Enhanced alkaline catalase production by Serratia marcescens FZSF01: enzyme purification, characterization, and recombinant expression

Background: Catalase (CAT) is an important enzyme that degrades H2O2 into H2O and O2. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized. Results: After optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20­70°C and pH 5.0­11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC­MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml. Conclusions: To our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.

Foxo3 gene expression and oxidative status in beta-thalassemia minor subjects

ABSTRACT Background: Oxidative stress may aggravate symptoms of hemolytic anemias such as beta-thalassemia. FoxO3 activation results in resistance to oxidative stress in fibroblasts and neuronal cell cultures. Objective: The purpose of this research was to study FoxO3 gene expression and oxidative status in beta-thalassemia minor individuals. Methods: Sixty-three subjects (42 apparently healthy individuals and 21 with beta-thalassemia minor) were analyzed at the Universidad Nacional de Tucumán, Argentina, between September 2013 and June 2014. A complete blood count, hemoglobin electrophoresis in alkaline pH and hemoglobin A2 levels were quantified. Moreover, thiobarbituric acid reactive species, erythrocyte catalase activity and iron status were evaluated. Beta-thalassemia mutations were determined by real-time polymerase chain reaction. FoxO3 gene expression was investigated by real-time reverse transcription-polymerase chain reaction using mononuclear cells from peripheral blood. Results: Subjects were grouped as children (≤12 years), and adult women and men. The analysis of erythrocyte catalase activity/hemoglobin ratio revealed a significant difference (p-value <0.05) between healthy and beta-thalassemia minor adults, but no significant difference was observed in the thiobarbituric acid reactive species levels and FoxO3 gene expression (p-value >0.05). Thiobarbituric acid reactive species and the erythrocyte catalase activity/hemoglobin ratio were not significantly different on comparing the type of beta-thalassemia mutation (β0 or β+) present in carriers. Conclusions: The lack of systemic oxidative imbalance demonstrated by thiobarbituric acid reactive species is correlated to the observation of normal FoxO3 gene expression in mononuclear cells of peripheral blood. However, an imbalanced antioxidant state was shown by the erythrocyte catalase activity/hemoglobin ratio in beta-thalassemia minor carriers. It would be necessary to study FoxO3 gene expression in reticulocytes to elucidate the role of FoxO3 in this pathology.

Dose related effects of nicotine on oxidative injury in young, adult and old rats.

Nicotine affects a variety of cellular process ranging from induction of gene expression to secretion of hormones and modulation of enzymatic activities. The objective of the present study was to study the dose dependent toxicity of nicotine on the oxidative stress in young, adult and old rats which were administered 0.75, 3 and 6 mg kg-1 nicotine as nicotine hydrogen tartarate intraperitoneally for a period of seven days. No changes were observed in blood catalase (CAT) activity and level of blood reactive oxygen species (ROS) in any of the age group at the lowest dose of nicotine. However, at the highest dose (6 mg kg-1 nicotine) ROS level increased significantly from 1.17to 1.41>M ml-1 in young rats and from 1.13 to 1.40 >M ml-1 in old rats. However, no change was observed in blood ROS levels of adult rats. Administration of 3 mg kg-1 nicotine resulted in an increase in level of reduced glutathione (GSH) in rats of all the age groups. The young animals were the most sensitive as a dose of 6 mg kg-1 resulted in decline in the levels of reduced GSH to 0.89 mg ml-1 as compared to normal control (1.03 mg ml-1). The antioxidant enzymes SOD and CAT were sensitive to a dose of 6 mg kg-1 as it resulted in decline of the enzymatic activity in all age group animals. Also, administration of nicotine at a lower dose of 3 mg kg-1 inhibited SOD activity from 1.48 to 1.20 units min-1 mg-1 protein in old rats. Catalase activity showed a similar trend at a dose of 3 mg kg-1. Administration of nicotine also increased the blood lipid peroxidation levels at all three doses in young and old rats dose dependently. Nicotine exposure also increased ROS in brain at the doses of 3 and 6 mg kg-1 in all the three age groups. Brain GSH decreased significantly at high dose of nicotine (6 mg kg-1 b.wt.) in adult rats (4.27 mg g-1) and old rats (3.68 mg g-1) but in young rats level increased to 4.40 mg g-1 at the lower dose (0.75 mg kg-1 nicotine). Brain lipid peroxidation increased at all three doses of nicotine in young as well as old rats as compared to their respective normal control. The SOD activity increased significantly in young (2.88 units min-1mg-1 protein) and old rats (1.81 units min-1mg-1 protein) as compared to their respective normal at a dose of 6 mg kg-1. Interestingly, the SOD activity decreased in adult rats (2.18 units min-1mg-1 protein) as compared to its normal control. Catalase activity decreased at the dose of 3 mg kg-1 and 6 mg kg-1 nicotine in young and old rats but no effect was observed in adult rats at any of the doses. Acetylcholine esterase (AchE) activity decreased in a dose dependent manner in adult and old rats. Overall, the results of the study indicate that young and old rats are more sensitive to nicotine induced oxidative stress as compared to the adult ones.

Animal Model for Oxidative Stress Research---Catalase Mutant Mice

Catalase-deficient mouse strains was initially established by Feinstein et al. through a large scale screening of the progeny of irradiated C3H mice in 1966. Later, Feinstein provided the mice of catalase mutant strain C3H/AnlCsaCsa (wild-type), C3H/AnlCsbCsb and C3H/AnlCscCs c to Okayama University Medical School in Japan. It is known that a point mutation at amino acid 11 (from glutamine to histidine) of acatalasemic mouse catalase and a point mutation at amino acid 439 (from asparagine to serine) of hypocatalasemic mouse catalase are responsible for the catalase deficiency of acatalasemic and hypocatalasemic mice, respectively. Recently, a liver cell line from an acatalasemic mouse and Escherichia coli (E. coli) strains with murine normal, hypocatalasemic, or acatalasemic catalase have been established. The construction of these new systems would be useful for studying the effects of oxidative stress at the cellular level. In this review, we give a brief overview of recent findings of studies in utilizing the catalase-deficient mice and evaluate the possibility of these mouse strains as a candidate animal model for oxidative stress research.

Animal model for oxidative stress research-Catalase mutant mice

Catalase-deficient mouse strains was initially established by Feinstein et al. through a large scale screening of the progeny of irradiated C3H mice in 1966. Later, Feinstein provided the mice of catalase mutant strain C3H/AnICs(a)Cs(a) (wild-type), C3H/AnICs(b)Cs(b) and C3H/AnlCs(c)Cs(c) to Okayama University Medical School in Japan. It is known that a point mutation at amino acid 11 (from glutamine to histidine) of acatalasemic mouse catalase and a point mutation at amino acid 439 (from as paragine to serine) of hypocatalasemic mouse catalase are responsible for the catalase deficiency of acatalasemic and hypocatalasemic mice, respectively. Recently, a liver cell line from an acatalasemic mouse andEscherichia coli (E. coli) strains with murine normal, hypocatalasemic, or acatalasemic catalase have been established. The construction of these new systems would be useful for studying the effects of oxidative stress at the cellular level. In this review, we give a brief overview of recent findings of studies in utilizing the catalase-deficient mice and evaluate the possibility of these mouse strains as a candidate animal model for oxidative stress research.

Blood Markers of Oxidative Stress in Dementia

OBJECTIVES: To identify changes in blood markers of oxidative stress among dementic and healthy aged persons, and to compare levels of peripheral markers of oxidative stress between dementia of the Alzheimer's type (DAT) and vascular dementia (VD), and finally to analyze factors affecting those. DESIGN: Some antioxidants had been known to reduce the risk of dementia, while increase of 4-hydroxy-nonenal and decrease of heme oxygenase in DAT had been reported. Recently increased free radical activities were also found in VD. Therefore, this study was tried to search for a blood markers to discriminate DAT and VD. MATERIALS AND METHOD: The blood levels of total antioxidant capacity (TAC) and catalase activity of 50 patients with DAT and 50 patients with VD were compared with those of 56 healthy aged controls. In addition, variables including risk factors of dementia, degree of dementia measured by MMSE, Hasegawa Dementia Scale, GDS and CDR, and activities of daily living were also assessed. RESULTS: 1) The levels of TAC were significantly (p<0.01) lower in dementia than normal aged controls (1.47+/-0.08 mmol/L), while the catalase activities were significantly (p<0.01) higher in dementia than normal aged controls (45.61+/-11.67 kU/L). 2) The TAC levels were significantly (p<0.01) lower while the catalase significantly (p<0.01) higher in DAT (1.24+/-0.09 mmol/L, 133.42+/-58.45 kU/L respectively) than VD (1.31+/-0.08 mmol/L, 81.33+/-24.65 kU/L respectively). 3) The TAC and catalase activities were significantly (p<0.01) correlated with duration of dementia, behavior symptoms, activities of daily living, and the severity of dementia. CONCLUSIONS: Peripheral blood markers of oxidative stress were significantly different in dementia from normal aged controls. Furthermore, the difference was more severe in DAT than VD, which means the effect of oxidative stress is more in DAT than in VD. Therefore, they might be clinically useful to assess and discriminate dementia.