Refine search
Results 1-2 of 2
Biochemical characterization of recombinant benzyl alcohol dehydrogenase from Rhodococcus ruber UKMP-5M
2017
Tavakoli, Arezoo | Hamzah, Ainon
Benzyl Alcohol Dehydrogenase (BADH) is an important enzyme for hydrocarbon degradation, which can oxidize benzyl alcohols to aldehydes, while being capable of catalyzing a reversible reaction by reducing benzaldehyde. BADH is a member of medium chain alcohol dehydrogenases, in which zinc and NAD are essential for enzyme activity. This paper describes the expression, purification, and characterization of recombinant benzyl alcohol dehydrogenase, encoded by xylB gene from Rhodococcus ruber UKMP-5M. The gene has been amplified and cloned into E. coli, and the recombinant plasmid pGEMT-xylB has been digested by NdeI and HindIII to construct plasmid pET28b-xylC and then ligated into E. coli BL21 (DE3), itself induced by 0.3 mM isopropyl β-D-thiogalactoside (IPTG) at 25°C. The expressed BADH has been 38 kDa, and is purified by affinity chromatography, in which the specific activity was 30 U/mg after 17 folds purification, leading to a NAD-dependent enzyme that uses benzyl alcohol as a substrate for enzyme characterization. The final metabolite is benzaldehyde, identified by gas chromatography mass spectrometry (GC-MS). The BADH activity has been 0.7 U/mL and the optimum pH and temperature, 9.5 and 30ºC, respectively. Also the Michaelis constant (Km) and maximum velocity (Vmax) have accounted to 705 µM and 1.3 U/mL, respectively. Benzyl alcohol dehydrogenase from R. ruber UKMP-5M can be used for hydrocarbon biodegradation in contaminated sites.
Show more [+] Less [-]Levofloxacin is phytotoxic and modifies the protein profile of lupin seedlings
2017
Orzoł, Aleksandra | Piotrowicz-Cieślak, AgnieszkaI.
The toxicity of levofloxacin to yellow lupin plants was evaluated in this study. Recommended indexes of plant (roots and shoots) growth were determined and new indexes were proposed which better characterise the phytotoxicity of levofloxacin. These were, in particular, the activity of antioxidative enzymes, the content of free radicals, as well as the root protein content and the root protein profile. The results showed that levofloxacin considerably affected EC₅₀, measured as the activity of catalase in roots, and leaves (1.05 and 0.069 mM, respectively). The activity of peroxidase in the roots and the dry weight of seedlings were the least sensitive parameters (EC₅₀ was 1.8 and 1.76 mM, respectively). Units of toxicity clearly showed that the activity of catalase is a better measure of toxicity for low concentrations of the drug, and it is a better index of plant physiological state than the morphological parameters of seedlings. Moreover, levofloxacin changed the location of free radicals and the protein profile in plants. The changes in location of reactive oxygen species in roots were an important symptom of the drug toxicity to lupin seedlings. Our results have shown that the toxicity of levofloxacin was manifested mainly by changes in the protein profile. The content of the glyceraldehyde-3-phosphate dehydrogenase, 14-3-3-like protein A, expansin-B3-like precursor, fructose-bisphosphate aldolase, lipoxygenase, nucleotide-binding subunit of vacuolar ATPase and pyruvate dehydrogenase were found to decrease. On the other hand, plant exposure to levofloxacin resulted in an increase in the content of enolase, protein LlR18A, class III chitinase, ascorbate peroxidase, aspartate aminotransferase, alcohol dehydrogenase 1, leghemoglobin reductase-like 17 and heat shock cognate protein 80-like.
Show more [+] Less [-]