High Antifungal Activity Research Articles

Morchella esculenta ‐based chitosan bionanocomposites: Evaluation as an antifungal agent

Considering the damage caused by fungicides to human health and problems such as microbial resistance, biological control against plant pathogens has started to gain importance worldwide. This research demonstrates a new, simple, cost-effective, and environmentally friendly method for synthesizing chitosan bionanocomposite (CBNC) from Morchella esculenta (L) Pers-extract (MEE). The antifungal property of the synthesized Morchella esculenta (L) Pers-chitosan bionanocomposite (MCBNC) against some plant pathogens was also evaluated. FTIR, XRD, FE-SEM, DSC, TGA, and BET were used to characterize the synthesized MCBNC. Mushroom-based chitosan nanoparticles were evaluated for antifungal activity against some fungal pathogens, including Neoscytalidium novaehollandiae, N. dimidiatum, Alternaria alternata, Verticillium dahliae, Bipolaris sorokiniana, and Colletotrichum sp. The findings obtained clearly showed that chitosan nanoparticles have antifungal activity. The results suggest that the chitosan nanoparticle can be used in the field to protect various crops from phytopathogens. Novelty impact statement Chitosan bionanocomposite (MCBNC) synthesis was performed for the first time using the wild mushroom Morchella esculenta, which has strong bioactive properties. It was observed that the bionanomaterial, which was characterized by FTIR, XRD, FE-SEM, DSC, TGA, and BET analyses, has high antifungal activity against plant pathogens such as Alternaria alternata and Bipolaris sorokiniana. MCBNCs synthesized by the green synthesis method can be an important area of use in the fight against plant pathogens, which corresponds to 1/3 of the world's agricultural production potential.

Bacillus velezensis strain Ag75 as a new multifunctional agent for biocontrol, phosphate solubilization and growth promotion in maize and soybean crops

Soybean and maize are some of the main drivers of Brazilian agribusiness. However, biotic and abiotic factors are of great concern, causing huge grain yield and quality losses. Phosphorus (P) deficiency is important among the abiotic factors because most Brazilian soils have a highly P-fixing nature. Thus, large amounts of phosphate fertilizers are regularly applied to overcome the rapid precipitation of P. Searching for alternatives to improve the use of P by crops is essential to reduce the demand for P input. The use of multifunctional rhizobacteria can be considered one of these alternatives. In this sense, the objective of the present work was to select and validate bacterial strains with triple action (plant growth promoter, phosphate solubilizer, and biocontrol agent) in maize and soybean, aiming to develop a multifunctional microbial inoculant for Brazilian agriculture. Bacterial strains with high indole acetic acid (IAA) production, phosphate solubilization, and antifungal activity against soil pathogenic fungi (Rhizoctonia solani, Macrophomina phaseolina, and Fusarium solani) were selected from the maize rhizosphere. Then, they were evaluated as growth promoters in maize under greenhouse conditions. Based on this study, strain 03 (Ag75) was selected due to its high potential for increasing biomass (root and shoot) and shoot P content in maize. This strain was identified through genomic sequencing as Bacillus velezensis. In field experiments, the inoculation of this bacterium increased maize and soybean yields by 17.8 and 26.5%, respectively, compared to the control (25 kg P2O5). In addition, the inoculation results did not differ from the control with 84 kg P2O5, indicating that it is possible to reduce the application of phosphate in these crops. Thus, the Ag75 strain has great potential for developing a multifunctional microbial inoculant that combines the ability to solubilize phosphate, promote plant growth, and be a biocontrol agent for several phytopathogenic fungi.

A comparative study of antimicrobial, anti-quorum sensing, anti-biofilm, anti-swarming, and antioxidant activities in flower extracts of pecan (Carya illinoinensis) and chestnut (Castanea sativa).

Antibiotic resistance, which has increased rapidly in recent years because of uncontrolled and unconscious antibiotic consumption, poses a major threat to public health. The inadequacy of existing antibiotics has increased the need for new, effective, and less toxic antibiotic raw materials or antibiotic derivatives. Pecan (Carya illinoinensis) and Chestnut (Castanea sativa) flowers possess abundant pollen contents and exhibit similar morphological features. The purpose of this study was to compare these two flower extracts in terms of their antimicrobial and antioxidant activities. Total phenolic content, total flavonoid contents, and phenolic components were also analyzed in aquatic and ethanolic extracts. Antioxidant activities were measured using ferric reducing/antioxidant capacity (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Antimicrobial and antifungal activities were compared by means of agar diffusion tests against bacteria including Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis, Acinetobacter haemolyticus, and Chromobacterium violaceum, and the yeasts Candida albicans and Candida parapsilosis. Anti-quorum sensing (anti-QS), anti-biofilm, and anti-swarming (SW) activities were also studied against Chromobacterium violaceum ATCC 31532, Chromobacterium violaceum ATCC 12472, and Pseudomonas aeruginosa PA01, respectively. Both extracts were rich in ellagic acid and gallic acid and exhibited similar antioxidant properties. Both flower extracts exhibited high antimicrobial and antifungal activities as well as anti-biofilm, anti-QS, and anti-SW activities.

Advances in the green synthesis and agrichemical applications of oxathiapiprolin derivatives.

Oxathiapiprolin was developed with high antifungal activity and novel target protein and is used in the oomycetes control for crop protection. The structural modifications of oxathiapiprolin are summarized. The achievements and challenges in the structural modification of oxathiapiprolin are also discussed in this mini review. The outlook in this field is perspected according to our own opinion and understanding on the development of oxysterol binding protein inhibition fungicides.

Vapor-Phase of Essential Oils as a Promising Solution to Prevent Candida Vaginal Biofilms Caused by Antifungal Resistant Strains.

Background: Vulvovaginal candidiasis (VVC) is a disease with high incidence, a huge impact on the quality of life and health of women, and which represents a great challenge to treat. The growing need to apply antifungal intensive therapies have contributed to an emergence of drug-resistant Candida strains. Thus, effective therapeutic options, to meet the antifungal-resistance challenge and to control high resilient biofilms, are urgently needed. This study aimed to investigate the antifungal activity of essentials oils (EOs) on drug-resistant Candida vaginal isolates. Method: Therefore, the antimicrobial effect of tea tree, niaouli, white thyme, and cajeput EOs on the planktonic growth of Candida isolates was initially evaluated by an agar disc diffusion method. Then, the vapor-phase effect of tea tree EO (VP-TTEO) on biofilm formation and on pre-formed biofilms was evaluated by crystal violet staining, XTT reduction assay, colony forming units’ enumeration, and scanning electron microscopy. Results: The results revealed high antifungal activity of EOs against drug-resistant Candida isolates. Additionally, the VP-TTEO showed a significant inhibitory effect on the biofilm formation of all tested isolates and was able to provoke an expressive reduction in mature Candida albicans biofilms. Conclusions: Overall, this study suggests that the VP-EO may be a promising solution that is able to prevent biofilm-related VVC caused by antifungal-resistant strains.

Eco-friendly synthesis, characterization, in-silico ADMET and molecular docking analysis of novel carbazole derivatives as antibacterial and antifungal agents

In search of novel biologically potent compounds, three novel organic molecules have been synthesized. The structures of the prepared compounds have been characterized by elemental analysis and 1H/13C NMR. The antimicrobial activity of this novel series of molecules was carried out against four bacterial and four fungal strains using the well diffusion method. The obtained results exhibited a high antibacterial and antifungal activity compared to that of streptomycin and Fluconazole (standard antibiotics). The presence of triazole in the DC3 compound increases the antibacterial activity against the selected strains compared to DC1 (dimethyl pyrazole) and DC2 (pyrazole). In silico ADME prediction shows that DC3 has a slightly better pharmacokinetic and druglikiness profile than DC1 and DC2. Otherwise, they might still be considered good drug candidates. Molecular docking investigations were also completed to explore the binding affinities and also the interactions of the examined molecules with the target proteins.

Isolation and Molecular Identification of Serratia Strains Producing Chitinases, Glucanases, Cellulases, and Prodigiosin and Determination of Their Antifungal Effect against Colletotrichum siamense and Alternaria alternata In Vitro and on Mango Fruit

Microorganisms represent a viable option for the control of phytopathogens. From the surface of healthy mangoes, different bacteria were isolated. For all isolated bacterial strains, we determined their antimicrobial activity against a fungal strain that caused anthracnose in mangoes and against Alternaria alternata, both in the culture medium and directly on mangoes. The bacterial strains with the highest antifungal activity were identified by sequencing the 16s rRNA gene. Two species of Serratia were identified: marcescens and nematodiphila. Finally, the chitinolytic, glucanolytic, and cellulolytic activity and prodigiosin production of bacteria with antifungal activity was determined. Five fungal strains were isolated from mangoes with anthracnose. Only one strain was responsible for anthracnose in mangoes. This fungal strain was identified as Colletotrichum siamense. Against C. siamense and A. alternata in vitro and in mango selected strains of Serratia showed antifungal activity. Finally, the Serratia strains produced chitinases, glucanases, cellulases and prodigiosin, and the two S. marcescens strains did not produce hemolysins. The three Serratia strains isolated in this study can potentially be used in the biological control of anthracnose caused by C. siamense and A. alternata on mango.

Lipase-Catalyzed Synthesis, Antioxidant Activity, Antimicrobial Properties and Molecular Docking Studies of Butyl Dihydrocaffeate.

Green chemistry approaches, such as lipase-catalyzed esterification, are promising methods for obtaining valuable chemical compounds. In the case of the use of lipases, unlike in aqueous environments, the processes of the ester bond formations are encountered in organic solvents. The aim of the current research was to carry out the lipase-catalyzed synthesis of an ester of dihydrocaffeic acid. The synthesized compound was then evaluated for antioxidant and antimicrobial activities. However, the vast majority of its antioxidant activity was retained, which was demonstrated by means of DPPH· (2,2-diphenyl-1-picrylhydrazyl) and CUPRAC (cupric ion reducing antioxidant capacity) methods. Regarding its antimicrobial properties, the antifungal activity against Rhizopus oryzae is worth mentioning. The minimum inhibitory and fungicidal concentrations were 1 and 2 mM, respectively. The high antifungal activity prompted the use of molecular docking studies to verify potential protein targets for butyl ester of dihydrocaffeic ester. In the case of one fungal protein, namely 14-α sterol demethylase B, it was observed that the ester had comparable binding energy to the triazole medication, isavuconazole, but the interacted amino acid residues were different.

Initial In Vitro Assessment of the Antifungal Activity of Aqueous Extracts from Three Invasive Plant Species

The development of new, safe, and effective methods of managing fungal pathogens is required. This study was conducted to perform an initial in vitro assessment of the antifungal activity of water-based plant extracts from three plants which are invasive in Egypt: Prosopis juliflora, Ipomoea carnea, and Leucaena leucocephala. These extracts were tested against three pathogenic fungi species that cause high crop losses in Egypt: Fusarium solani, Alternaria solani, and Colletotrichum circinans. Three extract concentrations, 10%, 20%, and 30%, were tested using a completely randomized design, with three replicates per treatment. Antifungal activity was determined based on the effects of plant extracts on fungal radial growth inhibition, average daily growth of fungi, spore formation, spore germination, and total biomass. Inhibition of the growth of fungal strains increased with increasing plant extract concentration, with the highest inhibitory rate at the 30% extract concentration. In addition, spore density, spore germination, and total biomass decreased significantly with increasing extract concentration. The three fungal pathogens differed in their inhibition and their response to these plant extracts. Prosopis juliflora had the highest inhibitory effect on the three fungal pathogens, compared to the extracts from the other two invasive plants. The results of this feasibility study indicate that P. juliflora extracts have high antifungal activity and follow-up in vivo assays should be conducted to determine their efficacy in the safe and sustainable management of these and other fungal pathogens.

Azadirachta indica A. Juss Fruit Mesocarp and Epicarp Extracts Induce Antimicrobial and Antiproliferative Effects against Prostate (PC-3), Breast (MCF-7), and Colorectal Adenocarcinoma (Caco-2) Cancer Cell Lines through Upregulation of Proapoptotic Genes.

Effective alternative strategies and methodological approaches are critically necessary for cancer prevention and therapy. In this study, we investigated the antitumor potential of neem fruit mesocarp and epicarp extracts. The chemical composition of the derived extracts was characterized using GC–MS. Data were collected on the antimicrobial activity of the extracts in addition to the cytotoxicity effect evaluated against PC-3, MCF-7, and Caco-2 cancer cell lines, compared with the normal Vero cells. Cell-cycle arrest, apoptosis, and expression of apoptosis-related genes were assessed on PC-3 cells. Both extracts had significant antiproliferative effects on all tested cell lines in a dose-dependent manner, with the mesocarp extract being more potent. Both extracts also showed high antibacterial and antifungal activities. These results were related to the chemical constituents of the extracts identified by the GC–MS analysis. The extract of neem fruit mesocarp caused cell-cycle arrest at G2/M phase of PC-3 cells. The cytotoxicity of neem mesocarp extract is strongly correlated with the induction of apoptosis, where it caused downregulation of the antiapoptotic BCL2 gene but upregulation of the proapoptotic P53 and BAX genes. This study showed that neem fruit extract is potential anticancer material in the future.

Design, Synthesis, and Antifungal Activities of Hymexazol Glycosides Based on a Biomimetic Strategy

Hymexazol (HYM) is irreplaceable for treating soil-borne diseases due to its high efficiency and low cost, as a broad-spectrum fungicide. However, when HYM is absorbed by plants, it is rapidly converted into two glycoside metabolites, and the antifungal activities of these glycosides are inferior to that of HYM. Therefore, in this study, to maintain strong antifungal activity in vitro and in vivo, HYM was glycosylated with amino sugars that have diverse biological activities to simulate plant glycosylation. The antifungal experiment proved that glycoside 15 has the highest antifungal activity, and N-acetyl glucosamine and HYM had obvious synergistic effects. According to the structure-activity relationship studies, glycoside 15 had greater numbers of active electron-rich regions and front-line orbital electrons due to the introduction of N-acetyl glucosamine. Moreover, glycoside 15 can significantly promote plant growth and induce an increase in plant defense enzyme activity. Additionally, compared to HYM, the results of electron microscopy and proteomics revealed that glycoside 15 has a unique antifungal mechanism. The promising antifungal activity and interactions with plants mean that glycoside 15 is a potential green fungicide candidate. Furthermore, this research conducted an interesting exploration of the agricultural applications of amino sugars.

Antifungal Activity of Endophytic Bacillus K1 Against Botrytis cinerea.

Gray mold caused by Botrytis cinerea is detrimental to plants and fruits. Endophytes have been shown to modify plant disease severity in functional assays. We conducted this study to investigate the endophytic strain Bacillus K1 with excellently antagonistic B. cinerea from the wild grape endosphere. We identified a wild grape endophytic strain K1 with high antifungal activity against B. cinerea both in vitro and in vivo. Combining the phylogenetic results based on 16S rDNA and genome sequencing, K1 was assigned as Bacillus subtilis. The in vitro results displayed that K1 and its volatile substances could significantly inhibit the mycelia growth of B. cinerea. Grape fruit inoculated with Bacillus K1 showed lower gray mold during treatment. The higher levels of defense-related enzymes, including peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase, were induced in grapes after inoculation. Scanning electron microscopy (SEM) suggested that K1 inhibited mycelial growth via bacterial colonization and antibiosis in grapes. The gas chromatography–mass spectrometry analysis identified 33 volatiles in which dibutyl phthalate was the major compound accounting for 74.28%. Dibutyl phthalate demonstrated strong activity in suppressing the mycelia growth of B. cinerea. Genome bioinformatics analysis revealed that the K1 chromosome harbored many known biosynthesis gene clusters encoding subtilosin, bacillaene, bacillibactin, bacilysin, and fengycin. This study provides a potential biological agent to control diseases of post-harvest grape fruit and improves our understanding of the possible biocontrol mechanisms of the Bacillus strain.

Effectiveness of different accelerated green synthesis methods in zinc oxide nanoparticles using red pepper extract: Synthesis and characterization

Abstract Potential ability of red pepper extract in zinc oxide nanoparticles (ZnO NPs) fabrication via three accelerated heating techniques, namely, conventional heating with stirring (at 100°C and 700 rpm, for 30 min), autoclave (at 15 psi and 121°C, for 15 min), and microwave irradiation (power of 800 W for 3 min) was assessed. Fourier transform infrared spectroscopy and gas chromatography mass spectroscopy indicated that the prepared extract contained 20 active compounds including alcohols, aldehydes, ketones, esters, and organic acids with several functional groups such as hydroxyls. Results indicated that ZnO NPs with spherical and hexagonal structures have been formed using three different heating methods, and the minimum crystal size for the fabricated ZnO NPs was 88.44 nm, which was attained using heating by autoclave. Antioxidant activity of the synthesized ZnO NPs was determined using the red pepper extract, and accuracy of this method was 80.21%. Finally, results indicated that the formed ZnO NPs had high antibacterial activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis and high antifungal activity against Aspergillus niger and Aspergillus flavus.

Pb (II) and Hg (II) thiosemicarbazones for inhibiting the broad‐spectrum pathogen Cladosporium sphaerospermum ASU18 (MK387875) and altering its antioxidant system

Pb (II) and Hg (II) ions were reacted in aqueous solution with two tridentate thiosemicarbazones, resulting in four complexes {[Pb(L1)2] 1, [Pb(L2)2] 2, [Hg(L1)2] 3, and [Hg(L2)2] 4; HL1 = 4‐(4‐nitrophenyl)‐1‐[(pyridin‐2‐yl)methylene]thiosemicarbazide and HL2 = 4‐(2‐chlorophenyl)‐1‐[(pyridin‐2‐yl)methylene]thiosemicarbazide}. The solid‐state structures of complexes 2 and 3 were determined by X‐ray crystallography, which also confirmed the monobasic tridentate NNS nature of the ligands, and the octahedral coordination geometry around the metal centers. Complexes 1–4 exhibited high antifungal activity against the pathogen Cladosporium sphaerospermum; all four complexes give rise to a reduction in the fungal dry mass and sugar consumption. Complexes 2 and 4 that inhibited C. sphaerospermum growth at the concentration of 1.8 mM are more efficient inhibitors than the free ligand and metal ions. Further, the complexes gave varied values of the fungal total antioxidants (TA), superoxide dismutase (SOD), and catalase (CAT) activities dependent on their concentrations.

Synthesis and Bioactivity of Novel Sulfonate Scaffold-Containing Pyrazolecarbamide Derivatives as Antifungal and Antiviral Agents.

Novel pyrazolecarbamide derivatives bearing a sulfonate fragment were synthesized to identify potential antifungal and antiviral agents. All the structures of the key intermediates and target compounds were confirmed by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The single-crystal X-ray diffraction of the compound T22 showed that pyrazole carbamide is a sulfonate. The in vitro antifungal activities of the target compounds against Colletotrichum camelliae, Pestalotiopsis theae, Gibberella zeae, and Rhizoctonia solani were evaluated at 50 μg/ml. Among the four pathogens, the target compounds exhibited the highest antifungal activity against Rhizoctonia solani. The compound T24 (EC50 = 0.45 mg/L) had higher antifungal activity than the commercial fungicide hymexazol (EC50 = 10.49 mg/L) against R. solani, almost similar to bixafen (EC50 = 0.25 mg/L). Additionally, the target compounds exhibited protective effects in vivo against TMV. Thus, this study reveals that pyrazolecarbamide derivatives bearing a sulfonate fragment exhibit potential antifungal and antiviral activities.

Identification of the Causal Agent of Brown Leaf Spot on Kiwifruit and Its Sensitivity to Different Active Ingredients of Biological Fungicides.

Kiwifruit (Actinidia chinensis) is an important commercial crop in China, and the occurrence of diseases may cause significant economic loss in its production. In the present study, a new pathogen that causes brown leaf spot disease on kiwifruit was reported. The fungus was isolated from an infected sample and identified as Fusarium graminearum based on morphological and molecular evaluation. Koch’s postulates were confirmed when the pathogen was re-isolated from plants with artificially induced symptoms and identified as F. graminearum. Based on the biological characteristics of the pathogen, it was determined that: its optimal growth temperature was 25 °C; optimal pH was 7; most suitable carbon source was soluble starch; most suitable nitrogen source was yeast powder; and best photoperiod was 12 h light/12 h dark. Further investigations were conducted by determining 50% effective concentrations (EC50) of several active ingredients of biological fungicides against F. graminearum. The results showed that among the studied fungicides, tetramycin and honokiol had the highest antifungal activity against this pathogen. Our findings provide a scientific basis for the prevention and treatment of brown leaf spot disease on kiwifruit.

Deverra triradiata Hochst. ex Boiss. from the Northern Region of Saudi Arabia: Essential Oil Profiling, Plant Extracts and Biological Activities.

Devrra triradiata Hochst. ex Boiss is an occasional plant species in the Northern region of Saudi Arabia. The shrub is favored on sandy desert wadis, gypsaceous substrate, and sandy gravel desert. In folk medicine, the plant is used for many purposes; to relieve stomach pains, against intestinal parasites, and for the regulation of menstruation. The present study describes the chemical composition of the essential oils (EOs) of different plant parts of D. triradiata. In vivo and in vitro biological activities of plant extracts and essential oils were also studied. Phenylpropanoids, elemicin (flowers: 100%), dillapiole (Stems: 82.33%; and seeds: 82.61%), and apiol (roots: 72.16%) were identified as the major compounds. The highest antioxidant activity was recorded for the EOs of roots and stems (IC50 = 0.282 µg/mL and 0.706 µg/mL, respectively). For plant extracts, ethyl acetate showed the highest antioxidant activities (IC50 = 2.47 and 3.18 µg/mL). EOs showed high antifungal activity against yeasts with low azole susceptibilities (i.e., Malassezia spp. and Candida krusei). The MIC values of EOs ranged between 3.4 mg/mL and 56.4 mg/mL. The obtained results also showed phytotoxic potential of plant extracts both on the germination features of Triticum aestivum seeds and the vegetative growth of seedlings.

Posaconazole-amino acid cocrystals for improving solubility and oral bioavailability while maintaining antifungal activity and low In vivo toxicity

The present work contributes to modulate the biopharmaceutical parameters through co-crystallization of posaconazole. The cocrystals of posaconazole were fabricated with amino acids as co-formers, which are zwitterionic, GRAS status, and biological non-hazardous in nature using solvent evaporation and liquid assistant grinding. Preliminary screening was done by using DSC for the suitability of the co-former. Structural characterizations DSC, PXRD, FT-IT, SEM, 1H FT-NMR were conducted to identify cocrystal formation. The equilibrium solubility, % drug release, and stability of cocrystals were explored. Among the two novel cocrystals, posaconazole-l-glutamine cocrystal showed higher solubility (92.42-fold) and dissolution (7.72-fold) enhancement compared to that of the parent molecule. This may be due to the less enthalpy and weaker crystallinity reported in DSC and PXRD respectively. Moreover, both cocrystals maintained their integrity under different conditions of stability studies after 90 days. FT-NMR results revealed that the hydroxyl and triazole group of posaconazole and carboxyl and amino group of amino acids participated in intermolecular ionic interaction. Under bioavailability study, AUC0.24 of Posaconazole-glutamine cocrystals exhibited 13.14-fold and 2.34-fold enhancement than the pure drug and noxafil suspension respectively. Furthermore, In vitro antifungal activity and In vivo acute toxicity studies on novel cocrystal found to have high antifungal activity and no signs of toxicity even at 5000 mg/kg of body weight respectively. These results indicate that the co-crystallization technique has great potential in modulating the physicochemical properties of poorly water-soluble drugs like posaconazole.

Variation in Anti-inflammatory, Anti-arthritic, and Antimicrobial Activities of Different Extracts of Common Egyptian Seaweeds with an Emphasis on Their Phytochemical and Heavy Metal Contents

The anti-inflammatory, anti-arthritic, and antimicrobial activities of some common Egyptian seaweeds in addition to their phytochemical and heavy metal contents were investigated. Phytochemical screening of the seaweed extracts showed the presence of different primary and secondary metabolites with different concentrations according to their species and the used solvent. The ethanolic extract of Colpmenia sinuosa (CSBE2) exhibited the maximum anti-inflammatory and anti-arthritic activity at 1000 μg/ml concentration compared to other seaweed extracts. The dichloromethane extract of Corallina officinalis (CORM) exerted the highest antimicrobial activity with an average inhibition zone diameter (AV) = 15.29 mm and activity index (AI) = 1.53 and with the highest antagonistic activity against Escherichia coli (28 mm). It is followed by Ulva linza ethanolic extract (ULGE2) which recorded (AV) of 14.71 mm and (AI) of 1.30 with the highest antifungal activity against Candida albicans (30 mm). The collected seaweeds would therefore be a very promising source for treating inflammatory, arthritic, and microbial diseases. Moreover, the investigated seaweeds showed variable concentrations of heavy metals among various species. The mean concentrations of the heavy metals took the following order: Fe > Zn > Mn > Ba > Cu > As > Cr > Ni > Pb > V > Cd > Se > Co > Mo. Based on the permissible limits set by the WHO and CEVA, Pb and Ni in the studied seaweeds were found to be within the permissible limits, whereas Cd and Zn contents were at the borderline. Significant correlations were observed between studied parameters. The estimated daily intakes for most heavy metals were lower than the recommended daily intakes.

Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.