Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering.

d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni2+. Its kcat and kcat/Km values toward d-fructose were measured to be 248.5 min-1 and 2.117 mM-1·min-1, respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.

Biochemical identification of D-mannose 2-epimerase from Cytophagaceae bacterium SJW1-29 for efficient bioconversion of D-glucose to D-mannose.

Enzymatic production of D-mannose attracts increasing attention because of the health effects and commercial values of D-mannose. Several kinds of epimerases or isomerases have been used for enzymatic production of D-mannose from D-glucose or D-fructose. D-Mannose epimerase (MEase), belonging to N-acyl-D-glucosamine 2-epimerase superfamily enzymes, catalyzes the C-2 epimerization between D-glucose and D-mannose. In this study, a novel MEase was identified from Cytophagaceae bacterium SJW1-29. Sequence and structure alignments indicate that it is highly conserved with the reported R. slithyformis MEase with the known crystal structure. It was a metal-independent enzyme, with an optimal pH of 8.0 and an optimal temperature of 40 °C. The specific activities on D-glucose and D-mannose were 2.90 and 2.96 U/mg, respectively. The Km, kcat, and kcat/Km on D-glucose were measured to be 194.9 mM, 2.72 s-1, and 0.014 mM-1 s-1, respectively. The purified enzyme produced 23.15 g/L of D-mannose from 100 g/L of D-glucose at pH 8.0 and 40 °C for 8 h, with a conversion rate of 23.15 %.

Statistical Optimization of Biosurfactant Production from Aspergillus niger SA1 Fermentation Process and Mathematical Modeling.

In this study, we sought to investigate the production and optimization of biosurfactants by soil fungi isolated from petroleum oil-contaminated soil in Saudi Arabia. Forty-four fungal isolates were isolated from ten petroleum oil-contaminated soil samples. All isolates were identified using the internal transcribed spacer (ITS) region, and biosurfactant screening showed that thirty-nine of the isolates were positive. Aspergillus niger SA1 was the highest biosurfactant producer, demonstrating surface tension, drop collapsing, oil displacement, and an emulsification index (E24) of 35.8 mN/m, 0.55 cm, 6.7 cm, and 70%, respectively. This isolate was therefore selected for biosurfactant optimization using the Fit Group model. The biosurfactant yield was increased 1.22 times higher than in the nonoptimized medium (8.02 g/l) under conditions of pH 6, temperature 35°C, waste frying oil (5.5 g), agitation rate of 200 rpm, and an incubation period of 7 days. Model significance and fitness analysis had an RMSE score of 0.852 and a p-value of 0.0016. The biosurfactant activities were surface tension (35.8 mN/m), drop collapsing (0.7 cm), oil displacement (4.5 cm), and E24 (65.0%). The time course of biosurfactant production was a growth-associated phase. The main outputs of the mathematical model for biomass yield were Yx/s (1.18), and µmax (0.0306) for biosurfactant yield was Yp/s (1.87) and Yp/x (2.51); for waste frying oil consumption the So was 55 g/l, and Ke was 2.56. To verify the model's accuracy, percentage errors between biomass and biosurfactant yields were determined by experimental work and calculated using model equations. The average error of biomass yield was 2.68%, and the average error percentage of biosurfactant yield was 3.39%.

Phylogenetic and Expression Studies of Small GTP-Binding Proteins in Solanum lycopersicum Super Strain B.

This investigation involved a comparative analysis of the small GTPase superfamily in S. lycopersicum super strain B compared to their analogues in leguminous and other non-leguminous species. The small GTPases superfamily members were recognized by tBLASTn searches. The sequences of amino acid were aligned using Clustal Omega and the analysis of phylogeny was performed with the MEGA7 package. Protein alignments were applied for all studied species. Three-dimensional models of RABA2, ROP9, and ROP10 from Solanum lycopersicum "Super strain B" were performed. The levels of mRNA of the Rab, Arf, Rop, and Ran subfamilies were detected in aerial tissues vs. roots. Significant divergences were found in the number of members and groups comprising each subfamily of the small GTPases and Glycine max had the highest count. High expression of Rab and Arf proteins was shown in the roots of legumes whilst in non-legume plants, the highest values were recorded in aerial tissues. S. lycopersicum super strain B had the highest expression of Rab and Arf proteins in its aerial tissues, which may indicate that diazotroph strains have supreme activities in the aerial tissues of strain B and act as associated N-fixing bacteria. The phylogenies of the small GTPase superfamily of the studied plants did not reveal asymmetric evolution of the Ra, Arf, Rop, and Ran subfamilies. Multiple sequence alignments derived from each of the Rab, Arf, and Rop proteins of S. lycopersicum super strain B showed a low frequency of substitutions in their domains. GTPases superfamily members have definite functions during infection, delivery, and maintenance of N2-fixing diazotroph but show some alterations in their function among S. lycopersicum super strain B, and other species.

Bioactive Levan-Type Exopolysaccharide Produced by Pantoea agglomerans ZMR7: Characterization and Optimization for Enhanced Production.

Levan is an industrially important, functional biopolymer with considerable applications in the food and pharmaceutical fields owing to its safety and biocompatibility. Here, levan-type exopolysaccharide produced by Pantoea agglomerans ZMR7 was purified by cold ethanol precipitation and characterized using TLC, FTIR, 1H, and 13C NMR spectroscopy. The maximum production of levan (28.4 g/l) was achieved when sucrose and ammonium chloride were used as carbon and nitrogen sources, respectively, at 35°C and an initial pH of 8.0. Some biomedical applications of levan like antitumor, antiparasitic, and antioxidant activities were investigated in vitro. The results revealed the ability of levan at different concentrations to decrease the viability of rhabdomyosarcoma and breast cancer cells compared with untreated cancer cells. Levan appeared also to have high antiparasitic activity against the promastigote of Leishmania tropica. Furthermore, levan had strong DPPH radical scavenging (antioxidant) activity. These findings suggest that levan produced by P. agglomerans ZMR7 can serve as a natural biopolymer candidate for the pharmaceutical and medical fields.

Assessment of fungal diversity in soil rhizosphere associated with Rhazya stricta and some desert plants using metagenomics.

This study aimed to compare the fungal rhizosphere communities of Rhazya stricta, Enneapogon desvauxii, Citrullus colocynthis, Senna italica, and Zygophyllum simplex, and the gut mycobiota of Poekilocerus bufonius (Orthoptera, Pyrgomorphidae, "Usherhopper"). A total of 164,485 fungal reads were observed from the five plant rhizospheres and Usherhopper gut. The highest reads were in S. italica rhizosphere (29,883 reads). Species richness in the P. bufonius gut was the highest among the six samples. Ascomycota was dominant in all samples, with the highest reads in E. desvauxii (26,734 reads) rhizosphere. Sordariomycetes and Dothideomycetes were the dominant classes detected with the highest abundance in C. colocynthis and E. desvauxii rhizospheres. Aspergillus and Ceratobasidium were the most abundant genera in the R. stricta rhizosphere, Fusarium and Penicillium in the E. desvauxii rhizosphere and P. bufonius gut, Ceratobasidium and Myrothecium in the C. colocynthis rhizosphere, Aspergillus and Fusarium in the S. italica rhizosphere, and Cochliobolus in the Z. simplex rhizosphere. Aspergillus terreus was the most abundant species in the R. stricta and S. italica rhizospheres, Fusarium sp. in E. desvauxii rhizosphere, Ceratobasidium sp. in C. colocynthis rhizosphere, Cochliobolus sp. in Z. simplex rhizosphere, and Penicillium sp. in P. bufonius gut. The phylogenetic results revealed the unclassified species were related closely to Ascomycota and the species in E. desvauxii, S. italica and Z. simplex rhizospheres were closely related, where the species in the P. bufonius gut, were closely related to the species in the R. stricta, and C. colocynthis rhizospheres.

Microbial sources of polyunsaturated fatty acids (PUFAs) and the prospect of organic residues and wastes as growth media for PUFA-producing microorganisms.

The discovery of non-fish sources of polyunsaturated fatty acids (PUFAs) is of great biotechnological importance. Although various oleaginous microalgae and fungi are able of accumulating storage lipids (single cell oils - SCOs) containing PUFAs, the industrial applications utilizing these organisms are rather limited due to the high-fermentation cost. However, combining SCO production with other biotechnological applications, including waste and by-product valorization, can overcome this difficulty. In the current review, we present the major sources of fungi (i.e. members of Mucoromycota, fungoid-like Thraustochytrids and genetically modified strains of Yarrowia lipolytica) and microalgae (e.g. Isochrysis, NannochloropsisandTetraselmis) that have come recently to the forefront due to their ability to produce PUFAs. Approaches adopted in order to increase PUFA productivity and the potential of using various residues, such as agro-industrial, food and aquaculture wastes as fermentation substrates for SCO production have been considered and discussed. We concluded that several organic residues can be utilized as feedstock in the SCO production increasing the competitiveness of oleaginous organisms against conventional PUFA producers.

Comparative metagenomics approaches to characterize the soil fungal communities of western coastal region, Saudi Arabia.

A total of 145007 reads were obtained from pyrosequencing for all the 4 samples. The total count ranged from 11,301,014 (Mecca old road) to 23,503,512 bp (Thuwal). A total of 460 fungal species belonging to 133 genera, 58 families, 33 orders, 13 classes and 4 phyla was identified across the four sites. The most abundant phylum at all four sites was Ascomycota followed by Basidiomycota. Four phyla (Ascomycota-99.31%, Basidiomycota-0.59%, Chytridiomycota-0.04%, Glomeromycota-0.03%) were detected in Khulais. Except for Glomeromycota, all phyla were detected at Mecca old road (Ascomycota-74.26%, Basidiomycota-25.71%, Chytridiomycota-0.01%) and Thuwal (Ascomycota-99.59%, Basidiomycota-0.40%, Chytridiomycota-0.002%); while only Ascomycota-90.98% and Basidiomycota-9.01% were detected in Asfan road. At the class level, Sordariomycetes was predominantly observed at Asfan road-59.88%, Khulais-68.26% and Thuwal-94.84%; while Pezizomycetes was dominant at Mecca old road-56.01%, was absent at Asfan road. Agaricomycetes was present only at Mecca old road-25.73%; while Tremellomycetes-5.77%, Malasseizomycetes-2.13% and Microbotryomycetes-1.10% were found only at Asfan road. The phylogenetic trees revealed that clear genus level differences are visible across all the four sites, with an overall predominance of Thielavia followed by Madurella, Aspergillus, and Gelasinospora. Chaetomium sp., Aspergillus caespitosus and Aspergillus sp. were found in moderate (Mecca old road and Thuwal) to abundant (Asfan road and Khulais) quantities. Thielavia sp., Thielavia hyalocarpa and Madurella sp. are found in moderate quantities at Khulais and Mecca old road, while in abundant levels at Asfan road and Thuwal. Fusarium equisati and F. oxysporum were detected at Thuwal and Khulais. Sordaria araneosa was present at Khulais, while Malasseiza globosa species was detected in moderate quantities across all sites except Khulais.

Origin and distribution of Sporothrix globosa causing sapronoses in Asia.

PURPOSE: The aim of the study was to evaluate the main sources and epidemiological patterns and speculate on the evolutionary origin of Sporothrix globosa in Asia. METHODOLOGY: Case and case series literature on sporotrichosis in Asia from January 2007 onwards were reviewed using meta-analysis. Phylogenetic analysis of relevant S. globosa was carried out on the basis of concatenated sequences of ITS, TEF3 and CAL. A haplotype network of CAL sequences of 281 Sporothrix isolates was analysed to determine the population structure of S. globosa. RESULTS: Nearly all cases of sporotrichosis caused by S. globosa in Asia were human. In contrast to the remaining pathogenic Sporothrix species, feline transmission was exceptional; nearly all regional cat-associated cases were caused by Sporothrix schenckii. While the latter species was highly variable and showed recombination, S. globosa seemed to be a clonal offshoot, as was Sporothrix brasiliensis. The origin of the segregants was located in an area of high variability in S. schenckii with a relatively high frequency of Asian strains. CONCLUSION: In Asia, S. globosa was the prevalent species. The low diversity of S. globosa suggested a recent divergence with a founder effect of low variability from the variable ancestral species, S. schenckii.

Two new species of the Fusarium fujikuroi species complex isolated from the natural environment.

Two new species in the Fusarium fujikuroi species complex (FFSC) are introduced. One of these, represented by strain CBS 454.97 was isolated from plant debris (Striga hermonthica) in the Sudan, while the second, represented by strains CBS 119850 and CBS 483.94, which originated from soil in Australia. Molecular analyses were performed including TEF1 spanning 576 bp region, 860 bp region of rPB2, and 500 bp BT2 region. Phylogenetic trees based on these regions showed that the two species are clearly distinct from all known taxa in the F. fujikuroi species complex. Based on phenotypic, physiological characters and molecular data, we introduce Fusarium sudanense and Fusarium terricola as novel species in the complex.

The genus Anthopsis and its phylogenetic position in Chaetothyriales.

The genus Anthopsis was introduced for a black fungus with peculiar, inverted phialides and triangular conidia. The genus accommodates, in addition to the type species Anthopsis deltoidea, which once was reported as a cause of human phaeohyphomycosis, two further taxa: A. catenata and A. microspora. Current taxonomy is mainly based on microscopic structures of phialides. To assess the phylogenetic position of the genus, sequences of the internal transcribed spacer region and partial LSU rDNA were obtained for Anthopsis spp. and compared with sequences from public databases. Phylogenetic analyses based on both loci were used to assess the evolutionary relationships of Anthopsis spp. at the family and ordinal levels. Anthopsis s.str. was found to cluster in Chaetothyriales, while A. catenata proved to be of helotialean affinity. Thermotolerance and morphology of each species were recorded.

Nomenclatural notes on Nadsoniella and the human opportunist black yeast genus Exophiala.

The opportunistic black yeast are particularly known through the genus Exophiala, characterised by annellidic budding cells. However, this phenotype is polyphyletic within the order Chaetothyriales. Seventeen generic names are available in the family Herpotrichiellaceae, one of which is Exophiala. Future taxonomy will be based on molecular phylogeny; each multi-species clade may qualify for one of these names. This paper focuses on the genus Nadsoniella, which is the oldest valid name in the Herpotrichiellaceae. Despite its exophiala-like phenotype, the type species of Nadsoniella clusters in the jeanselmei-clade, competing with the sympodial genus Rhinocladiella. In contrast, Exophiala competes with morphologically pronounced genera Thysanorea and Veronaea. Replacing the current phenotypic system for phylogenetic nomenclature requires highly stable phylogenies, which currently are not available.

Metagenomic analysis of fungal taxa inhabiting Mecca region, Saudi Arabia.

The data presented contains the sequences of fungal Internal Transcribed Spacer (ITS) and 18S rRNA gene from a metagenome of the Mecca region, Saudi Arabia. Sequences were amplified using fungal specific primers, which amplified the amplicon aligned between the 18S and 28S rRNA genes. A total of 460 fungal species belonging to 133 genera, 58 families, 33 orders, 13 classes and 4 phyla were identified in four contrasting locations. The raw sequencing data used to perform this analysis along with FASTQ file are located in the NCBI Sequence Read Archive (SRA) under accession numbers: SRR3150823, SRR3144873, SRR3150825 and SRR3150846.

Fatty acid constituents of Peganum harmala plant using Gas Chromatography-Mass Spectroscopy.

Fatty acid contents of the Peganum harmala plant as a result of hexane extraction were analyzed using GC-MS. The saturated fatty acid composition of the harmal plant was tetradecanoic, pentadecanoic, tridecanoic, hexadecanoic, heptadecanoic and octadecanoic acids, while the saturated fatty acid derivatives were 12-methyl tetradecanoic, 5,9,13-trimethyl tetradecanoic and 2-methyl octadecanoic acids. The most abundant fatty acid was hexadecanoic with concentration 48.13% followed by octadecanoic with concentration 13.80%. There are four unsaturated fatty acids called (E)-9-dodecenoic, (Z)-9-hexadecenoic, (Z,Z)-9,12-octadecadienoic and (Z,Z,Z)-9,12,15-octadecatrienoic. The most abundant unsaturated fatty acid was (Z,Z,Z)-9,12,15-octadecatrienoic with concentration 14.79% followed by (Z,Z)-9,12-octadecadienoic with concentration 10.61%. Also, there are eight non-fatty acid compounds 1-octadecene, 6,10,14-trimethyl-2-pentadecanone, (E)-15-heptadecenal, oxacyclohexadecan-2 one, 1,2,2,6,8-pentamethyl-7-oxabicyclo[4.3.1]dec-8-en-10-one, hexadecane-1,2-diol, n-heneicosane and eicosan-3-ol.

Arthrocladium, an unexpected human opportunist in Trichomeriaceae (Chaetothyriales).

The family Trichomeriaceae (Chaetothyriales) mainly comprises epiphytic and epilithic organisms. In some species elaborate ascomata are formed, but for the great majority the species no asexual conidium formation is known other than simple fragmentation of the thallus. The present paper re-establishes the genus Arthrocladium with three non-sporulating species. One of these is described for a strain causing a fatal infection in a human patient with a rare genetic immune disorder.

Dermatophytes and other associated fungi in patients attending to some hospitals in Egypt.

Dermatophytes are keratinophilic fungi that infect keratinized tissues causing diseases known as dermatophytoses. Dermatophytes are classified in three genera, Epidermophyton, Microsporum, and Trichophyton. This investigation was performed to study the prevalence of dermatomycosis among 640 patients being evaluated at the dermatology clinics at Kasr elainy, El-Husein and Said Galal hospitals in Cairo and Giza between January 2005 and December 2006. The patients were checked for various diseases. Tinea capitis was the most common clinical disease followed by tinea pedis and tinea corporis. Tinea cruris and tinea unguium were the least in occurrence. Tinea versicolor also was detected. The most susceptible persons were children below 10 years followed by those aged 31-40 years. Unicellular yeast was the most common etiological agent and T. tonsuranswas the second most frequent causative agent followed by M. canis.

Dermatophytes and other associated fungi in patients attending to some hospitals in Egypt

Dermatophytes are keratinophilic fungi that infect keratinized tissues causing diseases known as dermatophytoses. Dermatophytes are classified in three genera, Epidermophyton, Microsporum, and Trichophyton. This investigation was performed to study the prevalence of dermatomycosis among 640 patients being evaluated at the dermatology clinics at Kasr elainy, El-Husein and Said Galal hospitals in Cairo and Giza between January 2005 and December 2006. The patients were checked for various diseases. Tinea capitis was the most common clinical disease followed by tinea pedis and tinea corporis. Tinea cruris and tinea unguium were the least in occurrence. Tinea versicolor also was detected. The most susceptible persons were children below 10 years followed by those aged 31–40 years. Unicellular yeast was the most common etiological agent and T. tonsuranswas the second most frequent causative agent followed by M. canis.

Enteroaggregative Escherichia coli in diarrheic children in Egypt: molecular characterization and antimicrobial susceptibility.

INTRODUCTION: Little information is available regarding the significance of enteroaggregative Escherichia coli (EAEC) in pediatric diarrhea in Egypt. METHODOLOGY: Escherichia coli was isolated from stool samples of 62 diarrheic and 43 non-diarrheic (control) Egyptian children. Samples were screened for genes specific for enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), and enteroinvasive E. coli (EIEC) using polymerase chain reaction (PCR). Diarrheagenic E. coli were grouped phylogenetically using PCR and tested for their susceptibility to antibiotics using the disk diffusion method. Isolates designated as EAEC were examined for eight virulence factors (VFs) using PCR. RESULTS: EAEC was detected in 19 (30.7%) and 4 (9.3%), EPEC in 2 (3.2%) and 1 (2.3%), and ETEC in 2 (3.2%) and 0 (0.0%) diarrheic and control children, respectively; STEC and EIEC were not detected. Only EAEC was significantly isolated from diarrheic children compared with controls (p < 0.01, OR = 4.31).Three or more VFs (multivirulent isolates) were found in 52.6% and 50% of EAEC isolated from diarrheic children and controls, respectively. More than 73% (17/23) of EAEC isolates were identified as belonging to phylogenetic group D. Multiple-antibiotic resistance (resistance to three or more drugs) was observed in more than 91% of EAEC. CONCLUSIONS: Multivirulent EAEC is a significant causative agent of pediatric diarrhea in Egypt, with the majority of isolated EAEC belong to phylogenetic group D. Multiple-antibiotic resistance among EAEC has the potential to be a serious public health problem for the country.

Characterization and chemical composition of fatty acids content of watermelon and muskmelon cultivars in Saudi Arabia using gas chromatography/mass spectroscopy.

BACKGROUND: The growth in the production of biodiesel, which is principally fatty acid methyl esters (FAME), has been phenomenal in the last ten years because of the general desire to cut down on the release of greenhouse gases into the atmosphere, and also as a result of the increasing cost of fossil fuels. OBJECTIVE: Establish whether there is any relationship between two different species (watermelon and muskmelon) within the same family (Cucurbitaceae) on fatty acid compositions and enumerate the different fatty acids in the two species. MATERIALS AND METHODS: Extraction of fatty acids from the two species and preparation the extract to gas chromatography/mass spectroscopy analysis to determine the fatty acids compositions qualitatively and quantitatively. RESULTS: The analyzed plants (watermelon and muskmelon) contain five saturated fatty acids; tetrdecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid and octadecanoic acid with different concentrations, while muskmelon contains an extra saturated fatty acid named eicosanoic acid. The watermelon plant contains five unsaturated fatty acids while muskmelon contains three only, the two plants share in two unsaturated fatty acids named 9-hexadecenoic acid and 9-octadecenoic acid, the muskmelon plant contains higher amounts of these two acids (2.04% and 10.12%, respectively) over watermelon plant (0.88% and 0.25%, respectively). CONCLUSION: The chemical analysis of watermelon and muskmelon revealed that they are similar in saturated fatty acids but differ in unsaturated fatty acids which may be a criterion of differentiation between the two plants.

Molecular characterization of diarrheagenic Escherichia coli from Libya.

Diarrheagenic Escherichia coli (DEC) are important enteric pathogens that cause a wide variety of gastrointestinal diseases, particularly in children. Escherichia coli isolates cultured from 243 diarrheal stool samples obtained from Libyan children and 50 water samples were screened by polymerase chain reaction (PCR) for genes characteristic of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC). The DEC were detected in 21 (8.6%) children with diarrhea; 10 (4.1%) cases were identified as EAEC, 3 (1.2%) as EPEC, and 8 (3.3%) were ETEC; EHEC, and EIEC were not detected. All DEC were grouped phylogenetically by PCR with the majority (> 70%) identified as phylogenetic groups A and B1. The EAEC isolates were also tested for eight genes associated with virulence using PCR. Multi-virulence (≥ 3 virulence factors) was found in 50% of EAEC isolates. Isolated EAEC possessed different virulence traits and belonged to different phylogenetic groups indicating their heterogeneity.