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New safety and rapid method for extraction of genomic DNA from bacteria and yeast strains suitable for PCR amplifications

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Abstract

Molecular biology studies based on PCR amplification require the extraction of good quality genomic DNA. In this study, freeze- sudden thawing as a safety, new, quick and low-cost genomic DNA extraction technique from gram-negative and gram-positive bacteria, and Saccharomyces and non- Saccharomyces yeasts for PCR applications has been developed. This technique does not require any risky chemicals, enzymes and/or extra purification steps. DNA extracted by this method is suitable for PCR applications using random and specific primers such as RAPD-PCR for fingerprinting and DNA sequencing of the 16S rDNA or the D1/D2 domain of the 26S rDNA for molecular identification of bacterial and yeast isolates respectively.

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... The identification was performed based on 16S rRNA gene sequence analysis. Genomic DNA was extracted from the isolated bacterium strain according to our previously described method [16]. PCR amplification was performed using common primers: 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3). ...
... The 16S rRNA gene sequences and phylogenetic analysis were considered as powerful tools for the identification of bacterial isolates [16,[21][22][23]. ...
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Polyhydroxybutyrates (PHBs) are macromolecules synthesized by bacteria. Because of their fast degradability under natural environmental conditions, PHBs were selected as alternatives for the production of biodegradable plastics. Sixteen PHB-accumulating strains were selected and compared for their ability to accumulate PHB granules inside their cells. Isolate AS-02 was isolated from cattle manure and identified as Bacillus wiedmannii AS-02 OK576278 by means of 16S rRNA analysis. It was found to be the best producer. The optimum pH, temperature, and incubation period for the best PHB production by the isolate were 7, 35 °C, and 72 h respectively. PHB production was the best with peptone and glucose as nitrogen and carbon sources at a C/N ratio of (2:1). The strain was able to accumulate 423, 390, 249, 158, and 144 mg/L PHB when pretreated orange, mango, banana, onion peels, and rice straw were used as carbon sources, respectively. The extracted polymer was characterized by Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), and GC-MS spectroscopy, which confirmed the structure of the polymer as PHB. The isolate B. wiedmannii AS-02 OK576278 can be considered an excellent candidate for industrial production of PHB from agricultural wastes.
... Genomic DNA was extracted from fungus and bacterium isolates. The method described in detail by Hesham [11] was used for bacterial genomic extraction, while a DNeasy plant mini kit (Qiagen, USA) was used for fungal genomic DNA according to the company's instructions. The purity and quantity of DNA were examined by recording UV absorption spectra and performing 1% agarose gel electrophoresis. ...
... The series concentration of MR (17-3500 mM) was prepared to determine the azoreductase kinetics, and all other constituents of the reaction mixture were constant. Kinetic values such as Michaelis-Menten constant (K max) and maximal velocity (Vmax ) were calculated from Line weaver-Burk doubles reciprocal plots [11]. ...
Article
Background The presence of anthraquinone (Disperse blue 64) and azodyes (Acid yellow 17) in a waterbody are considered among the most dangerous pollutants. Methods In this study, two different isolated microbes, bacterium and fungus, were individually and as a co-culture applied for degradation of Disperse Blue 64 (DB 64) and Acid Yellow 17 (AY 17) dyes. The isolates were genetically identified based upon 16S (for bacteria) and ITS/5.8S (for fungus) rRNA genes sequences as Pseudomoans aeruginosa and Aspergillus flavus respectively. Results The fungal/bacterial consortium exhibited a higher percentage of dyes degradation than the individual strains even at high concentration of 300 mg/L. Azoreductase could be identified as the main catabolic enzyme and the consortium could induce azoreductase enzyme in the presence of both dyes. However, the specific substrate which achieved the highest azoreductase specific activity was Methyl red (MR) (3.5 U/mg protein). The tentatively proposed metabolites that were detected by HPLC/MS suggested that the reduction process catalyzed the degradation of dyes. The metabolites produced by the action consortium on two dyes were safe on Vicia faba and Triticum vulgaris germination and health of seedlings. Toxicity of the dyes and their degradation products on the plant was different according to the type and chemistry of these compounds as well as the type of irrigated seeds. Conclusion We submit that the effective microbial degradation of DB64 and AY17 dyes will lead to safer metabolic products.
... The identification was performed based on 16S rDNA sequence analysis. Genomic DNA was extracted from the isolated bacterium strain according to our previously described method [25]. The 16S rDNA PCR amplification was performed using universal primers: ...
... The sequences obtained were then aligned with known 16S rDNA sequences in GenBank database using the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/), the obtained 16S rDNA sequences were aligned and compared with the known 16S rDNA sequences in Genbank database to check the closest available database sequences. To determine the taxonomic position of the isolates, the phylogenetic tree was constructed with MEGA version 4.0 using a neighbor-joining algorithm, plus the Jukes Cantor distance estimation method with bootstrap analyses for 100 replicates was performed [25]. The sequence of the strain has been deposited in the GenBank nucleotide sequence database. ...
Article
Background Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies. Objective Detection the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determine the variation in the concentration of both nahAC and C23O catabolic genes along 15 days of the incubation period. Methods In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique on mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture was determined by quantitative PCR. Results Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of pheanthrene degradation. Conclusion The nahAc and C23O genes are the main marker genes for the phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.
... Genomic DNA was extracted from the isolated yeast strains according to our previously described method [18]. The D1/D2 domain of the 26S rRNA gene was amplified using universal primers NL1 (5 0 -GCATATCAATAAGCGGAGGAAAAG-3 0 ) and NL4 (5 0 -GGTCCGTGTTTCAAGACGG-3 0 ) and the PCR conditions and parameters described by Kurtzman and Robnett [19]. ...
... gov/BLAST/). To determine the exact taxonomic positions of the isolates, phylogenetic trees were constructed using MEGA version 4.0, as described previously [18]. The partial 26S rRNA gene sequences obtained in this study for strains KKU-L42 and KKU-L53 have been deposited in the DNA Data Bank of Japan (www.ddbj.nig.ac.jp/), the European Molecular Biology Laboratory (www.embl.de/), ...
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Citric acid is a commercially valuable organic acid widely used in food, pharmaceutical, and beverage industries. In this study, 260 yeast strains were isolated from soil, bread, juices, and fruits wastes and preliminarily screened using bromocresol green agar plates for their ability to produce organic acids. Overall, 251 yeast isolates showed positive results, with yellow halos surrounding the colonies. Citric acid production by 20 promising isolates was evaluated using both free and immobilized cell techniques. Results showed that citric acid production by immobilized cells (30–40 g/L) was greater than that of freely suspended cells (8–19 g/L). Of the 20 isolates, two (KKU-L42 and KKU-L53) were selected for further analysis based on their citric acid production levels. Immobilized KKU-L42 cells had a higher citric acid production rate (62.5%), while immobilized KKU-L53 cells showed an ∼52.2% increase in citric acid production compared with free cells. The two isolates were accurately identified by amplification and sequence analysis of the 26S rRNA gene D1/D2 domain, with GenBank-based sequence comparison confirming that isolates KKU-L42 and KKU-L53 were Candida tropicalis and Pichia kluyveri, respectively. Several factors, including fermentation period, pH, temperature, and carbon and nitrogen source, were optimized for enhanced production of citric acid by both isolates. Maximum production was achieved at fermentation period of 5 days at pH 5.0 with glucose as a carbon source by both isolates. The optimum incubation temperature for citric acid production by C. tropicalis was 32 °C, with NH4Cl the best nitrogen source, while maximum citric acid by P. kluyveri was observed at 27 °C with (NH4)2 SO4 as the nitrogen source. Citric acid production was maintained for about four repeated batches over a period of 20 days. Our results suggest that apple and banana wastes are potential sources of novel yeast strains; C. tropicalis and P. kluyveri which could be used for commercial citric acid production.
... 29 The robustness of the most parsimonious trees was evaluated by 100 bootstrap replications. 30,31 The best optimal model of nucleotide substitution for the ML analyses was determined using Smart Model Selection (SMS) version 1.8.1. 32 The phylogenetic tree was drawn using MEGA X, 33 and edited using Microsoft Power Point (2016) and saved as TIF file. ...
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Citation: Moharram AM, Zohri AA, Hesham AE, Maher MA, Al-Bedak OAMS. Production of Cocktail Enzymes by Three
... They were genetically identified by sequencing the D1/D2 domain of the 26S rDNA region and phylogenetic analysis. The extraction of total yeast genomic DNA was performed according to the procedures described by Hesham (2014). The DNA was amplified using primers described by Kurtzman and Robnett (1998). ...
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This research aimed at the production of bioethanol from a cheap and renewable resource (spoilage dates) by nonconventional yeasts to reduce total cost of the production. Chemical, physical or biological pretreatment of the spoilage date juice (SDJ) did not affect the availability of fermentable sugars significantly. The isolated osmotolerant yeast strains: Pichia kudriavzevii KKUY-0034, Hanseniaspora opuntiae KKUY-0152 and H. uvarum KKUY-0078, which were genetically identified based on sequences of D1/D2 domain 26S rRNA gene and phylogenetic analysis, were tested for their fermentability of the SDJ. The fermentation conditions were adjusted to induce the maximum production of ethanol. Results showed that the highest quantities of ethanol were obtained when the yeasts were grown on 20% of date juice at 30°C and when the pH was adjusted at 4-6 for 60 h. Addition of either Zn or Mg (0.4 g/L) and NH4H2PO4 (4 g/L) had a good impact on the ethanol productivity by the three species, however, H. uvarum KKUY-0078 was the leader that produced 60 g/L of ethanol. In 7-L fermentor, when the optimum conditions were kept constant, ethanol production reached to 80 g/L after 60 h. The study concludes that SDJ is a promising and costless substrate for production of the bioenergy and using the osomtolerant yeasts is an economic strategy. The partial 26S rRNA gene sequences of P. kudriavzevii KKUY-0034, H. uvarum KKUY-0078 and H. opuntiae KKUY-0152 were deposited in the DDBJ, EMBL, and GenBank database under the accession Nos. JQ690250, JQ690236 and KC110834, respectively.
... Determination. The genomic DNA was isolated from strain ASU-06 according to the method described by Hesham [16] and the 16S rRNA gene was amplified. Amplification was carried out with universal primers: 27F (5-AGAGTTTGATCCTGG-CTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) in a final volume of 50 L containing 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , each dNTP at a concentration of 0.2 mM, 1.25 IU of Taq polymerase, each primer at a concentration of 0.2 mM, and 1 L of the DNA template. ...
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Polycyclic aromatic hydrocarbons (PAHs) are serious pollutants and health hazards. In this study, 15 PAHs-degrading bacteria were isolated from Egyptian oily soil. Among them, one Gram-negative strain (ASU-06) was selected and biodegradation ability and initial catabolic genes of petroleum compounds were investigated. Comparison of 16S rRNA gene sequence of strain ASU-06 to published sequences in GenBank database as well as phylogenetic analysis identified ASU-06 as Sphingomonas koreensis. Strain ASU-06 degraded 100, 99, 98, and 92.7% of 100 mg/L naphthalene, phenanthrene, anthracene, and pyrene within 15 days, respectively. When these PAHs present in a mixed form, the enhancement phenomenon appeared, particularly in the degradation of pyrene, whereas the degradation rate was 98.6% within the period. This is the first report showing the degradation of different PAHs by this species. PCR experiments with specific primers for catabolic genes alkB, alkB1, nahAc, C12O, and C23O suggested that ASU-06 might possess genes for aliphatic and PAHs degradation, while PAH-RHDαGP gene was not detected. Production of biosurfactants and increasing cell-surface hydrophobicity were investigated. GC/MS analysis of intermediate metabolites of studied PAHs concluded that this strain utilized these compounds via two main pathways, and phthalate was the major constant product that appeared in each day of the degradation period.
... DNA isolation, amplification conditions with tem perature time profiles for different genes, and con struction of phylogenetic trees. The extraction of total bacterial genomic DNA was performed according to the procedures described by Hesham [19]. Molecular genetics identification of the isolate was done by the analysis of partial sequences of the two genes (16S rRNA gene and RNA polymerase β subunit gene (rpoB gene). ...
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Natural rubber latex is one of the problems that raises the environmental concerns. In this study the degrading ability of Ficus elastica rubber latex by a bacterium strain ASU-03, isolated from Egyptian soil was assessed. The strain was able to produce clear zone around its colony on latex rubber containing medium and was identified by conventional methods as Streptomyces sp. Phylogenetic analysis of 16S rRNA (16S rRNA) and RNA polymerase ß-subunit (rpoB) genes were applied. Results of the 16S rRNA gene analysis revealed that the strain was highly related to Streptomyces sp. (100% similarity), so the rpoB gene was partially sequenced to clarify the specific name of the isolate. Phylogenetic tree based on rpoB gene sequences indicated that strain ASU-03 was highly similar to the reference strain Streptomyces labedae and both were shared a one cluster. The current results demonstrated that the use of a rpoB gene-based method gives a better resolution in the species level identification. To our knowledge, this species has never been reported to be involved in natural rubber degradation. This was therefore the first report about the degradation of Ficus elastic by S. labedae. The degradation of Ficus elastica rubber latex was determined by measuring the increase in protein content of bacterium (mg/g dry wt), reduction in molecular weight (g/mol) and inherent viscosity (dL/g) of the latex. Moreover the degradation was also confirmed by formation of aldehyde or keto group by Schiff’s reagent and by observing the growth of the Streptomyces strain using scanning electron microscopy.
... The Genomic DNA from isolated bacteria was extracted according to the method described by (Hesham, 2014) and the 16S rRNA gene was amplified. Gene amplification was done with universal primers: forward 27F (5-AGAGTTTGATCCTGGCTCAG-3) and reverse 1492R (5-CGGCTACCTTGTTACGACTT-3) (Lane, 1991). ...
Article
Nowadays, most of the pathogenic bacteria become resistant to antibiotics. Therefore, the pharmaceutical properties of the natural plant extracts have become of interest to researchers as alternative antimicrobial agents. In this study, antibacterial activities of extract gained from Acacia etbaica, Acacia laeta, Acacia origena and Acacia pycnantha have been evaluated against isolated pathogenic bacteria (Strains MFM-01, MFM-10 and AH-09) using agar well diffusion methods. The bacterial strains were isolated from infected individuals, and their exact identification was detected on the basis of 16S rRNA gene amplification and sequence determination. Alignment results and the comparison of 16S rRNA gene sequences of the isolates to 16S rRNA gene sequences available in Gen Bank database as well as the phylogenetic analysis confirmed the accurate position of the isolates as Klebsiella oxytoca strain MFM-01, Staphylococcus aureus strain MFM-10 and Klebsiella pneumoniae strain AH-09. Except for cold water, all tested solvents (Chloroform, petroleum ether, methanol, diethyl ether, and acetone) showed variation in their activity against studied bacteria. GC-MS analysis of ethanol extracts showed that four investigated Acacia species have different phytocomponents. Eight important pharmaceutical components were found in the legume of Acacia etbaica, seven in the legume of Acacia laeta, fifteen in the legume of Acacia origena and nine in the leaves of Acacia pycnantha. A dendrogram was constructed based on chemical composition, revealed that Acacia laeta is more closely related to Acacia etbaica forming one clade, whereas Acacia origena less similar to other species. Our results demonstrated that, investigated plants and chemical compounds present could be used as promising antibacterial agents.
... The genomic DNA was isolated from the ASU-035 according to the method described by Hesham (2014). The 16S rRNA gene was amplified with universal primers: 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) (Lane 1991). ...
Article
The use of bacteria is a good method for biodegradation of polycyclic aromatic hydrocarbons due to their fast-growing and cost effectiveness. Bacteria can completely mineralize such toxic compounds to harmless by-products. ASU-035 is a bacterial strain, which was isolated from oil contaminated soil. It was identified as Achromobacter denitrificans on the basis of the nucleotide sequence of 16S rRNA gene, and deposited in GenBank under accession number KC342253. Achromobacter denitrificans have been tested for utilization of pyrene as a sole source of carbon and energy. Some pyrene degradative enzymes and ability of this strain for biosurfactants production have been studied. The results illustrated that the strain could utilize 76.5 mg/L of pyrene (100 mg/L) within 18 days with growth rate and mean generation time 0.033 h−1 and 30.3 h, respectively. The maximum specific activities (U/mg protein) of pyrene dioxygenase, catechol 1,2 dioxygenase (C12O), and catechol 2,3 dioxygenase (C23O) were 0.78 ± 0.12, 0.41 ± 0.05, and 0.2 ± 0.01, respectively. Achromobacter denitrificans could enhance the bioavailability of pyrene to get use of it as a sole carbon source by increasing the emulsification activity to 11.5 ± 2.1 U/mg proteins and cell-surface hydrophobicity to 39 ± 1.3 %.
... Molecular identification was done by 16S rRNA sequencing. The genomic DNA of strain JMG-01 was extracted according to the method described by Hesham (2014). Universal primer 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-CGGCTACCTTGTTACGACTT-3) were used for PCR amplification of the 16S rRNA gene. ...
Article
The present study displays the biodegradation capacities of native bacteria toward polycyclic aromatic hydrocarbons (PAHs) with particular emphasis to anthracene. A total of 23 bacterial strains were isolated from hydrocarbon contaminated sites of Guwahati city; using anthracene as the carbon source. Among all these isolates, one Gram-positive strain (JMG-01) was selected as an efficient anthracene degrader, based on its maximum growth ability in anthracene enriched medium (100 ppm – 700 ppm). At 500 ppm concentration, strain JMG-01 showed the maximum growth rate with 98% of anthracene degradation within 21 days of observation. The strain also demonstrated its potentiality by utilizing higher molecular hydrocarbons like naphthalene, pyrene, and benzo(a)pyrene at 500 ppm. The morphological, biochemical and molecular characterization identified the strain as Bacillus cereus. Surface morphology of the biomass, captured by Atomic Force Microscope (AFM) showed a distinctive modification, during the process of degradation. Study revealed that the effect of hydrocarbon exhibited the alteration, which concurrently enhanced the metabolic activity. Further, Gas chromatography-mass spectrometer (GC-MS) analysis elucidates the possible metabolic pathway of anthracene degradation, depending on the intermediate metabolites produced. The finding thus suggests the essence of Bacillus cereus strain JMG-01 in enhanced anthracene degradation along the utilization of other hydrocarbons.
... Genomic DNA of the strain was extracted using the method as described previously (Hesham, 2014). A universal primer set consisting of 27F and 1492R was used to amplify the 16S rRNA gene (Lane, 1991;Topp et al., 2000). ...
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Aflatoxin M1 (AFM1) is a potent mycotoxin which causes serious health concerns in developing countries, where it is mainly found in milk, meat, and other foods. Biological detoxification is a promising method for eliminating AFM1. The aim of this work was to search for AFM1‐degrading bacterial strains from animal waste, soil, and activated sludge. High‐performance liquid chromatography and Fourier‐transform infrared spectroscopy were used to analyze the AFM1 degradation products. A strain designated E‐1‐1‐1 was obtained from African elephants feces, with the degradation ratio of AFM1 reaching 89.55% in 12 hr. Based on morphology, physiological and biochemical tests, and 16S rRNA gene sequence analysis, strain E‐1‐1‐1 was identified as Bacillus pumilus. The culture supernatant of B. pumilus E‐1‐1‐1 degraded AFM1 effectively, whereas the cells and cell extracts of B. pumilus E‐1‐1‐1 were far less effective. Carbon and nitrogen sources had highly significant effects on the degradation of AFM1 by B. pumilus E‐1‐1‐1. The AFM1‐degrading strain, B. pumilus E1‐1‐1, could have great potential in industrial applications. The biological detoxification is a promising method for eliminating AFM1. The goals this work was to search for AFM1‐degradation strains from the animal waste, soil and activated sludge. A strain named as E‐1‐1‐1 was obtained from African Elephants, which the degradation ratio of AFM1 could reach 89.55% in 72 hr.
... 29 The robustness of the most parsimonious trees was evaluated by 100 bootstrap replications. 30,31 The best optimal model of nucleotide substitution for the ML analyses was determined using Smart Model Selection (SMS) version 1.8.1. 32 The phylogenetic tree was drawn using MEGA X, 33 and edited using Microsoft Power Point (2016) and saved as TIF file. ...
Article
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The current research demonstrates the biotechnological economization of accumulated and inefficiently used agro-industrial orange peel wastes to generate amylase, endoglucanase, exoglucanase, pectinase, and xylanase, industrially essential enzymes with growing demands in enzyme markets, from three Cladosporium isolates. In submerged fermentation (SmF) at 10°C, the isolate AUMC 10865 produced the highest level of amylase (4164 IU/gram dry substrate). Endoglucanase, exoglucanase and xylanase had development peaks (923 IU/gds, 2280 IU/gds, and 1646 IU/gds, respectively in case of Cladosporium sp. AUMC 11366. Pectinase produced the most (7840 IU/gds) in the strain AUMC 11340. At 30°C, the strain AUMC 11340 secretes the most amylase (4120 IU/gds), endoglucanase (2700 IU/gds) and xylanase (3220 IU/gds). Exoglucanase development reached the peak (8750 IU/gds) in the isolate AUMC 10865. The overall production (5570 IU/gds) was instead enhanced by pectinase in the AUMC 11366 isolate. In solid-state fermentation (SSF) at 10°C, the isolate AUMC 10865 outperformed the other two isolates producing 640.0 IU/gds amylase, 763.3 IU/gds endoglucanase, 771.0 IU/gds exoglucanase, 1273.23 IU/gds pectinase and 1062.0 IU/gds xylanase, while the isolate AUMC 11366 produced the least amount of 399.7 IU/gds, 410.0 IU/gds, 413.3 IU/gds, 558.7 IU/gds, and 548.0 IU/gds, respectively. At 30°C, the isolate AUMC 11340 was superiorly producing higher levels of amylase (973.3 IU/gds), endoglucanase (746.0 IU/gds), exoglucanase (1052.0 IU/gds), pectinase (1685.3 IU/gds) and xylanase (1340.0 IU/gds), whereas isolate AUMC 10865 generated the least amounts of amylase (556.7 IU/gds) and exoglucanase (452.7 IU/gfs), and the isolate AUMC 11366 produced the least endoglucanase (256.3 IU/gds), pectinase (857.7 IU/gfs) and xylanase (436.3 IU/gds) amounts.
... The extraction of total yeast genomic DNA was performed according to the procedures described by Hesham [10]. ...
... DNA extraction and 26S rRNA gene D1/D2 domain amplification for amylase producing yeast: Total genomic DNA from yeast isolate AUN-H100 with the most promising ability to produce -amylase was isolated 13 . The 26S rDNA D1/D2 domain region was amplified using the primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) 19 . ...
Article
A total of 12 yeast isolates recovered from rice were screened for their amylase activity. All isolates showed positive results and according to clear zone measurements and Enzyme Activity Index (EAI), 4 isolates were recorded as high amylase producers, 3 moderate and 5 low. Amylase activity for all isolates was estimated using the DNS method. A potent strain AUN-H100 was recorded and identified by sequencing the variable D1/D2 domain of the large subunit (26S) rDNA region as Candida tropicalis AUN-H100. The strain exhibited an enzyme index of 5.1 on the solid medium and showed amylase activity of 6.050 IU/ml/min in submerged fermentation. Amylase activity was also optimized at different pH values ranging from 3 to 10 and different nitrogen sources namely yeast extract peptone, sodium nitrate, sodium nitrite and ammonium chloride at five temperatures of 25, 30, 35, 40 and 45 °C respectively. C. tropicalis AUN-H100 showed its maximum enzyme activity of 21.123 ± 2.060 IU/ml/min at pH 5 and 30 °C using ammonium chloride as nitrogen source in submerged fermentation.
... Total genomic DNA from yeast isolate AUN-S18 with the most promising ability to biosynthesis of Ag NPs was isolated, according [20]. The 26S rDNA D1/D2 domain region was amplified using the primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 ...
... DNA extraction and 26S rRNA gene D1/D2 domain amplification for amylase producing yeast: Total genomic DNA from yeast isolate AUN-H100 with the most promising ability to produce -amylase was isolated 13 . The 26S rDNA D1/D2 domain region was amplified using the primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) 19 . ...
Article
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A total of 12 yeast isolates recovered from rice were screened for their amylase activity. All isolates showed positive results and according to clear zone measurements and Enzyme Activity Index (EAI), 4 isolates were recorded as high amylase producers, 3 moderate and 5 low. Amylase activity for all isolates was estimated using the DNS method. A potent strain AUN-H100 was recorded and identified by sequencing the variable D1/D2 domain of the large subunit (26S) rDNA region as Candida tropicalis AUN-H100. The strain exhibited an enzyme index of 5.1 on the solid medium and showed amylase activity of 6.050 IU/ml/min in submerged fermentation. Amylase activity was also optimized at different pH values ranging from 3 to 10 and different nitrogen sources namely yeast extract peptone, sodium nitrate, sodium nitrite and ammonium chloride at five temperatures of 25, 30, 35, 40 and 45 °C respectively. C. tropicalis AUN-H100 showed its maximum enzyme activity of 21.123 ± 2.060 IU/ml/min at pH 5 and 30 °C using ammonium chloride as nitrogen source in submerged fermentation.
... Ten-fold serial dilutions of spore suspension were prepared. An aliquot of 100µL was withdrawn and plated on sterilized yeast extract peptone dextrose (YEPD) agar plates as the method described by 20 . The plates were incubated at 28°C for 72 h. ...
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The xylanolytic and amylolytic yeasts were qualitatively determined by Cong red xylan agar and soluble starch agar plates, respectively. The most xylanase and α-amylase inducible strain (AUN-02) was selected and identified using PCR amplification of 26S rRNA gene and sequence analysis. The comparison of the alignment results and phylogenetic analysis of the sequences of the isolated yeast to published rRNA gene sequences in GenBank, confirmed the identification of the isolate as Pichia membranifaciens. Xylanase and α-amylase production by isolated P. membranifaciens were investigated at different pH values (4-8), temperature degrees (20-45°C), incubation time (1-7 days) and various substrates.A higher production of xylanase (38.8 U/mL) and a-amylase (28.7 U/mL) was obtained after 4 days of fermentation of P. membranifaciens. Higher activity of xylanase (36.83 U/mL) and a-amylase (27.7 U/mL) was obtained in the fermentation of P. membranifaciens in a culture medium adjusted to pH 7.0. The optimum temperature showed maximum xylanase and a-amylase activity (42.6 and 32.5 units/mL, respectively) was estimated at 35 °C. The xylanase and a-amylase activities of P. membranifaciens were estimated and compared for the different substrates tested. The strain revealed 100% relative activity of xylanase and a-amylase on beechwood and potato starch, respectively. The affinity of enzymes towards substrate was estimated using Km values. The Km values of xylanase and α-amylase increased in the order of pH’s 7.0, 6.0 and 4.5 (0.85, 1.6 and 3.4 mg xylan/mL and 0.22, 0.43 and 2.8 mg starch/mL, respectively). the yeast P. membranifaciensis is suitable for produce neutral xylanase and α-amylase enzymes. So, it could be used as a promising strain for production of these enzymes in industrial field.
... A possible reason may be because these detected dioxygenase genes are highly conserved among different gram negative bacteria. The nahAc gene is of particular interest as an indicator for PAHs degradation because the enzyme encoded by this gene not only degrades naphthalene, but also mediates degradation of other PAHs compounds [9,11,28]. Intradiol C12O and extradiol C23O dioxygen ases play a key role in the metabolism of aromatic rings by the bacteria. C12O and C23O are extradiol dioxyge nases, cleaving aromatic C-C bond at ortho or meta position of dihydroxylated aromatic substrates, cataly ses the convention of catechol to cis, cis dihydroxyl muconate or 2 hydroxymuconic semialdehyde [29]. ...
Article
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Polycyclic aromatic hydrocarbons (PAHs) are xenobiotic compounds, which being degraded by chemical, physical or biological methods. The latter is the safest and the cheapest one. Two bacterial strains ASU-01and ASU-016 were isolated from different Egyptian petroleum contaminated sites. They were genetically identified based on the analysis of the nucleotide sequences of the 16S ribosomal RNA gene and the phylogenetic tree as Enterobacter hormaechei and Pseudomonas pseudoalcaligenes respectively. When pyrene as high molecular weight (HMW)-PAH was added as a sole carbon source, both strains could degrade it with efficiency 77.7 and 83.7% within 15 days of incubation, respectively. However, when it was mixed with low molecular weight (LMW)-PAHs, two opposite phenomena appeared. The first one was enhancement, which occurred with ASU-01. This strain shifted pyrene efficiency to 98.5%. The second phenomenon was inhibition occurred with ASU-016 which completely retarded pyrene degradation. Naphthalene dioxygenase (nahAc), and Catechol dioxygenases (C12O &C23O) genes were detected in the two strains based on PCR. The detected genes were confirmed by determining the different specific activities of their translated protein (enzymes) on different PAHs. The maximum values of biosurfacatant production activity and cell- surface and percentage of cell surface hydrophobicity (CSH) were detected during the exponential phase. These latter factors increased the bioavailability and consequently, the assimilation of PAHs.
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