Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Design of Inhibitors Targeting Chitin-Degrading Enzymes by Bioisostere Substitutions and Scaffold Hopping for Selective Control of Ostrinia furnacalis.

Chitin-degrading enzymes are critical components in regulating the molting process of the Asian corn borer and serve as potential targets for controlling this destructive pest of maize. Here, we used a scaffold-hopping strategy to design a series of efficient naphthylimide insecticides. Among them, compound 8c exhibited potent inhibition of chitinase from OfChi-h and OfChtI at low nanomolar concentrations (IC50 = 1.51 and 9.21 nM, respectively). Molecular docking simulations suggested that 8c binds to chitinase by mimicking the interaction of chitin oligosaccharide substrates with chitinase. At low ppm concentrations, compound 8c performed comparably to commercial insecticides in controlling the highly destructive plant pest, the Asian corn borer. Tests on a wide range of nontarget organisms indicate that compound 8c has very low toxicity. In addition, the effect of inhibitor treatment on the expression of genes associated with the Asian corn borer chitin-degrading enzymes was further investigated by quantitative real-time polymerase chain reaction. In conclusion, our study highlights the potential of 8c as a novel chitinase-targeting insecticide for effective control of the Asian corn borer, providing a promising solution in the quest for sustainable pest management.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Co-overexpression of chitinase and ß-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium.

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Preliminary Study on the Pathogenic Mechanism of Jujube Flower Disease in Honeybees (Apis mellifera ligustica) Based on Midgut Transcriptomics.

Honeybees are prone to poisoning, also known as jujube flower disease, after collecting nectar from jujube flowers, resulting in the tumultuous demise of foragers. The prevalence of jujube flower disease has become one of the main factors affecting the development of the jujube and beekeeping industries in Northern China. However, the pathogenic mechanisms underlying jujube flower disease in honeybees are poorly understood. Herein, we first conducted morphological observations of the midgut using HE-staining and found that jujube flower disease-affected honeybees displayed midgut damage with peritrophic membrane detachment. Jujube flower disease was found to increase the activity of chitinase and carboxylesterase (CarE) and decrease the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and the content of CYP450 in the honeybee midgut. Transcriptomic data identified 119 differentially expressed genes in the midgut of diseased and healthy honeybees, including CYP6a13, CYP6a17, CYP304a1, CYP6a14, AADC, and AGXT2, which are associated with oxidoreductase activity and vitamin binding. In summary, collecting jujube flower nectar could reduce antioxidant and detoxification capacities of the honeybee midgut and, in more severe cases, damage the intestinal structure, suggesting that intestinal damage might be the main cause of honeybee death due to jujube nectar. This study provides new insights into the pathogenesis of jujube flower disease in honeybees.

Plasma-activated water improves the accessibility of chitinase to chitin by decreasing molecular weight and breaking crystal structure.

Chitooligosaccharides, the hydrolysis products of chitin, have superior biological activities and application value to those of chitin itself; however, the ordered and highly crystalline structure of chitin renders its degradation by chitinase difficult. Herein, the effects of plasma-activated water (PAW) pre-treatment on the physicochemical properties, crystal structure, and enzymatic hydrolysis of chitin were investigated. The hydrolysis of PAW-pre-treated chitin (PAW activation time of 5 min) using chitinase from Vibrio harveyi (VhChit2) yielded 71 % more reducing sugar, compared with that from untreated chitin, with the degree of chitin hydrolysis increasing from 13 % without pre-treatment to 23 % post-treatment. Moreover, the amount of VhChit2 adsorbed by chitin increased from 41.7 to 58.2 mg/g. Fourier transform infrared spectrometry revealed that PAW could break the ß-1,4-glycosidic bonds of chitin (but had no effects on the hydrogen and amido bonds), thereby decreasing the molecular weight and crystallinity of the polysaccharide, which caused its structural damage and enhanced its enzymatic hydrolysis by chitinase. Consequently, PAW pre-treatment can be considered a simple, effective, and environmentally-friendly method for the biotransformation of chitin as its easier hydrolysis yields high-value products.

Dissecting the role of salicylic acid in mediating stress response in mungbean cultivars concurrently exposed to Macrophomina phaseolina infection and drought stress.

The combined stress studies provide fundamental knowledge that could assist in producing multiple stress resilient crops. The fungal phytopathogen, Macrophomina phaseolina is a major limiting factor in the productivity of the crop, Vigna radiata (mungbean). This fungal species tends to flourish under hot and dry conditions. Therefore, in this study the salicylic acid (SA) mediated stress responses in contrasting mungbean cultivars (Shikha and RMG-975) exposed to combined M. phaseolina infection (F) and drought stress (D) have been elucidated. The combined stress was applied to ten days seedlings in three orders i.e. drought followed by fungal infection (DF), drought followed by fungal infection with extended water deficit (DFD) and fungal infection followed by drought stress (FD). The severity of infection was analyzed using ImageJ analysis. Besides, the concentration of SA has been correlated with the phenylpropanoid pathway products, expression of pathogenesis-related proteins (ß-1,3-glucanase and chitinase) and the specific activity of certain related enzymes (phenylalanine ammonia lyase, lipoxygenase and glutathione-S-transferase). The data revealed that the cultivar RMG-975 was relatively more tolerant than Shikha under individual stresses. However, the former became more susceptible to the infection under DFD treatment while the latter showed tolerance. Otherwise, the crown rot severity was reduced in both the cultivars under other combined treatments. The stress response analysis suggested that enhanced chitinase expression is vital for tolerance against both, the pathogen and drought stress. Also, it was noted that plants treat each stress combination differently and the role of SA was more prominently visible under individual stress conditions.

Boosting one-step degradation of shrimp shell waste to produce chitin oligosaccharides at smart nanoscale enzyme reactor with liquid-solid system.

Chitin oligosaccharides (CTOS) possess potential applications in food, medicine, and agriculture. However, lower mass transfer and catalytic efficiency are the main kinetic limitations for the production of CTOS from shrimp shell waste (SSW) and crystalline chitin. Chemical or physical methods are usually used for pretreatment to improve chitinase hydrolysis efficiency, but this is not eco-friendly and cost-effective. To address this challenge, a chitinase nanoreactor with the liquid-solid system (BcChiA1@ZIF-8) was manufactured to boost the one-step degradation of SSW and crystalline chitin. Compared with free enzyme, the catalytic efficiency of BcChiA1@ZIF-8 on colloidal chitin was significantly improved to 142 %. SSW and crystalline chitin can be directly degraded by BcChiA1@ZIF-8 without any pretreatments. The yield of N, N'-diacetylchitobiose [(GlcNAc)2] from SSW and N-acetyl-D-glucosamine (GlcNAc) from crystalline chitin was 2 times and 3.1 times than that of free enzyme, respectively. The reason was that BcChiA1@ZIF-8 with a liquid-solid system enlarged the interface area, increased the collision frequency between enzyme and substrate, and improved the large-substrates binding activity of chitinase. Moreover, the biphasic system exhibited excellent stability, and the design showed universal applicability. This strategy provided novel guidance for other polysaccharide biosynthesis and the conversion of environmental waste into carbohydrates.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii.

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul-1 h-1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol-1, 100.59 kJ mol-1, 102.64 kJ mol-1 and 5.95 J mol-1 K-1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2-4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2-4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

A Potential Multitarget Insect Growth Regulator Candidate: Design, Synthesis, and Biological Activity of Novel Acetamido Derivatives Containing Hexacyclic Pyrazole Carboxamides.

Insect growth regulators (IGRs) are important green insecticides that disrupt normal growth and development in insects to reduce the harm caused by pests to crops. The ecdysone receptor (EcR) and three chitinases OfChtI, OfChtII, and OfChi-h are closely associated with the molting stage of insects. Thus, they are considered promising targets for the development of novel insecticides such as IGRs. Our previous work identified a dual-target compound 6j, which could act simultaneously on both EcR and OfChtI. In the present study, 6j was first found to have inhibitory activities against OfChtII and OfChi-h, too. Subsequently, taking 6j as a lead compound, 19 novel acetamido derivatives were rationally designed and synthesized by introducing an acetamido moiety into the amide bridge based on the flexibility of the binding cavities of 6j with EcR and three chitinases. Then, their insecticidal activities against Plutella xylostella (P. xylostella), Ostrinia furnacalis (O. furnacalis), and Spodoptera frugiperda (S. frugiperda) were carried out. The bioassay results revealed that most of these acetamido derivatives possessed moderate to good larvicidal activities against three lepidopteran pests. Especially, compound I-17 displayed excellent insecticidal activities against P. xylostella (LC50, 93.32 mg/L), O. furnacalis (LC50, 114.79 mg/L), and S. frugiperda (86.1% mortality at 500 mg/L), significantly better than that of 6j. In addition, further protein validation and molecular docking demonstrated that I-17 could act simultaneously on EcR (17.7% binding activity at 8 mg/L), OfChtI (69.2% inhibitory rate at 50 µM), OfChtII (71.5% inhibitory rate at 50 µM), and OfChi-h (73.9% inhibitory rate at 50 µM), indicating that I-17 is a potential lead candidate for novel multitarget IGRs. This work provides a promising starting point for the development of novel types of IGRs as pest management agents.

Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts.

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.

Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

Antagonistic activity and mechanism of Bacillus subtilis CG-6 suppression of root rot and growth promotion in Alfalfa.

Root rot is a common disease, that severely affects the yield and quality of alfalfa. Biocontrol is widely used to control plant diseases caused by pathogenic fungi, however, biocontrol strains for alfalfa root rot are very limited. In this study, a Bacillus subtilis CG-6 strain with a significant biocontrol effect on alfalfa root rot was isolated. CG-6 secretes antibacterial enzymes and siderophore, phosphate solubilization and indoleacetic acid (IAA). The inhibition rate of strain CG-6 against Fusarium oxysporum was 87.33%, and it showed broad-spectrum antifungal activity. Inoculation with CG-6 significantly reduced the incidence of alfalfa root rot, the control effect of greenhouse cultivation reached 58.12%, and CG-6 treatment significantly increased alfalfa plant height, root length, fresh weight, and dry weight. The treatment with CG-6 significantly increased the levels of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and lipoxygenase) in alfalfa leaves by 15.52%-34.03%. Defensive enzymes (chitinase and ß-1,3-glucanase) increased by 24.37% and 28.08%, respectively. The expression levels of regulatory enzyme genes (MsCAT, MsPOD, MsCu, Zn-SOD1, MsCu, Zn-SOD2, MsCu, Zn-SOD3, and MsLOX2) and systemic resistance genes (MsPR1, MsPDF1.2, and MsVSP2) increased by 0.50-2.85 fold, which were higher than those in the pathogen treatment group. Therefore, CG-6 could be used as a potential strain to develop biopesticides against alfalfa root rot.

The biology of insect chitinases and their roles at chitinous cuticles.

Chitin is one of the most prevalent biomaterials in the natural world. The chitin matrix formation and turnover involve several enzymes for chitin synthesis, maturation, and degradation. Sequencing of the Drosophila genome more than twenty years ago revealed that insect genomes contain a number of chitinases, but why insects need so many different chitinases was unclear. Here, we focus on insect GH18 family chitinases and discuss their participation in chitin matrix formation and degradation. We describe their variations in terms of temporal and spatial expression patterns, molecular function, and physiological consequences at chitinous cuticles. We further provide insight into the catalytic mechanisms by discussing chitinase protein domain structures, substrate binding, and enzymatic activities with respect to structural analysis of the enzymatic GH18 domain, substrate-binding cleft, and characteristic TIM-barrel structure.

Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 µM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs.

Functional Comparison of Three Chitinases from Symbiotic Bacteria of Entomopathogenic Nematodes.

Xenorhabdus and Photorhabdus, bacterial symbionts of entomopathogenic nematodes Steinernema and Heterorhabditis, respectively, have several biological activities including insecticidal and antimicrobial activities. Thus, XnChi, XhChi, and PtChi, chitinases of X. nematophila, X. hominickii, and P. temperata isolated from Korean indigenous EPNs S. carpocapsae GJ1-2, S. monticolum GJ11-1, and H. megidis GJ1-2 were cloned and expressed in Escherichia coli BL21 to compare their biological activities. Chitinase proteins of these bacterial symbionts purified using the Ni-NTA system showed different chitobiosidase and endochitinase activities, but N-acetylglucosamidinase activities were not shown in the measuring of chitinolytic activity through N-acetyl-D-glucosarmine oligomers. In addition, the proteins showed different insecticidal and antifungal activities. XnChi showed the highest insecticidal activity against Galleria mellonella, followed by PtChi and XhChi. In antifungal activity, XhChi showed the highest half-maximal inhibitory concentration (IC50) against Fusarium oxysporum with 0.031 mg/mL, followed by PtChi with 0.046 mg/mL, and XnChi with 0.072 mg/mL. XhChi also showed the highest IC50 against F. graminearum with 0.040 mg/mL, but XnChi was more toxic than PtChi with 0.055 mg/mL and 0.133 mg/mL, respectively. This study provides an innovative approach to the biological control of insect pests and fungal diseases of plants with the biological activity of symbiotic bacterial chitinases of entomopathogenic nematodes.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum.

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

Polysaccharide degradation in Cellvibrionaceae: Genomic insights of the novel chitin-degrading marine bacterium, strain KSP-S5-2, and its chitinolytic activity.

In this study, we present the genome characterization of a novel chitin-degrading strain, KSP-S5-2, and comparative genomics of 33 strains of Cellvibrionaceae. Strain KSP-S5-2 was isolated from mangrove sediment collected in Balik Pulau, Penang, Malaysia, and its 16S rRNA gene sequence showed the highest similarity (95.09%) to Teredinibacter franksiae. Genome-wide analyses including 16S rRNA gene sequence similarity, average nucleotide identity, digital DNA-DNA hybridization, and phylogenomics, suggested that KSP-S5-2 represents a novel species in the family Cellvibrionaceae. The Cellvibrionaceae pan-genome exhibited high genomic variability, with only 1.7% representing the core genome, while the flexible genome showed a notable enrichment of genes related to carbohydrate metabolism and transport pathway. This observation sheds light on the genetic plasticity of the Cellvibrionaceae family and the gene pools that form the basis for the evolution of polysaccharide-degrading capabilities. Comparative analysis of the carbohydrate-active enzymes across Cellvibrionaceae strains revealed that the chitinolytic system is not universally present within the family, as only 18 of the 33 genomes encoded chitinases. Strain KSP-S5-2 displayed an expanded repertoire of chitinolytic enzymes (25 GH18, two GH19 chitinases, and five GH20 ß-N-acetylhexosaminidases) but lacked genes for agar, xylan, and pectin degradation, indicating specialized enzymatic machinery focused primarily on chitin degradation. Further, the strain degraded 90% of chitin after 10 days of incubation. In summary, our findings provided insights into strain KSP-S5-2's genomic potential, the genetics of its chitinolytic system, genomic diversity within the Cellvibrionaceae family in terms of polysaccharide degradation, and its application for chitin degradation.

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product.

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-ß-D-glucosamine from powdery chitin.

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously ß-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.