Characterization of Homocysteine γ-Lyase from Submerged and Solid Cultures of Aspergillus fumigatus ASH (JX006238)

( El Sayed Ashraf S ),( Salwa A Khalaf ),( Hani A Aziz ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.4

Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine γ- lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at 37-40℃, with a Tm value of 70.1oC. The enzyme showed clear catalytic and thermal stability below 40℃, with T1/2 values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at 30℃, 35℃, 40℃, 50℃, and 60℃, respectively. Additionally, the enzyme Kr values were 0.002, 0.054, 0.097, 0.184, and 0.341 S-1 at 30oC, 35oC, 40oC, 50℃, and 60℃, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuriarelated diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine (Km 2.46 mM, Kcat 1.39 × 10-3 s-1), methionine (Km 4.1 mM, Kcat 0.97 × 10-3 s-1), and cysteine (Km 4.9 m M, Kcat 0.77 × 10-3 s-1). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5`-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.

Purification and Characterization of a New L-Methioninase from Solid Cultures of Aspergillus flavipes

Ashraf S. A. El-Sayed 한국미생물학회 2011 The journal of microbiology Vol.49 No.1

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anionexchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8-8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg^(2+), Cu^(2+), and Fe^(2+), with slight inhibition by Triton X-100. A. flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S^(-1)) followed by a relative demethiolating activity to L-homocysteine (Km, 12 mM and Kcat, 9.3 S^(-1)). The enzyme has two absorption maxima at 280 and 420 nm,typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.

Biocidal Activity of Metal Nanoparticles Synthesized by Fusarium solani against Multidrug-Resistant Bacteria and Mycotoxigenic Fungi

Manal T. El Sayed,Ashraf S. A. El-Sayed 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.2

Antibiotic resistance by pathogenic bacteria and fungi is one of the most serious global public health problems in the 21st century, directly affecting human health and lifestyle. Pseudomonas aeruginosa and Staphylococcus aureus with strong resistance to the common antibiotics have been isolated from Intensive Care Unit patients at Zagazig Hospital. Thus, in this study we assessed the biocidal activity of nanoparticles of silver, copper and zinc synthesized by Fusarium solani KJ 623702 against these multidrug resistant-bacteria. The synthesized Metal Nano-particles (MNPs) were characterized by UV-Vis spectroscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and Zeta potential. The Fourier transform infrared spectroscopy (FTIR) result showed the presence of different functional groups such as carboxyl, amino and thiol, ester and peptide bonds in addition to glycosidic bonds that might stabilize the dispersity of MNPs from aggregation. The antimicrobial potential of MNPs by F. solani against the multidrug-resistant (MDR) P. aeruginosa and S. aureus in addition to the mycotoxigenic Aspergillus awamori, A. fumigatus and F. oxysporum was investigated, based on the visual growth by diameter of inhibition zone. Among the synthesized MNPs, the spherical AgNPs (13.70 nm) displayed significant effect against P. aeruginosa (Zone of Inhibition 22.4 mm and Minimum Inhibitory Concentration 21.33 μg/ml), while ZINC oxide Nano-Particles were the most effective against F. oxysporum (ZOI, 18.5 mm and MIC 24.7 μg/ml). Transmission Electron Microscope micrographs of AgNP-treated P. aeruginosa showed cracks and pits in the cell wall, with internalization of NPs. Production of pyocyanin pigment was significantly inhibited by AgNPs in a concentration-dependent manner, and at 5-20 μg of AgNPs/ml, the pigment production was reduced by about 15- 100%, respectively.

Purification and Characterization of Anabaena flos-aquae Phenylalanine Ammonia-Lyase as a Novel Approach for Myristicin Biotransformation

Asmaa M. Arafa,Afaf E. Abdel-Ghany,Samih I. El-Dahmy,Sahar Abdelaziz,Yassin El-Ayouty,Ashraf S. A. El-Sayed 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.4

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 μmol/mg/min) and A. flos-aquae (30.5 μmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30oC, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.

Fungal biopriming increases the resistance of wheat to abiotic stress

Ashraf S. A. El-Sayed,Hanan E. Dief,El-Sayed A. Hashem,Ahmed M. Desouky,Zamarud Shah,Salwa Fawzan 한국식물생명공학회 2022 JOURNAL OF PLANT BIOTECHNOLOGY Vol.49 No.2

Increasing soil salinity is one of the global challenges that the agriculture sector in Egypt has been facing; 33% of the cultivated land in Egypt, which includes merely 3% of the entire land area, is already salinized. The present review sheds light on the role of fungal biopriming, a technique in which hydrated seeds are inoculated with beneficial fungal flora, in mitigating the deleterious influence of NaCl tension. Endophytic fungi were recognized to be able to interact with several plant species, markedly contributing to the mitigation of NaCl stress in these plants, such that some plants get impoverished to their absent associated microbes under stressful conditions

Alleviation of salt stress in  Triticum aestivum by biopriming with  Phanerochaete chrysosporium

Hanan El-Sayed Dief,El-Sayed A. Hashem,Salwa Fawzan,Ashraf S. A. El-Sayed 한국작물학회 2021 Journal of crop science and biotechnology Vol.24 No.1

Wheat is one of the most important cereals, vital basic crop in Egypt, covering approximately 32.6% of the total winter land. However, sensitivity to abiotic stress especially salinity is one the major current hurdles that reduce the global yield of this crop. Plant biopriming with fungi is the recent technology that might alleviate the salt stress eff ect. Thus, the objective of this work was to examine the eff ect of Triticum aestivum grains biopriming with Phanerochaete chrysosporium on the response of wheat to salt stress. From the physiological and biochemical parameters, biopriming of wheat grains with P. chrysosporium significantly alleviates the salt stress and markedly increases the growth parameters, photosynthetic pigments and osmolytes (soluble sugars, soluble protein and proline) contents. In addition, it alleviates the oxidative damage, as indicated by the lower accumulation of malondealdehyde and increasing the activity of antioxidant enzymes; superoxide dismutase, catalase, and ascorbate peroxidase in wheat seedlings. Results indicate the potential of using P. chrysosporium biopriming for reducing the deteriorating eff ects of salinity.