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Chemical Treatment of Sludge
Published in Antoine Prota Trzcinski, Advanced Biological, Physical, and Chemical Treatment of Waste Activated Sludge, 2018
Figure 6.18 illustrates the effect of the addition of a commercial enzyme targeting protein in WAS. Proteinase K (EC3.4.21.64) was obtained from Sigma-Aldrich. Proteinase K exhibits broad substrate specificity. It degrades many proteins in their native state even in the presence of detergents. Proteinase K was isolated from a fungus, Engyodontium album (formerly Tritirachium album), which is able to grow on keratin. Consequently, Proteinase K is able to digest native keratin (hair), hence the name “Proteinase K.” The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha-amino groups. It is commonly used for its broad specificity. One unit of Proteinase K will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 mmole (181 mg) of tyrosine per minute at pH 7.5 at 37°C. Various volumes (0, 10, 50 and 100 µL) of a Proteinase K solution (600 U/mL) was added to 5 mL of sludge and incubated at three different temperatures for 24 h. After that, the SCOD was measured to determine the efficiency of solubilization due to enzymatic treatment.
Semen quality and sperm DNA damage associa –revised – final-finalted with oxidative stress in relation to exposure to polycyclic aromatic hydrocarbons
Published in Journal of Environmental Science and Health, Part A, 2018
Hueiwang Anna C. Jeng, Wen Y. Lin, Mu R. Chao, Wen Y. Lin, Chih H. Pan
Sperm DNA was isolated according to the procedure recommended by the European Standard Committee on Oxidative DNA Damage (ESCODD) with several modifications to minimize DNA oxidization during DNA isolation procedures.[25] Briefly, sperm samples (15 − 100 × 106 cells) were washed with 1% HSA in PBS and centrifuged at 3,000 g for 5 min. The resulting pellet was added to 600 μL of ice-cold extraction buffer, 70 μL of 10% (w/v) SDS and 30 μL of DTT (1M). After 30 μL of proteinase K was added, the samples were incubated at 55 °C for 1 h. Then, 30 μL of RNase A (10 mg mL−1) and 8 μL of RNase T1 (1 U μL–1) were added. The mixture was incubated at 37 °C for 1 h and then cooled to 4 °C for 5 min. Subsequently, 1.2 mL of NaI solution and 2 mL of 2-propanol were added. After centrifugation at 5,000 g for 5 min, the DNA pellet was washed with 1 mL of ice-cold 40% (v/v) 2-propanol. Finally, the DNA pellet was collected by centrifugation (5,000 g for 5 min) and dissolved in 200 μL of 0.1 mM DFO overnight. DNA concentration was measured by the amount of absorbance at 260 nm.
In vitro investigation of hypoglycemic and oxidative stress properties of fava bean (Vicia faba L.) seed extract in Saccharomyces cerevisiae 2376
Published in Preparative Biochemistry and Biotechnology, 2018
Dhiraj Kumar Choudhary, Abha Mishra
DNA fragmentation was analyzed by agarose gel electrophoresis as described by Moongkarndi et al.[20] with slight modifications. Cells (1 × 106 cells) were incubated with extract and H2O2 (1%, 2%) for 60 min. It was scraped and harvested by centrifugation. The cell pellets were incubated for 60 min 50 °C in 100 µl lysis buffer (100 mM Tris–HCl pH 8, 100 mM NaCl and 10 mM EDTA). Proteinase K (10 µl of 20 mg/ml) was added and further incubated for 30 min at 50 °C. RNase (3 µl of 10 mg/ml) was then added and incubated for 2 hrs at 50 °C. The DNA was extracted with phenol–chloroform–isoamyl alcohol, subjected to 2.0% of agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light transilluminator. Band intensity was calculated by image J softwere.