Hydrolysis Of Chitin Research Articles

Assessment of Morita-Baylis-Hillman adducts as novel modulators of quorum sensing phenotypes in Chromobacterium CV026 by targeting CviR

This work presents Morita-Baylis-Hillman adducts (MBHA) as new modulators of the violacein biosynthesis in the biosensor strain Chromobacterium CV026. Seven adducts displayed violacein reduction higher than 50±1% when tested at concentrations lower than 625 μM. The most active MBHA inhibited hydrolysis of chitin concomitantly with inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. One MBHA induced the violacein biosynthesis in CV026 in the absence of the cognate AI, also supporting its role as a QS modulator of this QS system. Further, RT-qPCR and competition experiments characterize these two molecules as transcriptional modulators of the QS-regulated operon vioABCDE. Blind docking studies for MBHA showed a strong preference for adducts positioned within the CviR autoinducer-binding domain (AIBD). Our results show MBHA as a promising chemical scaffold for the development of novel QS inhibitors affecting LuxR/LuxI-systems.

Bioconversion of chitin into chitin oligosaccharides using a novel chitinase with high chitin-binding capacity

Chitin is the second largest renewable biomass resource in nature, it can be enzymatically degraded into high-value chitin oligosaccharides (CHOSs) by chitinases. In this study, a chitinase (ChiC8–1) was purified and biochemically characterized, its structure was analyzed by molecular modeling. ChiC8–1 had a molecular mass of approximately 96 kDa, exhibited its optimal activity at pH 6.0 and 50 °C. The Km and Vmax values of ChiC8–1 towards colloidal chitin were 10.17 mgmL−1 and 13.32 U/mg, respectively. Notably, ChiC8–1 showed high chitin-binding capacity, which may be related to the two chitin binding domains in the N-terminal. Based on the unique properties of ChiC8–1, a modified affinity chromatography method, which combines protein purification with chitin hydrolysis process, was developed to purify ChiC8–1 while hydrolyzing chitin. In this way, 9.36 ± 0.18 g CHOSs powder was directly obtained by hydrolyzing 10 g colloidal chitin with crude enzyme solution. The CHOSs were composed of 14.77–2.83 % GlcNAc and 85.23–97.17 % (GlcNAc)2 at different enzyme-substrate ratio. This process simplifies the tedious purification and separation steps, and may enable its potential application in the field of green production of chitin oligosaccharides.

Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage. Here, we present atomic-level insights into how the structure of the chitin-active SmLPMO10A is affected by oxidative damage using NMR and circular dichroism spectroscopy. Incubation with ascorbic acid could lead to rearrangements of aromatic residues, followed by more profound structural changes near the copper-active site and loss of activity. Longer incubation times induced changes in larger parts of the structure, indicative of progressing oxidative damage. Incubation with ascorbic acid in the presence of chitin led to similar changes in the observable (i.e., not substrate-bound) fraction of the enzyme. Upon subsequent addition of H2O2, which drastically speeds up chitin hydrolysis, NMR signals corresponding to seemingly intact SmLPMO10A reappeared, indicating dissociation of catalytically competent LPMO. Activity assays confirmed that SmLPMO10A retained catalytic activity when pre-incubated with chitin before being subjected to conditions that induce oxidative damage. Overall, this study provides structural insights into the process of oxidative damage of SmLPMO10A and demonstrates the protective effect of the substrate.

Bioproduction of Chitin Hydrolysate Containing N-Acetylglucosamine by Serratia marcescens PT6 Crude Chitinase and Its Effects on Bacterial Growth Inhibition in Various Temperature

N-acetylglucosamine (GlcNAc), a chitin monomer, can be used as a natural preservative to ensure food quality and safety. Combining natural preservatives with low storage temperature offers physical hurdles to bacterial growth in food. This study aimed to produce chitin hydrolysate containing GlcNAc using Serratia marcescens PT6 crude chitinase and investigate its effect on bacterial growth rate as a function of temperature. Crude chitinase from partial purification was used to hydrolyze 1.3% colloidal chitin. The optimal enzymatic conditions were pH 6 and 45˚C for 120 min, at an enzyme:substrate ratio of 1:1, yielding a 65.6 µg/mL GlcNAc. Inhibitory activity of hydrolysate containing 2.5-7.5 ppm GlcNAc on Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus was measured at 4, 15, and 30oC in nutrient broth. Bacterial growth was measured using of optical density for each combination of GlcNAc concentration and temperature. Growth curves fitted by the Baranyi and Roberts model were developed using DMFit software. The growth rate was converted to the square root and then modeled as a function of temperature using the Ratkowsky square root model. Incubation temperature exerted a pronounced effect on the inhibition of all bacterial species (P<0.0001), with the greatest effect observed for E. coli at 30°C (P<0.0001), and the least effect for V. parahaemolyticus (P=0.0878). The inhibitory effect of GlcNAc in chitin hydrolysate was only significant for E. coli (P<0.0001) and S. aureus (P=0.0041). This study revealed that the effect of temperature in growth inhibition was more significant than GlcNAc addition. However, a reduction in bacterial growth with the addition of GlcNAc at 30°C was observed, which may be effective for food encountered thermal abuse conditions. Further investigation of the effect of GlcNAc on bacteria structure and metabolism is required to elucidate the mechanism of GlcNAc as a food preservative.

Family 92 carbohydrate-binding modules specific for β-1,6-glucans increase the thermostability of a bacterial chitinase

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10–15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

Simultaneous Preparation of Chitin and Flavor Protein Hydrolysates from the By-Products of Shrimp Processing by One-Step Fermentation with Lactobacillus fermuntum.

One-step fermentation, inoculated with Lactobacillus fermentum (L. fermentum) in shrimp by-products, was carried out to obtain chitin and flavor protein hydrolysates at the same time. The fermentation conditions were optimized using response surface methodology, resulting in chitin with a demineralization rate of 89.48%, a deproteinization rate of 85.11%, and a chitin yield of 16.3%. The surface of chitin after fermentation was shown to be not dense, and there were a lot of pores. According to Fourier transform infrared spectroscopy and X-ray diffraction patterns, the fermented chitin belonged to α-chitin. More than 60 volatiles were identified from the fermentation broth after chitin extraction using gas chromatography-ion transfer spectrometry analysis. L. fermentum fermentation decreased the intensities of volatile compounds related to unsaturated fatty acid oxidation or amino acid deamination. By contrast, much more pleasant flavors related to fruity and roasted aroma were all enhanced in the fermentation broth. Our results suggest an efficient one-step fermentation technique to recover chitin and to increase aroma and flavor constituents from shrimp by-products.

Recombinant production, characterization and industrial application testing of a novel acidic exo/endo-chitinase from Rasamsonia emersonii.

An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1kDa, which sharpens into bands at 46.0kDa, 48.4kDa and a smear above 60kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50°C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.

Establishment of the colloidal chitin enzymatic hydrolysis conditions to obtain N-acetyl glucosamine

The wastes generated by the shrimp industry are approximately between 50 and 60% of the catch volume. These residues such as head, viscera, and shell are potential pollutants if they are not treated for proper disposal. One way to solve this problem is to use the residues as functional food ingredients. In this regard, shrimp residues are rich in chitin, the second most abundant biopolymer on the planet after cellulose. Chitin is composed of N-acetyl glucosamine, a molecule used as a sweetener in the food industry and as an aid in the treatment of coronary diseases and gonarthrosis. N-acetyl glucosamine can be obtained by the hydrolysis of colloidal chitin using chemical or enzymatic methods; however, chemical methods are associated with pollution. In this study, we determined the hydrolysis conditions of shrimp colloidal chitin for obtaining N-acetyl glucosamine, using the extracellular enzymes produced by a marine bacterium isolated in the coastal zone of Progreso, Yucatan, Mexico. The best N-acetyl glucosamine yield obtained was 2.65%, using 10 mg/mL colloidal chitin, at 60°C, and pH 8.9 with 3.5% NaCl.

Functional genomics gives new insights into the ectomycorrhizal degradation of chitin.

Ectomycorrhizal (EcM) fungi play a crucial role in the mineral nitrogen (N) nutrition of their host trees. While it has been proposed that several EcM species also mobilize organic N, studies reporting the EcM ability to degrade N-containing polymers, such as chitin, remain scarce. Here, we assessed the capacity of a representative collection of 16 EcM species to acquire 15 N from 15 N-chitin. In addition, we combined genomics and transcriptomics to identify pathways involved in exogenous chitin degradation between these fungal strains. Boletus edulis, Imleria badia, Suillus luteus, and Hebeloma cylindrosporum efficiently mobilized N from exogenous chitin. EcM genomes primarily contained genes encoding for the direct hydrolysis of chitin. Further, we found a significant relationship between the capacity of EcM fungi to assimilate organic N from chitin and their genomic and transcriptomic potentials for chitin degradation. These findings demonstrate that certain EcM fungal species depolymerize chitin using hydrolytic mechanisms and that endochitinases, but not exochitinases, represent the enzymatic bottleneck of chitin degradation. Finally, this study shows that the degradation of exogenous chitin by EcM fungi might be a key functional trait of nutrient cycling in forests dominated by EcM fungi.

Chitin Hydrolysis Using Zeolites in Lithium Bromide Molten Salt Hydrate

N-Acetyl-d-glucosamine (NAG), the monomer of chitin, is used as a food supplement and a key platform chemical to access a range of organonitrogen chemicals. However, efficient and environmentally benign catalytic systems to depolymerize raw chitin into monomers are still limited. In this regard, here we report hydrolysis of untreated chitin using low-cost and recyclable zeolites in LiBr molten salt hydrate (MSH). The chabazite (CHA) zeolites (silicoaluminophosphate SAPO-34 and aluminosilicate SSZ-13) exhibited superior performance compared with other zeolites (e.g., Hβ, HZSM-5, and HUSY). Under optimized conditions, nanosheet-like SAPO-34 crystals (NS-0.1) and commercial microporous SAPO-34 achieved 61 and 63% NAG yield, respectively. H+ in zeolites exchanges with Li+ in LiBr MSH, which then promotes chitin hydrolysis. Thus, the formation rate of NAG was not dependent on the textural properties of zeolites (e.g., pore size, surface area, pore volume) but rather correlated to the acid site properties. In particular, a strong negative correlation between Lewis acid density and NAG yield was observed. The work establishes a pathway to hydrolyze chitin using solid heterogeneous catalysts.

King crab gills as a new source of chitin/chitosan and protein hydrolysates

This is the first report on the physicochemical properties of chitin obtained from gills of the king crab Paralithodes camtschaticus. In the present study, we investigated the chemical composition of red king crab gills and considered methods of its complex processing to obtain chitin and enzymatic protein hydrolysates. The gills contained approximately 21 % chitin in terms of dry matter. For the first time, the gills of the king crab were investigated as a source of chitin and chitosan.Chitin was isolated from crab gills using chemical and enzymatic methods. The physicochemical properties of chitin and chitosan from the gills were investigated. By performing infrared spectroscopy and X-ray phase analyses, the chitin present in the gills was established to be α-chitin. The physical and chemical properties (degree of deacetylation, molecular weight and crystal structure) of gill chitin and chitosan were absolutely similar to those of crab shell.Crab gills can be used as an additional source of chitin in the integrated processing of king crabs. The yield of chitin from the gills is up to 45 % of the yield of chitin from the crab carapace.

Acid hydrolysis of chitin in calcium chloride solutions

This work describes a mild aging-hydrolysis integrated approach to selectively produceN-acetyl-d-glucosamine from chitin in cheap and abundant CaCl2-based molten salt hydrate solvents.

α-Alkylidene δ-lactones inhibit quorum sensing phenotypes in Chromobacterium strain CV026 showing interaction with the CviR receptor.

Disruption of bacterial quorum sensing (QS) is presented as a promising strategy to overcome clinically relevant and phytopathogenic bacteria. This work presents α-alkylidene δ-lactones as new chemical scaffolds that inhibit the biosynthesis of violacein in the biosensor strain Chromobacterium CV026. Three molecules displayed higher than 50% violacein reduction when tested at concentrations lower than 625 µM. The most active α-alkylidene δ-lactone inhibited the hydrolysis of chitin concomitantly with the inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. Further, RT-qPCR and competition experiments showed this molecule to be a transcriptional inhibitor of the QS-regulated operon vioABCDE. Docking calculations suggested a good correlation between binding affinity energies and inhibition effects, with all molecules positioned within the CviR autoinducer-binding domain (AIBD). The most active lactone yielded the best binding affinity energy, most probably due to its unprecedented binding with the AIBD. Our results show α-alkylidene δ-lactones as promising chemical scaffolds for the development of new QS inhibitors affecting LuxR/LuxI-systems.

Titelbild: Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst (Angew. Chem. 3/2023)

Titelbild: Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst (Angew. Chem. 3/2023)

Cover Picture: Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst (Angew. Chem. Int. Ed. 3/2023)

Cover Picture: Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst (Angew. Chem. Int. Ed. 3/2023)

Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon‐Based Catalyst

AbstractOligosaccharides possess fascinating functions that are applicable in a variety of fields, such as agriculture. However, the selective synthesis of oligosaccharides, especially chitin‐oligosaccharides, has remained a challenge. Chitin‐oligosaccharides activate the plant immune system, enabling crops to withstand pathogens without harmful agrichemicals. Here, we demonstrate the conversion of chitin to chitin‐oligosaccharides using a carbon catalyst with weak acid sites and mechanical milling. The catalyst produces chitin‐oligosaccharides with up to 94 % selectivity in good yields. Monte‐Carlo simulations indicate that our system preferentially hydrolyzes larger chitin molecules over oligomers, thus providing the desired high selectivity. This unique kinetics is in contrast to the fact that typical catalytic systems rapidly hydrolyze oligomers to monomers. Unlike other materials carbons more strongly adsorb large polysaccharides than small oligomers, which is suitable for the selective synthesis of small oligosaccharides.

Selective Synthesis of Oligosaccharides by Mechanochemical Hydrolysis of Chitin over a Carbon-Based Catalyst.

Oligosaccharides possess fascinating functions that are applicable in a variety of fields, such as agriculture. However, the selective synthesis of oligosaccharides, especially chitin-oligosaccharides, has remained a challenge. Chitin-oligosaccharides activate the plant immune system, enabling crops to withstand pathogens without harmful agrichemicals. Here, we demonstrate the conversion of chitin to chitin-oligosaccharides using a carbon catalyst with weak acid sites and mechanical milling. The catalyst produces chitin-oligosaccharides with up to 94 % selectivity in good yields. Monte-Carlo simulations indicate that our system preferentially hydrolyzes larger chitin molecules over oligomers, thus providing the desired high selectivity. This unique kinetics is in contrast to the fact that typical catalytic systems rapidly hydrolyze oligomers to monomers. Unlike other materials carbons more strongly adsorb large polysaccharides than small oligomers, which is suitable for the selective synthesis of small oligosaccharides.

Synergic chitin degradation by Streptomyces sp. SCUT-3 chitinases and their applications in chitinous waste recycling and pathogenic fungi biocontrol

The genus Streptomyces comprises the most important chitin decomposers in soil and revealing their chitinolytic machinery is beneficial for the conversion of chitinous wastes. Streptomyces sp. SCUT-3, a chitin-hydrolyzing and a robust feather-degrading bacterium, was isolated previously. The potential chitin-degrading enzymes produced by SCUT-3 were analyzed in the present study. Among these enzymes, three chitinases were successfully expressed in Pichia pastoris at comparatively high yields of 4.8 U/mL (SsExoChi18A), 11.2 U/mL (SsExoChi18B), and 17.8 U/mL (SsEndoChi19). Conserved motifs and constructive 3D structures of these three exo- and endochitinases were also analyzed. These chitinases hydrolyzed colloidal chitin to chitin oligomers. SsExoChi18A showed apparent synergic effects with SsEndoChi19 in colloidal chitin and shrimp shell hydrolysis, with an improvement of 29.3 % and 124.9 %, respectively. Compared with SsExoChi18B and SsEndoChi19, SsExoChi18A exhibited the strongest antifungal effects against four plant pathogens by inhibiting mycelial growth and spore germination. This study provided good candidates for chitinous waste-processing enzymes and antifungal biocontrol agents. These synergic chitin-degrading enzymes of SCUT-3 are good targets for its further genetical modification to construct super chitinous waste-degrading bacteria with strong abilities to hydrolyze both protein and chitin, thereby providing a direction for the future path of the chitinous waste recycling industry.

Genome-wide characterization of the chitinase gene family in wild apple (Malus sieversii) and domesticated apple (Malus domestica) reveals its role in resistance to Valsa mali.

Chitinases are responsible for catalyzing the hydrolysis of chitin and contribute to plant defense against fungal pathogens by degrading fungal chitin. In this study, genome-wide identification of the chitinase gene family of wild apple (Malus sieversii) and domesticated apple (Malus domestica) was conducted, and the expression profile was analyzed in response to Valsa mali infection. A total of 36 and 47 chitinase genes belonging to the glycosyl hydrolase 18 (GH18) and 19 (GH19) families were identified in the genomes of M. sieversii and M. domestica, respectively. These genes were classified into five classes based on their phylogenetic relationships and conserved catalytic domains. The genes were randomly distributed on the chromosomes and exhibited expansion by tandem and segmental duplication. Eight of the 36 MsChi genes and 17 of the 47 MdChi genes were differentially expressed in response to V. mali inoculation. In particular, MsChi35 and its ortholog MdChi41, a class IV chitinase, were constitutively expressed at high levels in M. sieversii and domesticated apple, respectively, and may play a crucial role in the defense response against V. mali. These results improve knowledge of the chitinase gene family in apple species and provide a foundation for further studies of fungal disease prevention in apple.

Screening of chitin degrading bacteria from the gut of Asian common toad Duttaphrynus melanostictus: Implication for valorization of chitin rich seafood waste

Chitin is a structural component of many invertebrates and the second most abundant biopolymer after cellulose. Owing to its high insolubility, waste management of chitin rich seafood has severe environmental consequences. Despite the value added bioconversion of waste chitin into commodity products like reducing sugars, its biodegradation by bacteria is still lacking. To this end, we have isolated and characterized chitin degrading bacteria from the gut of common Asian toad, Duttaphrynus melanostictus . The isolates, FG4 and ND3 demonstrated highest chitin degradation activities of 1.44 ± 0.129 U/mL and 1.269 ± 0.138 U/mL respectively. Molecular phylogeny using 16S rRNA gene sequence affiliated FG4 to Stenotrophomonas maltophila and ND3 to Bacillus paramycoides , respectively. Among the tested substrates, highest chitinase activities of 4.210 ± 0.070 U/mL and 1.724 ± 0.494 U/mL were exhibited by S. maltophila FG4 on prawn shells (PS) and chitin flakes (CF), respectively. Similarly, the highest substrate degradation ratio of prawn shells and chitin flakes were 49.52% and 38.15% by S. maltophila FG4. Furthermore, scanning electron microscopy revealed adhesion of bacteria and creation of pores on the substrates. The Fourier transform infrared spectroscopy (FTIR) and X - ray diffraction analysis revealed hydrolysis of prawn shells and chitin flakes by bacteria. Further, the high-resolution mass spectrometric (HRMS) analysis confirmed production of N-acetyl D glucosamine by S. maltophila FG4 on prawn shells. Thus, the present study identifies D. melanostictus gut as a unique reservoir to isolate chitinolytic bacteria that may have potential industrial and therapeutic applications. • First report of bioprospection of amphibian guts for bacteria capable of chitin hydrolysis. • Gut chitinolytic isolates could utilize sea food wastes as sole carbon source. • Applications in production of valuable byproducts such as N-acetyl-D-glucosamine.