Catalases are well studied enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. The ubiquity of the enzyme and the availability of substrates made heme catalases the focus of many biochemical and molecular biology studies over 100 years. In human, this has been implicated in various physiological and pathological conditions. Advancement in proteomics revealed many of novel and previously unknown features of this mysterious enzyme, but some functional aspects are yet to be explained. Along with discussion on future research area, this mini-review compile the information available on the structure, function and mechanism of action of human catalase.
Catalase ; chemistry ; metabolism ; physiology ; Heme ; chemistry ; Humans ; Hydrogen Peroxide ; metabolism

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca₃(PO₄)₂ hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca₃(PO₄)₂ hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca₃(PO₄)₂ hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca₃(PO₄)₂ hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The K_m of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the k_cat/K_m was (13 151.53± 299.19) L/(mmol·s). The K_m of the KatA/Ca₃(PO₄)₂ hybrid nanoflowers was (32.75±2.96) mmol/L, and the k_cat/K_m was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca₃(PO₄)₂ hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca₃(PO₄)₂ hybrid nanoflowers using Ca²⁺ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.
Catalase ; Nanostructures/chemistry* ; Hydrogen Peroxide/metabolism* ; Enzymes, Immobilized/chemistry* ; Catalysis

OBJECTIVETo investigate the influence of a broad range of environmental conditions on initial rates of hydrogen peroxide produced by Streptococcus oralis (S. oralis).

METHODSFor each rate measurement, 1 ml aliquots of 10(12) cells/L mid-logarithmic phase S. oralis in TSBY were centrifuged and respectively washed by phosphate buffer containing 0.01-10 mmol/L glucose or sucrose, phosphate buffer with 5.0-7.5 pH or Bis-Tris buffer containing 0.01-100 mmol/L Ca(2+), 0.01-100 mmol/L F(-) or 0.01-100 mmol/L HFPO(3)(-). After S. oralis was cultured in respective buffer for 10, 20 and 30 min at 37 degrees C, the concentration of hydrogen peroxide in supernatant was assayed spectrophotometrically in 96-well micro-plate by ABTS-HRP at A(405).

RESULTSSynthesis rate of hydrogen peroxide by S. oralis was 7.48 micromol/L per minute without carbohydrate, the synthesis rate of hydrogen peroxide by S. oralis increased with 0.01-10 mmol/L glucose and 0.01-10 mmol/L sucrose, but there was no statistically significant difference in synthesis rate among the carbohydrate groups. The rates of H2O2 synthesis were inhibited in the buffer at pH 5.0-6.0, compared with pH 7.0 (P < 0.05). Ca(2+) had little influence on the rate of H2O2 synthesis. IC(50) of H2O2 synthesis rates by S. oralis responded to FHPO(3)(-) and F(-) were 12.65 mmol/L and 1.90 mmol/L respectively.

CONCLUSIONSEnvironmental conditions may influence the synthesis rate of H2O2 by S. oralis.

Culture Media ; chemistry ; Glucose ; Hydrogen Peroxide ; metabolism ; Hydrogen-Ion Concentration ; Metals, Heavy ; Streptococcus oralis ; metabolism

5-aminovalanoic acid (5AVA) can be used as the precursor of new plastics nylon 5 and nylon 56, and is a promising platform compound for the synthesis of polyimides. At present, the biosynthesis of 5-aminovalanoic acid generally is of low yield, complex synthesis process and high cost, which hampers large-scale industrial production. In order to achieve efficient biosynthesis of 5AVA, we developed a new pathway mediated by 2-keto-6-aminohexanoate. By combinatory expression of L-lysine α-oxidase from Scomber japonicus, α-ketoacid decarcarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli, the synthesis of 5AVA from L-lysine in Escherichia coli was achieved. Under the initial conditions of glucose concentration of 55 g/L and lysine hydrochloride of 40 g/L, the final consumption of 158 g/L glucose and 144 g/L lysine hydrochloride, feeding batch fermentation to produce 57.52 g/L of 5AVA, and the molar yield is 0.62 mol/mol. The new 5AVA biosynthetic pathway does not require ethanol and H₂O₂, and achieved a higher production efficiency as compared to the previously reported Bio-Chem hybrid pathway mediated by 2-keto-6-aminohexanoate.
Nylons ; Lysine/metabolism* ; Hydrogen Peroxide/metabolism* ; Metabolic Engineering ; Plastics/metabolism* ; Fermentation ; Escherichia coli/metabolism* ; Aminocaproates/metabolism*

OBJECTIVETo investigate the effect of environmental oxygen on the inhibition between Streptococcus oligofermentans (So) and Streptococcus mutans (Sm) and the producibilities of hydrogen peroxide by So.

METHODSThe aerobic and anaerobic environment was established by the carbon dioxide cultivation. The inhibition between So and Sm was observed by plating method. The production and synthesis rates of hydrogen peroxide by So were determined in both aerobic and anaerobic environment by 4-ATTP-horseradish peroxidase method at A(510).

RESULTSWhen both Sm and So were inoculated at the same time, Sm was not inhibited under the anaerobic environment, vice versa. Sm was slightly inhibited by So under the aerobic environment, the inhibition area was 1/5 of all bacterial membrane. When So was cultivated first and then Sm applied, So could inhibite Sm growth under both anaerobic and aerobic conditions. The inhibition area was 1/5 of bacterial membrane under the anaerobic environment, and 4/5 under the aerobic environment. When Sm was cultivated first and then So applied, So was unable to proliferate under both conditions. During the logarithmic phase, the production of H2O2 by So under the aerobic environment was higher than under the anaerobic environment (P < 0.05). The initial synthesis rate of H2O2 by So during growth cycle under the anaerobic condition was (11.84 ± 3.97) µmol/L per minute, which was only 49% of that under the aerobic environment [(24.13 ± 4.46) µmol/L per minute].

CONCLUSIONSThe oxygen has the effect on the inhibition between So and Sm, and the inhibition in the aerobic environment is much stronger than in the anaerobic environment. The synthesis ability of hydrogen peroxide by So under the aerobic environment is higher than under the anaerobic environment.

Aerobiosis ; Hydrogen Peroxide ; metabolism ; Oxygen ; metabolism ; Streptococcus ; growth & development ; metabolism ; Streptococcus mutans ; growth & development ; metabolism

Animals ; Humans ; Hydrogen Peroxide ; metabolism ; Liver ; cytology ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

Extracellular matrix (ECM) stiffness is closely related to the physiological and pathological states of breast tissue. The current study was aimed to investigate the effect of silk fibroin/collagen composite hydrogels with adjustable matrix stiffness on the growth and phenotype of normal breast epithelial cells. In this study, the enzymatic reaction of horseradish peroxidase (HRP) with hydrogen peroxide (H₂O₂) was used to change the degree of cross-linking of the silk fibroin solution. The rotational rheometer was used to characterize the composite hydrogel's biomechanical properties. Human normal mammary epithelial cell line MCF-10A were inoculated into composite hydrogels with various stiffness (19.10-4 932.36 Pa) to construct a three dimensional (3D) culture system of mammary epithelial cells. The CCK-8 assay was applied to detect the cell proliferation rate and active states in each group. Hematoxylin-Eosin (HE) staining and whole-mount magenta staining were used for histological evaluation of cell morphology and distribution. The results showed that with the increase of matrix stiffness, MCF-10A cells exhibited inhibited proliferation rate, decreased formation of acinus structures and increased branching structures. Meanwhile, with the increase of matrix stiffness, the polarity of MCF-10A cells was impeded. And the increase of matrix stiffness up-regulated the expression levels of mmp-2, mmp-3, and mmp-9 in MCF-10A cells. Among the genes related to epithelial-mesenchymal transition (EMT), the expression level of the epithelial marker gene E-cadherin was significantly down-regulated, while the interstitial cell marker gene Vimentin was up-regulated, and the expression levels of Snail, Wnt5b and Integrin β1 in the Wnt pathway were up-regulated. These results suggest that the silk fibroin/collagen composite hydrogels with adjustable matrix stiffness regulates the proliferation and the phenotype of MCF-10A cells. The effects of increased matrix stiffness may be closely related to the changes of the polar structures and function of MCF-10A cells, as well as the occurrence of ECM-remodeling and EMT.
Collagen/metabolism* ; Epithelial Cells/metabolism* ; Fibroins/pharmacology* ; Humans ; Hydrogels/metabolism* ; Hydrogen Peroxide ; Phenotype

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.
Animal ; Calcium/*metabolism ; Cells, Cultured ; Hydrogen Peroxide/*pharmacology ; Osteoblasts/*drug effects/*metabolism ; Oxidants/*pharmacology ; Rats

BACKGROUNDGlucocorticoid is speculated to be able to have Aspergillus fumigatus (A. fumigatus) being more susceptible to reactive oxygen species (ROS) by inhibiting Afyap1, the transcription factor activating protein-1 (AP-1) homologue in A. fumigatus, which may provide a clue to expand the clinical use of glucocorticoid in patients with fungal infections. In this study, we used dexamethasone to determine the direct effect on oxidative killing susceptibility of A. fumigatus in vitro, as well as the expression level of Afyap1 gene and its target genes (catalase and superoxide dismutase (SOD) genes).

METHODSA. fumigatus spores were treated with different concentrations (0, 0.02, 0.2 mg/ml) of glucocorticoids and assigned to four groups (A: 0.5 hour, B: 2 hours, C: 7 hours, D: 16 hours) according to the time of treatment. The H2O2 oxidative killing assay was done, using the standard method-spot test, in each group of A. fumigatus. We measured the oxidative killing susceptibility as well as the expression level of the gene Afyap1, CATA, SOD1 and SOD2 in A. fumigatus at each group. The antifungal susceptibility to itraconazole and amphotericin B in each group of A. fumigatus was also measured with M38-A2 method.

RESULTSThe oxidative killing susceptibility of A. fumigatus was increased, consistent with the reduction of Afyap1, CATA, SOD1 and SOD2 gene expression level after being treated with dexamethasone for 0.5 hours. However, these observations were disappeared along with being treated for longer time. The antifungal susceptibility to itraconazole and amphotericin B in the A. fumigatus strains treated with dexamethasone indicated no change, compared with those without dexamethasone treatment.

CONCLUSIONDexamethasone can have A. fumigatus being more susceptible to ROS when treated for shorter period (0.5 to 2 hours) via the reduction of Afyap1 gene expression as well as the down-stream enzyme-coding gene expression.

Aspergillus fumigatus ; drug effects ; genetics ; metabolism ; Dexamethasone ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Hydrogen Peroxide ; pharmacology

Harmaline and harmine are β-carboline alkaloids with effective pharmacological effects. Harmaline can be transformed into harmine after oral administration. However, enzymes involved in the metabolic pathway remain unclear. In this study, harmaline was incubated with rat liver microsomes (RLM), rat brain microsomes (RBM), blood, plasma, broken blood cells, and heme peroxidases including horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO). The production of harmine was determined by a validated UPLC-ESI-MS/MS method. Results showed that heme peroxidases catalyzed the oxidative dehydrogenation of harmaline. All the reactions were in accordance with the Hill equation. The reaction was inhibited by ascorbic acid and excess H₂O₂. The transformation of harmaline to harmine was confirmed after incubation with blood, plasma, and broken blood cells, rather than RLM and RBM. Harmaline was incubated with blood, plasma, and broken cells liquid for 3 h, and the formation of harmine became stable. Results indicated an integrated metabolic pathway of harmaline, which will lay foundation for the oxidation reaction of dihydro-β-carboline. Moreover, the metabolic stability of harmaline in blood should not be ignored when the pharmacokinetics study of harmaline is carried out.
Animals ; Harmaline/metabolism* ; Harmine/metabolism* ; Heme ; Hydrogen Peroxide ; Rats ; Tandem Mass Spectrometry