Chitin Substrates Research Articles

Integrating Cu2O Colloidal Mie Resonators in Structurally Colored Butterfly Wings for Bio-Nanohybrid Photonic Applications.

Colloidal Cu2O nanoparticles can exhibit both photocatalytic activity under visible light illumination and resonant Mie scattering, but, for their practical application, they have to be immobilized on a substrate. Butterfly wings, with complex hierarchical photonic nanoarchitectures, constitute a promising substrate for the immobilization of nanoparticles and for the tuning of their optical properties. The native wax layer covering the wing scales of Polyommatus icarus butterflies was removed by simple ethanol pretreatment prior to the deposition of Cu2O nanoparticles, which allowed reproducible deposition on the dorsal blue wing scale nanoarchitectures via drop casting. The samples were investigated by optical and electron microscopy, attenuated total reflectance infrared spectroscopy, UV-visible spectrophotometry, microspectrophotometry, and hyperspectral spectrophotometry. It was found that the Cu2O nanoparticles integrated well into the photonic nanoarchitecture of the P. icarus wing scales, they exhibited Mie resonance on the glass slides, and the spectral signature of this resonance was absent on Si(100). A novel bio-nanohybrid photonic nanoarchitecture was produced in which the spectral properties of the butterfly wings were tuned by the Cu2O nanoparticles and their backscattering due to the Mie resonance was suppressed despite the low refractive index of the chitinous substrate.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

A new strain of Rhodococcus indonesiensis T22.7.1T and its functional potential for deacetylation of chitin and chitooligsaccharides.

Chitin, abundant in marine environments, presents significant challenges in terms of transformation and utilization. A strain, T22.7.1T, with notable chitin deacetylation capabilities, was isolated from the rhizosphere of Acanthus ebracteatus in the North Sea of China. Comparative 16S rDNA sequence analysis showed that the new isolate had the highest sequence similarity (99.79%) with Rhodococcus indonesiensis CSLK01-03T, followed by R. ruber DSM 43338T, R. electrodiphilus JC435T, and R. aetherivorans 10bc312T (98.97%, 98.81%, and 98.83%, respectively). Subsequent genome sequencing and phylogenetic analysis confirmed that strain T22.7.1T belongs to the R. indonesiensis species. However, additional taxonomic characterization identified strain T22.7.1T as a novel type strain of R. indonesiensis distinct from CSLK01-03T. This study refines the taxonomic description of R. indonesiensis and investigates its application in converting chitin into chitosan. The chitin deacetylase (RiCDA) activity of strain T22.7.1T was optimized, and the enzyme was isolated and purified from the fermentation products. Through optimization, the RiCDA activity of strain T22.7.1T reached 287.02 U/mL, which is 34.88 times greater than the original enzyme's activity (8.0 U/mL). The natural CDA enzyme was purified with a purification factor of 31.83, and the specific activity of the enzyme solution reached 1200.33 U/mg. RiCDA exhibited good pH and temperature adaptability and stability, along with a wide range of substrate adaptabilities, effectively deacetylating chitin, chitooligosaccharides, N-acetylglucosamine, and other substrates. Product analysis revealed that RiCDA treatment increased the deacetylation degree (DD) of natural chitin to 83%, surpassing that of commercial chitosan. Therefore, RiCDA demonstrates significant potential as an efficient deacetylation tool for natural chitin and chitooligosaccharides, highlighting its applicability in the biorefining of natural polysaccharides.

Tunable dye adsorbing materials from crab and shrimp waste shells for water remediation

Crab and shrimp shells are waste byproducts from shellfish farming and processing. In this study, we demonstrated that it is possible to obtain individual shell components either separately or in combinations. These diverse materials were composed of calcium carbonate and organic molecules inter- and intra-mineral, calcium carbonate and organic molecules intra-mineral, organic molecules such as chitin and proteins, only calcium carbonate, and only chitin. These substrates were tested for the adsorption of Methylene Blue and Eosin dyes. The goal is to demonstrate that the organic dye adsorption capacity can be modified by controlling the degree of deconstruction of the starting material. The best adsorption for the positively charged Methylene Blue was achieved on the chitin-calcium carbonate substrates, while the negatively charged Eosin adsorbed best on the chitin substrate. In conclusion, this research demonstrates that it is possible to obtain different materials by deconstructing the shells from crabs and shrimps at various levels reducing the generation of additional wastes. Moreover, these materials exhibit features that are related to the original material and possess new properties that can be also exploited in other fields like material sciences.

Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils

BackgroundMicrobial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp.ResultsFive heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%.ConclusionThis direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

Internal Transcribed Spacers (ITS) Based Identification of Trichoderma Isolates and Biocontrol Activity against Fusarium oxysporum and Chitinase Activity

Twelve Trichoderma spp. isolated from different locations in Uttar Pradesh, India. Internal transcribed spacer (ITS) amplification and sequencing were used to identify isolates of Trichoderma that showed 99–100% identification with Trichoderma harzianum, Trichoderma asperellum, and Trichoderma longibrachiatum, the three species of Trichoderma. Tests were conducted in Vitro to evaluate the biocontrol potential of Trichoderma isolates against Fusarium species. After seven days of incubation, the isolate TBT6 (T. harzianum) exhibited the greatest antagonism against Fusarium oxysporum, with inhibition rates of 87.1%) by TBT7 (82.2%), and the least amount of inhibition (59.7%) by T. longibrachiatum isolate TBT10. Under field conditions, the antagonistic T. harzianum isolate TBT6 can be employed as a bio-control agent against Fusarium oxysporum. and for the development of formulations based on Trichoderma. Using the dual plate approach, twelve highly antagonistic Trichoderma isolates were selected for chitinolytic activity. Observing the breakdown of chitin substrates, Trichoderma isolates TBT6 was shown to have strong chitinolytic enzyme activity, making it a good candidate for endochitinase gene isolation. Using two particular primers, the genomic DNA of the Trichoderma isolate TBT6 was isolated and amplified.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 μmole min−1 and a Km of 4.6 mg mL−1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg−1 and 625 U mg−1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

Transmission Electronic Microscopy of Vibrio cholerae Biofilms on Chitin-Containing Substrates

Introduction. The evolutionary association of Vibrio cholerae with chitin provided resistance to stress and protection from predators. The most important mechanism that provided V. cholerae with the effectiveness of association with chitin is biofilm formation. The ability to form a biofilm in V. cholerae depends on the presence of a factor, toxin-corrected adhesion pili (TCP), which are synthesized by the tcp A-F genes. One of the key methods for studying biofilms is microscopy. It allows one to visualize the structural elements and study various parameters of biofilms and the effects of various factors on them. Aim. To determine the epidemiological significance of the biofilm-forming ability of toxigenic strains by their morphological characteristics on chitin-containing substrates. Study of structural differences in biofilms of Vibrio cholerae tcpA+– and tcpA– strains on chitin-containing substrates. Results. It has been shown that Vibrio cholerae tcpA+– and tcpA– strains are able to form biofilms on the surface of chitin-containing substrates. The intensity of biofilm formation is more pronounced in tcpA+ strains, because V. cholerae ctxA+ tcpA+ cells in the biofilm are predominantly singly located and the surface of the chitinous exoskeleton with which they are in contact is intact, V. cholerae ctxA– tcpA– cells form chains in the biofilm, which indicates division processes, and scattered chitin of the endocuticle indicates activity of metabolic processes. Conclusion. The strains of V. cholerae used in the work, regardless of the presence or absence of the ctx and tcp genes, form bioplecs on a chitin substrate. The indicator of biofilm formation in terms of the thickness of the biofilm matrix is higher in V. cholerae ctxA+ tcpA+ , in terms of the degree of degradation of the chitin substrate it is higher in V. cholerae ctxA– tcpA– .

Engineering the Relatively Conserved Residues in Active Site Architecture of Thermophilic Chitinase SsChi18A Enhanced Catalytic Activity.

Chitinase plays a vital role in the efficient biotransformation of the chitin substrate. This study aimed to modify and elucidate the contribution of the relatively conserved residues in the active site architecture of a thermophilic chitinase SsChi18A from Streptomyces sp. F-3 in processive catalysis. The enzymatic activity on colloidal chitin increased to 151%, 135%, and 129% in variants Y286W, E287A, and K186A compared with the wild type (WT). Also, the apparent processive parameter G2/G1 was lower in the variants compared to the WT, indicating the essential role of Tyr-286, Glu-287, and Lys-186 in processive catalysis. Additionally, the enzymatic activity on the crystalline chitin of F48W and double mutants F48W/Y209F and F48W/Y286W increased by 35%, 16%, and 36% compared with that for WT. Molecular dynamics simulations revealed that the driving force of processive catalysis might be related to the changes in interaction energy. This study provided a rational design strategy targeting relatively conserved residues to enhance the catalytic activity of GH18 processive chitinases.

Deacetylation of chitin oligomers by Fusarium graminearum polysaccharide deacetylase suppresses plant immunity.

Chitin is a long-chain polymer of β-1,4-linked N-acetylglucosamine that forms rigid microfibrils to maintain the hyphal form and protect it from host attacks. Chitin oligomers are first recognized by the plant receptors in the apoplast region, priming the plant's immune system. Here, seven polysaccharide deacetylases (PDAs) were identified and their activities on chitin substrates were investigated via systematic characterization of the PDA family from Fusarium graminearum. Among these PDAs, FgPDA5 was identified as an important virulence factor and was specifically expressed during pathogenesis. ΔFgpda5 compromised the pathogen's ability to infect wheat. The polysaccharide deacetylase structure of FgPDA5 is essential for the pathogenicity of F. graminearum. FgPDA5 formed a homodimer and accumulated in the plant apoplast. In addition, FgPDA5 showed a high affinity toward chitin substrates. FgPDA5-mediated deacetylation of chitin oligomers prevented activation of plant defence responses. Overall, our results identify FgPDA5 as a polysaccharide deacetylase that can prevent chitin-triggered host immunity in plant apoplast through deacetylation of chitin oligomers.

Degradation Patterns of Chitin Substrates and Reaction Production Obtained from Paenibacillus lautus JS-1 Using Different Culture Media

Degradation Patterns of Chitin Substrates and Reaction Production Obtained from Paenibacillus lautus JS-1 Using Different Culture Media

Isolation and screening of a chitin deacetylase producing Bacillus cereus and its potential for chitosan preparation.

Chitosan is a biopolymer material extracted from marine biomass waste such as shrimp and crab shells, which has good biocompatibility and degradability with great potential for application in the field of wastewater treatment and soil remediation. The higher the degree of deacetylation (DD), the better the adsorption performance of chitosan. Chitin deacetylase (CDA) can specifically catalyze the deacetylate of chitin in a green reaction that is environmentally friendly. However, the scarcity of high yielding chitin deacetylase strains has been regarded as the technical bottleneck of chitosan green production. Here, we screened a natural chitin degrading bacterium from coastal mud and identified it as Bacillus cereus ZWT-08 by re-screening the chitin deacetylase activity and degree of deacetylation values. By optimizing the medium conditions and enzyme production process, ZWT-08 was cultured in fermentation medium with 1% (m/V) glucose and yeast extract at pH 6.0, 37°C, and a stirring speed of 180 r/min. After fermenting in 5L fermenter for 48 h, the deacetylation activity of the supernatant reached 613.25 U/mL. Electron microscopic examination of the chitin substrate in the fermentation medium revealed a marshmallow-like fluffy texture on its structural surface. Meanwhile, 89.29% of the acetyl groups in this chitin substrate were removed by enzymatic digestion of chitin deacetylase produced by ZWT-08, resulting in the preparation of chitosan a degree of deacetylation higher than 90%. As an effective strain for chitosan production, Bacillus cereus ZWT-08 plays a positive role in the bioconversion of chitin and the upgrading of the chitosan industry.

Conservation of cholera vibrios in complex microcosm containing green microalgae

BACKGROUND: In the summer-autumn period, the peak of phytoplankton reproduction and flowering occurs and results in the deterioration of the quality of river water is noted. At the same time, cases of gastrointestinal diseases in humans are recorded, and epidemic complications of cholera may occur in endemic areas. V. cholerae form biofilm on the surface of chitinous hydrobionts and plastic components can lead to the spread of V. cholerae, possibly explaining the autochthonous mechanism of their existence in water bodies.
 AIM: This study determines the duration of Vibrio cholerae preservation on biotic (chitin) and abiotic (plastic) substrates in the presence of green unicellular algae with a change in cultivation temperature under experimental conditions.
 MATERIALS AND METHODS: This study used bacteriological and molecular genetic methods to achieve its goal.
 RESULTS: The preservation of toxigenic and non-toxigenic strains of V. cholerae O1 El Tor and V. cholerae O139 (ctxАВ+tcpА+csh1 and ctxАtcpАcsh1+) for six months, including three months at a low temperature simulating the autumn-winter period, as part of biofilms in microcosms, where one component is green microalgae. We noted an excess of the concentration of V. cholerae O1 and O139 serogroups by two orders of magnitude in samples where one of the components is chitin and the reproduction of green microalgae in the presence of a chitin substrate. This is probably one stage in the food chain in the ecology of water bodies and, accordingly, can be a reservoir for biofilm forms of cholera vibrios. Preservation of V. cholerae O1 El Tor non-toxigenic strain in a viable state in biofilm samples on plastic at decreased temperatures 82C for three months is possibly due to the presence of a cold shock gene in its genome.
 CONCLUSION: Without a substrate for adhesion, colonization, and biofilm formation, vibrios are incapable of long-term persistence at low temperatures.

The potential of degrading natural chitinous wastes to oligosaccharides by chitinolytic enzymes from two Talaromyces sp. isolated from rotten insects (Hermetia illucens) under solid state fermentation.

It is difficult to produce chitin oligosaccharides by hydrolyzing untreated natural chitinous waste directly. In this study, two fungi Talaromyces allahabadensis Hi-4 and Talaromyces funiculosus Hi-5 from rotten black soldier fly were isolated and identified through multigene phylogenetic and morphological analyses. The chitinolytic enzymes were produced by solid state fermentation, and the growth conditions were optimized by combining single-factor and central composite design. The best carbon sources were powder of molting of mealworms (MMP) and there was no need for additional nitrogen sources in two fungi, then the maximum chitinolytic enzyme production of 46.80 ± 3.30 (Hi-4) and 55.07 ± 2.48 (Hi-5) U/gds were achieved after analyzing the 3D response surface plots. Pure chitin (colloidal chitin) and natural chitinous substrates (represented by MMP) were used to optimize degradation abilities by crude enzymes obtained from the two fungi. The optimum temperature for hydrolyzing MMP (40°C both in two fungi) were lower and closer to room temperature than colloidal chitin (55°C for Hi-4 and 45°C for Hi-5). Then colloidal chitin, MMP and the powder of shrimp shells (SSP) were used for analyzing the products after 5-day degradation. The amounts of chitin oligosaccharides from SSP and MMP were about 1/6 (Hi-4), 1/17 (Hi-5) and 1/8 (Hi-4), 1/10 (Hi-5), respectively, in comparison to colloidal chitin. The main components of the products were GlcNAc for colloidal chitin, (GlcNAc)2 for MMP, and oligosaccharides with higher degree of polymerization (4-6) were obtained when hydrolyzing SSP, which is significant for applications in medicine and health products.

Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

Structure-Function Insights into the Fungal Endo-Chitinase Chit33 Depict its Mechanism on Chitinous Material.

Chitin is the most widespread amino renewable carbohydrate polymer in nature and the second most abundant polysaccharide. Therefore, chitin and chitinolytic enzymes are becoming more importance for biotechnological applications in food, health and agricultural fields, the design of effective enzymes being a paramount issue. We report the crystal structure of the plant-type endo-chitinase Chit33 from Trichoderma harzianum and its D165A/E167A-Chit33-(NAG)4 complex, which showed an extended catalytic cleft with six binding subsites lined with many polar interactions. The major trait of Chit33 is the location of the non-conserved Asp117 and Arg274 acting as a clamp, fixing the distorted conformation of the sugar at subsite –1 and the bent shape of the substrate, which occupies the full catalytic groove. Relevant residues were selected for mutagenesis experiments, the variants being biochemically characterized through their hydrolytic activity against colloidal chitin and other polymeric substrates with different molecular weights and deacetylation percentages. The mutant S118Y stands out, showing a superior performance in all the substrates tested, as well as detectable transglycosylation capacity, with this variant providing a promising platform for generation of novel Chit33 variants with adjusted performance by further design of rational mutants’. The putative role of Tyr in binding was extrapolated from molecular dynamics simulation.

Probing the Structural Details of Chitin Nanocrystal–Water Interfaces by Three‐Dimensional Atomic Force Microscopy

Chitin is one of the most abundant and renewable natural biopolymers. It exists in the form of crystalline microfibrils and is the basic structural building block of many biological materials. Its surface crystalline structure is yet to be reported at the molecular level. Herein, atomic force microscopy (AFM) in combination with molecular dynamics simulations reveals the molecular-scale structural details of the chitin nanocrystal (chitin NC)-water interface. High-resolution AFM images reveal the molecular details of chitin chain arrangements at the surfaces of individual chitin NCs, showing highly ordered, stable crystalline structures almost free of structural defects or disorder. 3D-AFM measurements with submolecular spatial resolution demonstrate that chitin NC surfaces interact strongly with interfacial water molecules creating stable, well-ordered hydration layers. Inhomogeneous encapsulation of the underlying chitin substrate by these hydration layers reflects the chitin NCs' multifaceted surface character with different chain arrangements and molecular packing. These findings provide important insights into chitin NC structures at the molecular level, which is critical for developing the properties of chitin-based nanomaterials. Furthermore, these results will contribute to a better understanding of the chemical and enzymatic hydrolysis of chitin and other native polysaccharides, which is also essential for the enzymatic conversion of biomass.

Expression, purification, and basic properties of a novel domain structure possessing chitinase from Escherichia coli carrying the family 18 chitinase gene of Bacillus velezensis strain RB.IBE29.

Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.

Enhancing nonspecific enzymatic hydrolysis of chitin to oligosaccharides pretreated by acid and green solvents under simultaneous microwave-radiation

It is hard to degrade untreated highly crystalline chitin. In this study, two solvents pretreatment chitin (acid swollen chitin (AC), super fine chitin (FC)) and microwave-heating method were used to enhance nonspecific enzymatic hydrolysis (lysozyme and pepsin), which obviously improved the enzymolysis rates by at least 1.31 times. Characterizations of chitin substrates (Mv, SEM, XRD) showed that calcium solvent pretreatment (obtained FC) was milder but effective than phosphoric acid pretreatment (obtained AC). The highest yield of chitin oligosaccharides (37.58 mg/g) were obtained after hydrolyzing AC under five-hour simultaneous microwave radiation by pepsin, among them, the content of N-acetylglucosamine was 13.76 mg/g. While, more chitin oligosaccharides with DP (degree of polymerization) 3–4 and lower DA (degree of acetylation) were obtained when using lysozyme than pepsin. Significantly, the conversion rate of chitin to oligosaccharides went best only when microwave and enzymes acting together (simultaneous strategy), which were at least 35.59% higher than separately pretreatment enzymes and substrates by microwave. The damages of microwave radiation on lysozyme and chitin substrates were revealed, and the operating principle of the whole enzyme reaction system heated by microwave was preliminatively explored.

Screening of Trichoderma spp. isolates based on antagonism and chitinolytic index against Xylaria sp.

Sugarcane disease disturbance is one of the limiting factors in achieving high productivity. Xylaria sp. has become endemic to sugarcane plantations in Lampung and South Sumatra. The intensity of disease attacks in Indonesia by 25% and 26% can reduce sugar production 12.3 and 15.4%, respectively. Losses due to disease attacks are greater in ratoons because plant cane sugarcane which is attacked by this disease, in ratoon stage, there will be a decrease in the number of shoots. The aim of this study was to obtain isolates of Trichoderma spp. which have antagonistic abilities and have chitinase enzymes so that they are effectively used as biological agents against Xylaria sp. The research method of antagonist test used dual culture test and qualitative test of chitin substrate was used to assess the chitinolytic index of four isolates of Trichoderma spp. The parameters were the inhibition of Trichoderma spp. isolates and the chitinolytic index of the four isolates moreover the value of the chitinase enzyme activity of the selected isolates. Mechanism of antagonist T10 are competition, antibiosis and mycoparasitism. The results showed that the Indonesian Sugar Research Institute (ISRI) T10 isolate could be used as a biological agent against Xylaria sp. The isolate had an inhibitory ability 73.33% with a chitinolytic index value 1.15 and the activity value of the chitinase enzyme crude extraction at a dilution 10-1 was 10.99 units/mL.