Comparison of in vitro Antifungal Activity Methods Using Extract of Chitinase-producing Aeromonas sp. BHC02.

Biological control to prevent fungal plant diseases offers an alternative approach to facilitate sustainable agriculture. Since the chitin in fungal cell walls is a target for biocontrol agents, chitinases are one of the important antifungal molecules. In this study, the aim was to investigate a new chitinase isolated from a fluvial soil bacterium and to show the antifungal activity of the characterized chitinase by comparing the three common methods. The bacterium with the highest chitinase activity was identified as Aeromonas sp. by 16 S rRNA sequence analysis. Following the determination of the optimum enzyme production time, the enzyme was partially purified, and the physicochemical parameters of the enzyme were investigated. In the antifungal studies, direct Aeromonas sp. BHC02 cells or partially purified chitinase were used. As a result, in the first method in which the Aeromonas sp. BHC02 cells were spread on the surface of petri dishes, no zone formation was observed around the test fungi spotted on the surface. However, zone formation was observed in the methods in which the antifungal activity was investigated using the partially purified chitinase enzyme. For example, in the second method, the enzyme was spread on the surface of PDA, and zone formation was observed only around Penicillum species among the test fungi spotted on the surface. In the third method, in which the necessary time was given for the formation of mycelium of the test fungi, it was observed that the growth of Fusarium solani, Alternaria alternata and Botrytis cinerea was inhibited by the partially purified chitinase. This study concludes that the results of the antifungal activities depend on the method used and all fungal chitins cannot be degraded with one strain's chitinase. Depending on the variety of chitin, some fungi can be more resistant.

Structure of intact chitinase with hevein domain from the plant Simarouba glauca, known for its traditional anti-inflammatory efficacy.

Chitinase from the leaves of Simarouba glauca, a plant used in traditional anti-inflammatory therapy is purified and characterized. Peptide mass finger print analysis revealed the protein as an endo-chitinase which was further confirmed using chitin-agar assay. The enzyme exhibited significant anti-fungal efficacy against phyto-pathogens such as Macrophomina phaseolina, Fusarium oxysporum and Sclerotium rolfsii. Chitinolysis was also examined against insoluble chitin using SEM. Using X-ray diffraction data up to 1.66 Å, the structure was determined by Molecular Replacement using crystal structure of GH19 Chitinase-like protein from Hevea brasiliensis. During structure refinement, an extra domain could be traced and identified as hevein domain. To our knowledge, this is the first report of any chitinase with intact hevein domain. The GH19 chitinase and hevein domains though connected by a lengthy loop, are restricted to be close by disulfide bridges. These bridges connecting each domain with the loop may be important for proper chitin feeding into the active site. By considering reports on hevein and chitinase domains as well as the traditional use of the plant, this report of an intact hevein-chitinase protein and their relative orientation may add further insights for the usefulness of this protein.

Antifungal Proteins from Plant Latex.

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants' defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action.

A chitinase with antifungal activity from naked oat (Avena chinensis) seeds.

A chitinase was purified from naked oat (Avena chinensis) seeds using simple chromatographic techniques. Its molecular weight and isoelectric point were determined as 35 kDa and 8.9, respectively. The purified chitinase exhibited specific activity of 3.6 U/mg and 15.6% yield using colloidal chitin as substrate. Partial amino acid sequence analysis and homology search indicated that it probably belonged to Class I plant chitinase, glycosyl hydrolase family 19. With chitin as substrate, the optimum pH and temperature of the chitinase were pH 7.0 and 40°C, respectively. The chitinase was remarkably stable from 30°C up to 50°C, but was inactivated at high temperatures above 85°C. Antifungal activity in vitro tests demonstrated this purified chitinase had potent, dose-dependent inhibitory activity against the fungi Panus conchatus and Trichoderma reesei. PRACTICAL APPLICATIONS: Chitinase has broad applications in many fields including the food industry and is recognized as one of the antifungal substances with potential use in plant disease resistance or biological control in agriculture. This study developed cost-effective purification methods for producing chitinase from naked oat (Avena chinensis) seeds, which may favor large-scale production of the enzyme. The remarkable stability of the chitinase at moderate temperatures (30°C-50°C), makes it a potentially useful enzyme in bioprocessing to produce chitooligosaccharides for various applications in the food, health, and agriculture sectors.

Cytotoxicity against tumor cell lines and anti-inflammatory properties of chitinases from Calotropis procera latex.

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 µg/mL but induced high messenger RNA expression of IL-6, IL1-ß, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 µg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-ß were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

Purification and characterization of a novel chitinase from Trichosanthes dioica seed with antifungal activity.

Chitinases are a group of enzymes that show differences in their molecular structure, substrate specificity, and catalytic mechanism and widely found in organisms like bacteria, yeasts, fungi, arthropods actinomycetes, plants and humans. A novel chitinase enzyme (designated as TDSC) was purified from Trichosanthes dioica seed with a molecular mass of 39±1 kDa in the presence and absence of ß-mercaptoethanol. The enzyme was a glycoprotein in nature containing 8% neutral sugar. The N-terminal sequence was determined to be EINGGGA which did not match with other proteins. Amino acid analysis performed by LC-MS revealed that the protein was rich in leucine. The enzyme was stable at a wide range of pH (5.0-11.0) and temperature (30-90 °C). Chitinase activity was little bit inhibited in the presence of chelating agent EDTA (ethylenediaminetetraaceticacid), urea and Ca(2+). A strong fluorescence quenching effect was found when dithiothreitol and sodium dodecyl sulfate were added to the enzyme. TDSC showed antifungal activity against Aspergillus niger and Trichoderma sp. as tested by MTT assay and disc diffusion method.

Anti-fungal potentials of extracellular metabolites of Western Ghats isolated Streptomyces sp. NII 1006 against moulds and yeasts.

Realization of hazardious effects of chemical fungicides has led to an interest in the usage of biocontrol agents. The present study, therefore, evaluates the biocontrol efficacy of Western Ghats (India) soil bacterial isolates. A potential strain NII 1006 was evaluated for its antagonistic property against a diverse range of moulds and yeasts. The strain was characterized morphologically, biochemically and molecularly, which revealed the isolate belonged to Streptomyces genus. Organic solvent extracts of NII 1006 culture filtrates inhibited the growth of the test pathogens indicating that growth suppression was due to extracellular anti-fungal metabolites present in the culture filtrates. The strain produced extracellular chitinase enzyme in addition to some stable partially purified anti-fungal compounds. Morphological changes such as hyphae degradation into debris and abnormal shapes were observed in test fungi and yeast grown on potato dextrose broth that contained the NII 1006 culture filtrate. The cell free supernatant has a tolerance to wide range of pH, temperature and enzymes such as lipase and protease. The biocontrol potential of NII 1006 strain may be correlated significantly with their ability to produce antibiotics as well as extracellular hydrolytic enzymes particularly chitinolytic enzyme.

Purification, characterization of a CkChn134 protein from Cynanchum komarovii seeds and synergistic effect with CkTLP against Verticillium dahliae.

Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic, and antidiarrheal, but also as herbal medicine to treat cholecystitis in people. In this work, an antifungal protein with sequence homology to chitinase was isolated from C. komarovii seeds and named CkChn134. The three-dimensional structure prediction of CkChn134 indicated that the protein has a loop domain formed a thin cleft, which is able to bind molecules and substrates. The protein and CkTLP synergistically inhibited the fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea, and Valsa mali in vitro. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed that the transcription level of CkChn134 had a significant increase under the stress of ethylene, NaCl, low temperature, drought, and pathogen infection, which indicates that CkChn134 may play an important role in response to abiotic and biotic stresses. The CkChn134 protein was located in the extracellular space/cell wall by CkChn134::GFP fusion protein in transgenic Arabidopsis. Furthermore, overexpression of CkChn134 significantly enhanced the resistance of transgenic Arabidopsis against V. dahliae. Interestingly, the coexpression of CkChn134 and CkTLP showed substantially greater protection against the fungal pathogen V. dahliae than either transgene alone. The results suggest that the CkChn134 is a good candidate protein or gene, and it had a potential synergistic effect with CkTLP for contributing to the development of disease-resistant crops.

A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination.

BACKGROUND: Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris. RESULTS: A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (ß/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 µg/µL inhibited the germination of spores of Phakopsora pachyrhizi by 45%. CONCLUSIONS: Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.

A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry.

Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low-molecular-weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH(4))(2)SO(4) precipitation, ion-exchange chromatography and hydrophobic-interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N-terminal amino acid sequencing revealed that PMAPI is a hevein-like protein, and PMAPII is a class I chitinase. They both had a half-maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high-molecular-weight extracts with antifungal activity from paper mulberry.

Cloning and expression of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field.

A chitinase producing Bacillus subtilis CHU26 was isolated from Taiwan potato field. This strain exhibited a strong extra-cellular chitinase activity on the colloidal chitin containing agar plate, and showed a potential inhibit activity against phytopathogen, Rhizoctonia solani. The gene encoding chitinase (chi18) was cloned from the constructed B. subtilis CHU26 genomic DNA library. The chi18 consisted of an open reading frame of 1791 nucleotides and encodes 595 amino acids with a deduced molecular weight of 64kDa, next to a promoter region containing a 9 base pair direct repeat sequence (ATTGATGAA). The deduced amino acid sequence of the chitinase from Bacillus subtilis CHU26 exhibits 62% and 81% similarity to those from B. circulans WL-12 and B. licheniformis, respectively. Subcloned chi18 into vector pGEM3Z and pYEP352 to construct recombinant plasmid pGCHI18 and pYCHI18, respectively, chitinase activity could be observed on the colloidal chitin agar plate from recombinant plasmid containing Escherichia coli transformant. Cell-free culture broth of pYCHI18 containing E. coli transformant decreased R. solani pathogenic activity more than 90% in the antagonistic test on the radish seedlings (Raphanus sativus Linn.).

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linné) and in vitro.

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown.

A chitinase, purified to homogeneity from ethylene-treated bean leaves, was applied to actively growing mycelial cells of Rhizoctonia solani to evaluate a potential antifungal activity. Light microscopic investigations at 30-min intervals following enzyme exposure revealed the induction of morphological changes such as swelling of hyphal tips and hyphal distortions. More precise information concerning fungal cell alteration was obtained by ultrastructural observation and cytochemical detection of chitin distribution in fungal cell walls. Chitin breakdown was found to be an early event preceding wall disruption and cytoplasm leakage. The large amounts of chitin present in the walls of control R. solani cells and the rapid chitin hydrolysis upon chitinase treatment lead us to suggest that this polysaccharide is one of the main components of this fungal cell wall and is readily accessible to chitinase, especially in the apical zone. By 60 min after enzyme treatment, labeled molecules were observed in the vicinity of some fungal cells, suggesting the release of chitin oligosaccharides from fungal cell walls. The antifungal activity of the bean chitinase on cells of R. solani grown in culture is discussed in relation to the potential of genetically modified transgenic plants to resist attack by R. solani through an antimicrobial activity in planta.

Degradation of melanin by Aspergillus fumigatus.

A strain of Aspergillus fumigatus from composted coffee and garden wastes utilized natural deproteinized insect, banana, hair, octopus, and synthetic tyrosine and dopa melanins as sole sources of carbon. With a sucrose supplement, degradation was essentially complete after 50 days in Czapek medium pH 6.5 at 30 degrees C. The catabolic rate differed for each substrate pigment, as did the molecular weight distribution of products accumulating in the medium. After incubation with L-[U-14C]melanin, over 50% was recovered in a dark fungal pigment, the remainder appearing as cell protein, chitin, lipid, CO2, and polar metabolites. When grown on melanin, the normally pale mycelia darkened with the production of a fungal allomelanin, with infrared spectrum and alkali fusion products differing from those of the substrate pigment. Isotope distribution in amino acids for A. fumigatus grown on labeled melanin supplemented with sucrose suggested separate pools for synthesis of cell proteins and melanoproteins. Deposition of allomelanin increased resistance of conidia, sterigma, and conidiophores to lytic carbohydrases as judged by scanning electron microscopy.