A carboxymethylcellulose (CMC)-degrading bacterium was isolated from soil, identified as Bacillus methylotrophicus according to the physiological properties and analyses of 16S rRNA and a partial sequence of the gyrase A (gyr A) gene, and named as B. methylotrophicus Y37. The CMCase enzyme was purified to homogeneity by 20.4-fold with 21.73% recovery using single-step hydrophobic interaction chromatography and biochemically characterized. CMCase showed a molecular weight of approximately 50 kDa as determined by SDS-PAGE. The activity profile of the CMCase enzyme exhibited optimum activity at 45 • C and pH 5.0. The activity was highly stable at alkaline pH levels. More than 90% of the original CMCase activity was maintained at relatively high temperatures ranging from 55 to 65 • C. The enzyme activity was induced by Ca 2+ , Cd 2+ , Co 2+ , K + , Mg 2+ , and Na 1+ , whereas it was strongly inhibited by phenylmethanesulfonyl fluoride and iodoacetic acid. The enzyme tolerated Hg 2+ up to 10 mM and presented hydrolytic activity towards glucan, filter paper, laminarin, and CMC but not o-nitrophenyl β-D-galactopyranoside. Kinetic analysis of the purified enzyme showed K m and V max values of 0.19 mg mL −1 and 7.46 U mL −1 , respectively. The biochemical properties of this CMCase make the enzyme a good candidate for many industrial applications.
This study aimed to investigate the effects of drought stress on Amsonia orientalis, an endangered ornamental plant with a limited natural distribution in Europe. Effects of polyethylene glycol (PEG)-mediated drought stress (-0.15, -0.49, -1.03 and -1.76 MPa osmotic potentials) were tested on in vitro cultures. In general, root lengths and numbers, total protein, chlorophyll a and carotenoid contents were negatively influenced at elevated levels of the stress factor. The successive decrease in the tested osmotic potentials resulted in gradually higher H2O2, malondialdehyde (MDA) and proline contents. Activities of the antioxidant enzymes, peroxidase (POD) and catalase (CAT), were found to be enhanced in response to the decreasing osmotic potential tested, whereas increased superoxide dismutase (SOD) activity was observed at the -0.15 MPa osmotic potential. Strong activation of POD enzymes under drought stress suggests that POD enzymes might have a major role in regulating the H2O2 content, while CAT has only a supplementary role in A. orientalis. These results indicated that although A. orientalis is susceptible to long-term drought, the species may survive during mild drought stress because the development of the plant was not totally inhibited but only limited. Nevertheless, the species should be introduced to well-irrigated lands, after evaluation of the soil’s water status, in order to ensure the continuation of its generations.
The structure of Scytalidium thermophilum catalase in complex with its well known inhibitor 3-amino-1,2,4-triazole revealed that the inhibitor occupies a surface pocket at the end of the lateral channel. This pocket corresponds to the site of NADPH binding in mammalian catalases. Peroxide-independent phenolic substrate oxidation is likely to occur in a similar manner to NADPH oxidation.
Polyphenol oxidase (PPO) has been purified from the rosemary plant (Rosmarinus officinalis L.) through three-phase partitioning (TPP) and has been biochemically characterized. The optimized TPP consisted of 50% (w/v) ammonium sulfate and equal volumes of crude extract and tert-butanol prepared at pH 6.5 and room temperature. Using this system, PPO was purified 14-fold, with 230% recovery of activity from the middle phase. The partitioned enzyme had a molecular mass of 53 kDa. The highest enzyme activity was detected at 30 °C and pH 7.0 against catechol. In substrate specificity tests, the enzyme displayed activity towards catechol, 4-methylcatechol, caffeic acid, hydroquinone, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), pyrogallol, syringaldezine, and 3,4-dihydroxy-L-phenylalanine but no activity towards L-tyrosine. The enzyme was inhibited by the common PPO inhibitors; salicylhydroxamic acid (SHAM), cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and the organic solvent dimethyl sulfoxide (DMSO). Enzyme activity increased in the presence of the organic solvents acetone, ethanol, and methanol.
This study aimed to investigate the effects of possible zinc (Zn) and molybdenum (Mo) contaminations on the critically endangered European Bluestar (Amsonia orientalis). The effects of Zn and Mo were tested in a dose-dependent manner on in vitro cultures. Zn at 0.1 mM in the medium inhibited root development whereas Mo showed the same effect only at ≥2.5 mM concentration. Gradual inhibition of shoot development was observed after treatment with both metals. Protein contents were also negatively affected by increasing metal concentrations, while proline levels increased gradually. Successive increases in metal concentrations resulted in higher hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations. The activity of the antioxidant enzymes, peroxidase (POD) and catalase (CAT), were found to be enhanced in response to increasing metal concentrations. Superoxide dismutase (SOD) activity decreased after Zn treatment but increased after Mo treatment. A marked increase in POD and CAT in response to metal stress suggests that these enzymes might have a significant cooperative role in regulating H2O2 production, although CAT, in response to drought and salt stress, has been reported to only play a supplementary role in A. orientalis. These results indicated that A. orientalis is susceptible to long-term Zn stress but can tolerate up to 2.5 mM Mo in the long-term. Deficiency of Mo is more common than high toxic concentrations in the environment. Therefore Zn contamination should be considered as one of the major threats for A. orientalis in its native habitat.
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