Synthesis of N-benzoyl Amino Esters and N-benzoyl Amino Acids and their Antifungal Activity

A series of N-benzoyl amino esters and N-benzoyl amino acids were synthesized from commercially-available amino acids (Val, Ile, Leu, Ala, Phe, Trp) and were evaluated for their antifungal activity against two filamentous fungi, A. fumigatus and F. temperatum. According to the in vitro assays, five compounds (5-7, 10, 13) exhibited relevant antifungal activity against F. temperatum and two compounds (5 and 7) showed remarkable activity against both fungi strains. Some structure-activity relationships were established regarding the side chain at Ca and the type of substituents on the aromatic ring in the benzoyl moiety. Docking calculations were performed in order to predict binding affinities between compounds prepared herein and fungal chitinase, a potential target against fungi; interactions involving the aromatic rings, the influence on the number of methyl substituents, and configurations on the a-carbon have been analyzed. Resumen. Una serie de derivados N-benzoilamino ésteres y N-benzoilaminoácidos, sintetizados a partir de aminoácidos disponibles comercialmente (Val, Ile, Leu, Ala, Phe, Trp), se evaluaron como agentes antifúngicos frente a dos hongos filamentosos, A. fumigatus y F. temperatum. De acuerdo con los ensayos in vitro, cinco compuestos (5-7, 10, 13) exhibieron una actividad relevante contra F. temperatum y dos derivados (5 y 7) mostraron una actividad notable contra ambas cepas. Algunas relaciones de estructura actividad permitieron observar el efecto de la cadena lateral del aminoácido, y de los sustituyentes del grupo benzoílo, en la actividad biológica. Se realizaron cálculos de acoplamiento molecular con el propósito de predecir afinidades de enlace entre los compuestos sintetizados y la enzima quitinasa, considerada un blanco molecular potencial. Se analizaron las interacciones que involucran anillos aromáticos, la influencia de los sustituyentes metilo, así como la configuración del Ca.

Molecular Analysis of Genes Involved in Chitin Degradation from the Chitinolytic Bacterium Bacillus Velezensis

Bacillus velezensis RB.IBE29 is a potent biocontrol agent with high chitinase activity isolated from the rhizosphere of black pepper cultivated in the Central Highlands, Vietnam. Genome sequences revealed that this species possesses some GH18 chitinases and AA10 protein(s); however, these enzymes have not been experimentally characterized. In this work, three genes were identified from the genomic DNA of this bacterium and cloned in Escherichia coli. Sequence analysis exhibited that the ORF of chiA consists of 1,203 bp and encodes deduced 45.46 kDa-chitinase A of 400 aa. The domain structure of chitinase A is composed of a CBM 50 domain at the N-terminus and a catalytic domain at the C-terminus. The ORF of chiB includes 1,263 bp and encodes deduced 47.59 kDa-chitinase B of 420 aa. Chitinase B consists of two CBM50 domains at the N-terminus and a catalytic domain at the C-terminus. The ORF of lpmo10 is 621 bp and encodes a deduced 22.44 kDa-AA10 protein, BvLPMO10 of 206 aa. BvLPMO10 contains a signal peptide and an AA10 catalytic domain. Chitinases A and B were grouped into subfamily A of family 18 chitinases. Amino acid sequences in their catalytic domains lack aromatic residues (Trp, Phe, Tyr) probably involved in processivity and substrate binding compared with well-known bacterial GH18 chitinases. chiB was successfully expressed in E. coli. Purified rBvChiB degraded insoluble chitin and was responsible for inhibition of fungal spore-germination and egg hatching of plant-parasitic nematode. This is the first report describing the analysis of the chitinase system from B. velezensis.

GH18 family glycoside hydrolase Chitinase A of Salmonella facilitates bacterial invasion and survival by modulating host immune responses

Salmonella is a facultative intracellular pathogen that has co-evolved with its host and has also developed various strategies to evade the host immune responses. Salmonella recruits an array of virulence factors to escape from host defense mechanisms. Previously chitinase A (chiA) was found to be upregulated in intracellular Salmonella. Although studies show that chitinases and chitin binding proteins (CBP) of many human pathogens have a profound role in various aspects of pathogenesis, like adhesion, virulence and immune evasion, the role of chitinase in strict intravacuolar pathogen Salmonella has not yet been elucidated. In this study, we deciphered the role of chitinase of Salmonella in the pathogenesis of the serovars, Typhimurium and Typhi. Our data propose that ChiA mediated modification of the glycosylation on the epithelial cell surface facilitates the invasion of the pathogen into the epithelial cells. Further we found that ChiA aids in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production in phagocytes, leading to MHCII downregulation followed by suppression of antigen presentation and antibacterial responses. In continuation of the study in animal model C. elegans, Salmonella Typhi ChiA was found to facilitate attachment to the intestinal epithelium, gut colonization and persistence by downregulating antimicrobial peptides.

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Maize Endochitinase Expression in Response to Fall Armyworm Herbivory

A large percentage of crop loss is due to insect damage yearly, especially caterpillar damage. Plant chitinases are considered excellent candidates to combat these insects since they can catalyze chitin degradation in peritrophic matrix (PM), an important protective structure in caterpillar midgut. Compared to chemical insecticides, chitinases could improve host plant resistance and be both economically and environmentally advantageous. The focus of this research was to find chitinase candidates that could improve plant resistance by effectively limiting caterpillar damage. Five classes of endochitinase (I-V) genes were characterized in the maize genome, and we further isolated and cloned four chitinase genes (chitinase A, chitinase B, chitinase I, and PRm3) present in two maize (Zea mays L.) inbred lines Mp708 and Tx601, with different levels of resistance to caterpillar pests. Further, we investigated the role of these maize chitinases in response to fall armyworm (Spodoptera frugiperda, FAW) attacks. Results from gene expression and enzyme assays from maize leaves indicated that both chitinase transcript abundance and enzymatic activity increased in response to FAW feeding and mechanical wounding. Furthermore, chitinase retained activity inside the caterpillar’s midgut since specific activity was detected in both the food bolus and frass. When examined under scanning electron microscopy, PMs from Tx601-fed caterpillars showed structural damage when compared to diet controls. Analysis of chitinase transcript abundance after caterpillar feeding and proteomic analysis of maize leaf trichomes in the two inbreds suggested that the chitinase PRm3 in Tx601 has potential insecticidal properties.

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

Chitinase and chitin-oligosaccaride can be used in multiple field, so it is important to develop a high-yield chitinase producing strain. Here, a recombinant Pichia pastoris with 4 copies of ChiA gene from Bacillus licheniformis and co-expression of molecular chaperon HAC1 was constructed. The amount of recombinant ChiA in the supernatant of high-cell-density fermentation reaches a maximum of 12.7 mg/mL, which is 24-fold higher than that reported in the previous study. The recombinant ChiA can hydrolyze 30% collodidal chitin with 74% conversion ratio, and GlcNAc is the most abundant hydrolysis product, followed by N, N′-diacetylchitobiose. Combined with BsNagZ, the hydrolysate of ChiA can be further transformed into GlcNAc with 88% conversion ratio. Additionally, the hydrolysate of ChiA can obviously accelerate the germination growth of rice and wheat, increasing the seedling height and root length by at least 1.6 folds within 10 days.

Genome-wide identification and expression analysis of chitin-binding gene family in Brassica oleracea L. reveals its role in different disease resistance

Background Chitinase, a category of pathogenesis-related proteins, is thought to play an important role in defending external stress in plants. However, comprehensive analysis of chitin-binding gene family has not yet been reported in cabbage (Brassica oleracea L.), especially their roles in response to different diseases. Result in this study, A total of 20 chitinase genes were identified using a genome-wide search method. Phylogenetic analysis classified these genes into two groups. They were distributed unevenly across six chromosomes in cabbage, and all of them contained few introns (≤ 2). The results of colinear analysis showed that the cabbage genome contained 1–5 copies of each chitinase gene (excluding Bol035470) found in Arabidopsis. The heatmap of the chitinase gene family showed that these genes were expressed in various tissues and organs. In addition, under four different stresses of Fusarium wilt, powdery mildew, black spot and downy mildew, we detected 9, 5, 8 and 8 genes with different expression, respectively. Conclusions Our results provide insights for further understanding the role of chitinase in host plants response to different diseases.