expressed by Escherichia coli BL21 (DE3) and purified by affinity chromatography. The enzymatic properties of the purified enzyme were studied by chitin oligosaccharides. Waste mycelium was pre-treated by alkaline. The optimal conditions for hydrolysis of fungal chitin by recombinant chitinase were determined by Schales method. HPLC/ESI-MS was used to determine the content of N-acetylglucosamine and chitooligosaccharides after hydrolysis. The level of reducing sugar released from pretreated mycelium by chitinase increased with the reaction time during 6 days. The main product in the hydrolysates was N,N′-diacetylchitobiose. After hydrolysis by chitinase for 5 d, the yield of N,N′-diacetylchitobiose from waste mycelium was around 10% with estimated purity of around 70%. Combination of chitinase and snailase remarkably increased the yield to 24% with purity of 78%. Fungal mycelium which contains chitin is a new potential source for obtaining food grade chitooligosaccharides.

Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides

The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans.

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50–80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 μmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21–94%). The enzyme hydrolyzed 70–84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6.

We have previously reported a non-processive endo-type chitinase, ChiA, from a newly isolated marine psychrophilic bacterium, Pseudoalteromonas sp. DL-6. In this study, a processive exo-type chitinase, ChiC, was cloned from the same bacterium and characterized in detail. ChiC could hydrolyze crystalline chitin into (GlcNAc)2 as the only observed product. It exhibited high catalytic activity even at low temperatures, e.g. close to 0°C, or in the presence of 5M NaCl, suggesting that ChiC was a cold-adapted and highly salt-tolerant chitinase. ChiC could also hydrolyze other substrates, including chitosan and Avicel, indicating its broad substrate specificity. Sequence features indicated that ChiC was a multi-domain protein having a deep substrate-binding groove that was regarded as characteristic of processive exo-chitinases. Enzymatic hydrolysis of chitin by ChiC could be remarkably boosted in the presence of ChiA, suggesting the synergy of ChiC and ChiA. This work provided a new evidence to prove that marine psychrophilic bacteria utilized a synergistic enzyme system to degrade recalcitrant chitin.