Purification and biochemical characterization of a new thermostable laccase from Enterococcus faecium A2 by a three-phase partitioning method and investigation of its decolorization potential.

Three-phase partitioning (TPP) is a simple, fast, cost-effective, and highly efficient process that can be used in the purification of laccases. In this study, microorganisms with laccase activity were isolated from water samples collected from the Agri-Diyadin hot spring. The isolate with the highest laccase activity was found to be the A2 strain. As a result of molecular (16S rRNA sequence) and conventional (morphological, biochemical, and physiological) analyses, it was determined that the A2 isolate was 99% similar to Enterococcus faecium (Genbank number: MH424896). The laccase was purified to 4.9-fold with 110% recovery using the TPP. The molecular mass of the enzyme was found by SDS-PAGE to be 50.11 kDa. Optimum pH 6.0 and optimum temperature for laccase were determined as 80 °C. The laccase exhibited pH stability over a wide range (pH 3.0-9.0) and a high thermostability, retaining over 90% of its activity after 1 h of incubation at 20-90 °C. The laccase exhibited high thermostability, with a heat inactivation half-life of approximately 24 h at 80 °C. The enzyme remained highly stable in the presence of surfactants and increased its activity in the presence of organic solvents, Cr2+, Cu2+, and Ag+ metal ions. The Km, Vmax, kcat, and kcat/Km values of laccase for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate were 0.68 mM, 5.29 µmol mL-1 min-1, 110.2 s-1, and 162.1 s-1 mM-1, respectively.

An in vivo and in vitro comparison of the effects of amoxicillin, gentamicin, and cefazolin sodium antibiotics on the mouse hepatic and renal glutathione reductase enzyme.

Despite the fact that the use of antibiotics is increasing worldwide, it is clear that antibiotics can lead to oxidative stress. This is the first study to make a comparison of the impact of frequently prescribed antibiotics, including amoxicillin, gentamicin, and cefazolin sodium, on the gene, protein, and activity of glutathione reductase (GR), which is one of the primary antioxidant enzymes, in mouse liver and kidney tissues. First, the GR enzyme was purified by the 2',5'-ADP Sepharose 4B affinity chromatography with a specific activity of 84.615 EU/mg protein and 9.63 EU/mg protein from the mouse liver and kidney, respectively. The in vitro inhibitory effects of the antibiotics in question was determined. While cefazolin sodium did not exhibit any inhibitory effect, gentamicin and amoxicillin inhibited GR activity in both tissues. Furthermore, the in vivo effects of these drugs were investigated, and amoxicillin and cefazolin sodium-inhibited GR activity in both liver and kidney tissues, while gentamicin did not have any effect on the kidney. Besides, while gentamicin downregulated and cefazolin sodium upregulated Gr gene expression, amoxicillin did not alter it. Protein expression was only affected by the administration of cefazolin sodium in the kidney. This study is important as it demonstrates that while amoxicillin and gentamicin showed parallel effects on the GR activity in liver and kidney tissues both in vitro and in vivo, cefazolin sodium had a very strong effect on hepatic and renal GR in vivo. Furthermore, the antibiotics used in this study induced oxidative stress in both tissues.

The Effects of Amoxicillin, Cefazolin, and Gentamicin Antibiotics on the Antioxidant System in Mouse Heart Tissues.

BACKGROUND: Free radicals lead to destruction in various organs of the organism. The improper use of antibiotics increases the formation of free radicals and causes oxidative stress. OBJECTIVE: In this study, it was aimed to determine the effects of gentamicin, amoxicillin, and cefazolin antibiotics on the mouse heart. METHODS: 20 male mice were divided into 4 groups (1st control, 2nd amoxicillin, 3rd cefazolin, and 4th gentamicin groups). The mice in the experimental groups were administered antibiotics intraperitoneally at a dose of 100 mg / kg for 6 days. The control group received normal saline in the same way. The gene expression levels and enzyme activities of SOD, CAT, GPx, GR, GST, and G6PD antioxidant enzymes were investigated. RESULTS: GSH levels decreased in both the amoxicillin and cefazolin groups, while GR, CAT, and SOD enzyme activities increased. In the amoxicillin group, Gr, Gst, Cat, and Sod gene expression levels increased. CONCLUSION: As a result, it was concluded that amoxicillin and cefazolin caused oxidative stress in the heart, however, gentamicin did not cause any effects.

A novel endo-ß-1,4-xylanase from Pediococcus acidilactici GC25; purification, characterization and application in clarification of fruit juices.

A novel extracellular xylanase was purified and characterized from Pediococcus acidilactici GC25 (GenBank number: MF289522). The purification was 4.6-fold with a yield of 43.61% through acetone precipitation, Q-Sepharose, and CM-Sepharose ion change chromatography. The molecular weight of the enzyme was 48.15 kDa, and the optimum pH and temperature were 7.0 and 40 °C, respectively. The maximum activity was observed between 20 and 50 °C. Although it was active within a wide pH range (pH 2.0-9.0), it retained over 85% of its activity after 24 h incubation; and over 70% of its activity after 168 h incubation in neutral and alkaline pH. It was observed that the enzyme showed high stability with K+, Ba2+, Cd2+, Co2+, Sr2+, Mg2+, Ca2+, Al3+, Zn2+, and Ni2+ ions. The Km and Vmax for the xylanase were 3.10 mg mL-1 and 4.66 U/mg protein, respectively. It was determined that treatment of different fruit juices with P. acidilactici GC25 xylanase improved the clarification. The highest increase in the reducing sugar amount and decrease in the turbidity was 24.47 ±â€¯1.08 and 21.22 ±â€¯0.58 for peach juice at 0.15 U/mL enzyme concentration. These results showed that the xylanase purified from P. acidilactici GC25 may have a wide potential in biotechnological processes of the food and baking industry.

New xylanolytic enzyme from Geobacillus galactosidasius BS61 from a geothermal resource in Turkey.

In this study, isolation, conventional and molecular characterizations of ten thermophilic bacteria from Rize/Ayder were carried out. Xylanase from Geobacillus galactosidasius BS61 (GenBank number: KX447660) was purified by acetone precipitation, Diethylaminoethyl-cellulose and Sephadex G-100 chromatographies. The xylanase of G. galactosidasius BS61 in clarifying fruit juice was also investigated. Enzyme was purified 29.80-fold with 75.18% yield; and molecular weight was determined as 78.15 kDa. The optimum temperature of xylanase was 60 °C. The enzyme activity was maintained fully after 24 h and over 50% after 168 h at pH 4.0-10.0, while optimum pH was 7.0. Km and Vmax for beech wood xylan were measured as 3.18 mg mL-1, 123 U mg protein-1. In addition, Ca2+, Na+, Al3+, Zn2+, Cd2+, Mg2+, Ni2+, Cu2+ had decreasing effect on enzyme activity, while enzyme activity had been protected against anions, especially HSO3- and HPO42- stimulated enzyme activity. Xylanase applications (with 15 U/mL enzyme activity) in orange and pomegranate juices were increased; and the sugar and turbidity amounts were reduced 17.36% ±â€¯1.18 and 30.52 ±â€¯1.23, respectively. These results indicated that the xylanase of G. galactosidasius BS61 has biotechnological potential in juice clarification due to its stability against metal ions, chemicals and high pH-values.

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 µmol.mL-1.min-1, respectively.

Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification, characterization, effects of surfactants and organic solvents.

In this study, the extracellular thermostable alkaline protease out of A10 strain was purified 1.38-fold with 9.44% efficiency through the ammonium sulfate precipitation-dialysis and DE52 anion exchange chromatography methods. The molecular weight of the enzyme in question along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be approximately 40.55 kDa, whereas the optimum pH and temperature ratings were identified as 9.0 and 70 °C, respectively. It was seen that the enzyme had remained stable between pH 7.5-10.5 range, protecting more than 90% of its activity in the wake of 1 h incubation at 60-70 °C. It was also observed that the enzyme enhanced its activity in the presence of Mg(2+), Mn(2+), K(+), while Fe(2+), Ni(2+), Zn(2+), Ag(+ )and Co(2+ ) decreased the activity. Ca(2+), however, did not cause any change in the activity. The enzyme was seen to have been totally inhibited by phenylmethylsulfonyl fluoride, therefore, proved to be a serine alkaline protease.

A compendium of expression patterns of cholesterol biosynthetic enzymes in the mouse embryo.

Cholesterol and its biosynthetic pathway intermediates and derivatives are required for many developmental processes including membrane biogenesis, transmembrane receptor signaling, steroid biogenesis, nuclear receptor activation, and posttranslational modification of hedgehog (Hh) proteins. To perform such multifaceted tasks depends on stringent regulation of expression of cholesterol biosynthetic enzymes (CBEs). We established for a whole organism, for the first time, the 3D expression pattern of all genes required for cholesterol biosynthesis (CBS), starting from acetyl-CoA and ending with cholesterol. This data was produced by high-throughput in situ hybridization on serial sections through the mouse fetus. The textually annotated image data were seamlessly integrated into the METscout and GenePaint public databases. This novel information helps in the understanding of why CBEs are expressed at particular locations within the fetus. For example, strong CBE expression is detected at sites of cell proliferation and also where cell growth increases membrane surface, such as in neurons sprouting axons and forming synapses. The CBE data also sheds light on the spatial relationship of cells and tissue that express sonic Hh (Shh) and produce cholesterol, respectively. We discovered that not all cells expressing Shh are capable of CBS. This finding suggests novel ways by which cholesterylation of Shh is regulated.

Neurotoxic effects of nickel chloride in the rainbow trout brain: Assessment of c-Fos activity, antioxidant responses, acetylcholinesterase activity, and histopathological changes.

The aim of this study was to determine the biochemical, immunohistochemical, and histopathological effects of nickel chloride (Ni) in the rainbow trout brain. Fish were exposed to Ni concentrations (1 mg/L and 2 mg/L) for 21 days. At the end of the experimental period, brain tissues were taken from all fish for c-Fos activity and histopathological examination and determination of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) enzyme activities, lipid peroxidation (LPO), and glutathione (GSH) levels. Our results showed that Ni treatment caused a significant increase in the brain SOD activity and in LPO and GSH levels (p < 0.05), but it significantly decreased AChE and CAT enzyme activities (p < 0.05). Strong induction in c-Fos was observed in some cerebral and cerebellar regions of fish exposed to Ni concentrations when compared with the control group. However, c-Fos activity was decreased in necrotic Purkinje cells. Brain tissues were characterized by demyelination and necrotic changes. These results suggested that Ni treatment causes oxidative stress, changes in c-Fos activity, and histopathological damage in the fish brain.

Inhibition profile of a series of phenolic acids on bovine lactoperoxidase enzyme.

Lactoperoxidase (LPO) catalyzes the oxidation of numerous of organic and inorganic substrates by hydrogen peroxide. It has very vital activity in the innate immune system by decreasing or stopping the activation of the bacteria in milk and mucosal secretions. This study's purpose was to investigate in vitro effect of some phenolic acids (ellagic, gallic, ferulic, caffeic, quercetin, p-coumaric, syringic, catechol and epicatechin) on the purified LPO. This enzyme was purified from milk by using different methods such as Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange and Sephadex G-100 gel filtration chromatography. LPO was purified 28.7-fold with a yield of 20.03%. We found phenolic acids have inhibition effects on bovine LPO enzyme to different concentrations. Our study showed lower concentrations of caffeic acid, ferulic acid and quercetin exhibited much higher inhibitory effect on enzyme, so these three of them were clearly a more potent inhibitor than the others were. All of compounds were non-competitive inhibitors.

Purification of acetylcholinesterase by 9-amino-1,2,3,4-tetrahydroacridine from human erythrocytes.

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and L-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5'-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K(m), V(max), optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25-35 °C. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V(max) was 0.090 µmol/mL/min. Maximum binding was achieved at 2 °C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.

Interactions of melatonin and serotonin with lactoperoxidase enzyme.

Melatonin is the chief secretory product of the pineal gland and is synthesized enzymatically from serotonin. These indoleamine derivatives play an important role in the prevention of oxidative damage. Lactoperoxidase (LPO; EC 1.11.1.7) was purified from bovine milk with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange, and Sephadex G-100 gel filtration chromatography, respectively. LPO was purified with a yield of 21.6%, a specific activity of 34.0 EU/mg protein, and 14.7-fold purification. To determine the enzyme purity, SDS-PAGE was performed and a single band was observed. The R(z) (A(412)/A(280)) value for LPO was 0.9. The effect of melatonin and serotonin on lactoperoxidase was determined using ABTS as chromogenic substrate. The half-maximal inhibitory concentration (IC(50)) values for melatonin and serotonin were found to be 1.46 and 1.29 µM, respectively. Also, the inhibition constants (K(i)) for melatonin and serotonin were 0.82 ± 0.28 and 0.26 ± 0.04 µM, respectively. Both melatonin and serotonin were found to be competitive inhibitors.

The inhibitory effect of propofol on bovine lactoperoxidase.

Propofol (2,6-diisopropylphenol) is a hypnotic intravenous agent with in vivo antioxidant properties. This study was undertaken to examine the in vitro effect of propofol on lactoperoxidase (LPO; E.C. 1.11.1.7) obtained from bovine milk. Lactoperoxidase was purified with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange chromatography and Sephadex G-100 gel filtration chromatography, respectively. Lactoperoxidase was purified with a yield of 21.6%, a specific activity of 34 EU/mg proteins and 14.7-fold purification. One enzyme unit is defined as the oxidation of 1 micromol ABTS per min under the assay condition (25( degrees )C, pH: 6.0). To determine enzyme purity, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The effect of propofol on lactoperoxidase were determined using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a chromogenic substrate. The IC(50) value of propofol was found as 15.97 microM. Also, K(i) constant for propofol was 3.72 microM and propofol was found as competitive inhibitor.

Effects of some vitamins on lactoperoxidase enzyme activity.

OBJECTIVE: The present paper investigates the in vitro effect of L-ascorbic acid (vitamin C), menadione sodium bisulfate (vitamin K3), and folic acid on purified lactoperoxidase (LPO). METHODS: This enzyme was purified from bovine milk by Amberlite CG 50 resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. RESULTS: Rz (A412/A280) value for the purified LPO was found to be 0.8. Lactoperoxidase was purified 20.45-fold with a yield of 28.8 %. Purity of enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method and a single band was observed. All tested vitamins caused inhibition of the enzyme activity and displayed a competitive type of inhibition mechanism. IC(50) values of these three vitamins were 2.03 microM, 0.025 mM, and 0.0925 mM, and the K(i) constants were 0.508+/-0.257 microM, 0.0107+/-0.0044 mM, and 0.0218+/-0.0019 mM respectively. CONCLUSION: The vitamins discussed here displayed inhibition-type competition with LPO enzyme at varying concentrations. Our study showed that L-ascorbic acid exhibited a much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than other vitamins tested.