Some enzymes belonging to the multicopper oxidase (MCO) family can degrade the hazardous biogenic amine (BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability, resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum (MCOF) was fused with a Bacillus subtilis catalase (CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2O2 increased by 51%-68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate, the substrate affinity (Km), the catalytic efficiency (kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions (pH 2.5-4.5) and moderate temperatures (35-55 °C) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.
Biogenic Amines ; Cadaverine ; Catalase/genetics* ; Escherichia coli/genetics* ; Hydrogen Peroxide

OBJECTIVETo construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.

METHODSTotal RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.

RESULTHigh expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.

CONCLUSIONAd/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

Adenoviridae ; genetics ; Catalase ; genetics ; metabolism ; Cell Line ; Genetic Vectors ; Humans ; Transfection

The effects of genetic factors on the noise-induced hearing loss (NIHL) are still unclear. In the present study, eight single-nucleotide polymorphisms (SNPs) included rs1227049 and rs3802711 (CDH23), rs1695 (GSTP1), rs137852540 (GJB2), rs2289274 (PMCA2), rs4880 (SOD2), rs7943316, and rs769214 within CAT that might associated with NIHL were further validated in Chinese workers. The results showed that the carriers of the T allele (AT+TT) of rs7943316 and A allele (GA+AA) of rs769214, were significantly associated with an increased risk of NIHL compared to those with AA genotype (P<0.05) and GG genotype (P<0.05). Moreover, a significant three-locus model (P=0.0107) involving rs2016520, rs9794, and rs1805192 were observed that might associated with NIHL, with 53.95% of testing accuracy. Thus, our present study provided the evidence that GJB2, SOD2, and CAT genes might account for the NIHL development in independently and/or in an interactive manner.
Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Catalase ; genetics ; China ; Connexin 26 ; Connexins ; genetics ; Genetic Predisposition to Disease ; Hearing Loss, Noise-Induced ; genetics ; Humans ; Male ; Superoxide Dismutase ; genetics

Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.
Bacillus subtilis ; genetics ; metabolism ; Bacteria, Aerobic ; enzymology ; genetics ; Catalase ; biosynthesis ; genetics ; Fermentation ; Genetic Engineering ; methods ; Industrial Microbiology ; Recombinant Proteins ; biosynthesis ; genetics ; Textile Industry ; methods

Bacterial Proteins ; genetics ; Catalase ; genetics ; Drug Resistance, Bacterial ; Humans ; Mutation ; Mycobacterium tuberculosis ; genetics ; Oxidoreductases ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

Catalase ; genetics ; Mycelium ; growth & development ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Sporothrix ; enzymology ; growth & development

BACKGROUND/AIMS: Many patients with acute paraquat (PQ) intoxication die even at low PQ concentrations, whereas others with similar concentrations recover. Therefore, it is possible that individual differences in antioxidant capacity are responsible for the variable clinical outcome in patients with acute PQ intoxication. METHODS: We investigated whether there was a relationship between the genetic polymorphisms of SOD (V16A), catalase (C262T), and GPX1 (C593T) in 62 patients with acute PQ intoxication and the clinical outcomes of these patients. RESULTS: The frequency of the Mn-SOD V/V, V/A, and A/A genotypes were 56.3, 43.5, and 0% in survivors and 86.9, 13.1, and 0% in non-survivors (p > 0.05). The GPX1 C/C, C/T, and T/T genotypes were present in 100, 0, and 0% of all subjects. The catalase C/C, C/T, and T/T genotypes were present in 100, 0, and 0% of survivors, and in 82.6, 17.4, and 0% of non-survivors. Neither erythrocyte SOD activity nor catalase activity were significantly different between survivors and non-survivors. CONCLUSIONS: No association was found between clinical outcome of acute PQ intoxication and the genetic polymorphism of GPX1 (C593T) or the genetic polymorphisms or enzyme activity of superoxide dismutase (V16A) or catalase (C262T).
Acute Disease ; Adult ; Aged ; Catalase/*genetics ; Female ; Genotype ; Glutathione Peroxidase/genetics ; Humans ; Male ; Middle Aged ; Paraquat/*poisoning ; Poisoning/mortality ; *Polymorphism, Genetic ; Superoxide Dismutase/*genetics

This study was aimed to investigate the growth and multiple differentiation potential of human umbilical cord tissue derived mesenchymal stem cells (UC-MSCs) transfected by a retroviral vector with catalase (CAT) gene. The UC-MSCs cultured in vitro were transfected by using pMSCV carrying GFP (pMSCV-GFP) and pMSCV carrying CAT (pMSCV-GFP-CAT) respectively, then the MSC-GFP cell line and MSC-GFP-CAT cell line were obtained by sorting of flow cytometry. The GFP expression was observed by a fluorescent microscopy at 48 hours after CAT gene transfection. The GFP+ cells were sorted by flow cytometry. The activity of CAT in GFP+ cells was detected by catalase assay kit. The proliferative capacity of transfected UC-MSCs was determined by cell counting kit-8. The differentiation ability of gene-transfected GFP+ cells into osteogenesis and adipogenesis was observed by von Kossa and oil red O staining. The results indicated that green fluorescence in UC-MSCs was observed at 48 hours after transfection, and the fluorescence gradually enhanced to a steady level on day 3. The percentage of MSCs-GFP was (25.54+/-8.65)%, while the percentage of MSCs-GFP-CAT was (35.4+/-18.57)%. The activity of catalase in UC-MSCs, MSCs-GFP, MSCs-GFP-CAT cells were 19.5, 20.3, 67.2 U, respectively. The transfected MSCs-GFP-CAT could be induced into osteoblasts and adipocytes. After 21 days, von Kossa staining showed induced osteoblasts. Many lipid droplets with high refractivity occurred in cytoplasm of the transfected UC-MSCs, and showed red fat granules in oil red O staining cells. There were no significant differences between transfected and non-transfected UC-MSCs cells (p>0.05). It is concluded that UC-MSCs are successfully transfected by retrovirus carrying GFP or CAT gene, the activity of catalase increased by 3.4-fold. The transfected UC-MSCs maintain proliferation potential and ability of differentiation into osteoblasts and adipocytes.
Catalase ; genetics ; metabolism ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Retroviridae ; genetics ; Transfection ; Umbilical Cord ; cytology

OBJECTIVETo construct a red fluorescent protein reporter gene driven by human catalase gene promoter.

METHODSThe red fluorescent protein reporter gene plasmid pDsRed-CATp containing human catalase gene promoter was constructed by gene recombination technique. The plasmid was transiently transfected into NIH/3T3 cells to observe their response to H(2)O(2) stimulation.

RESULTSThe plasmid was constructed correctly as verified by double enzyme digestion and sequence analysis. The plasmid was lowly expressed in resting NIH/3T3 cells, but the expression level increased obviously after stimulation by H(2)O(2). CONCLSIONS: A red fluorescent protein reporter gene plasmid driven by human catalase gene promoter has been constructed successfully with a sensitive response to H(2)O(2) stimulation. This system provides a convenient tool for the study of the regulatory mechanism of catalase gene expression.

3T3 Cells ; Animals ; Catalase ; genetics ; Gene Expression ; Genes, Reporter ; genetics ; Genetic Vectors ; biosynthesis ; genetics ; Humans ; Hydrogen Peroxide ; pharmacology ; Luminescent Proteins ; biosynthesis ; genetics ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; Transfection

Arylalkylamine N-acetyltransferase (AANAT) and Hydroxyindole O-methyltransferase(HIOMT) are the key regulation enzymes in the melatonin biosynthesis pathway in mammals. The AANAT and HIOMT genes were constructed into a binary plant expression vector YXu55. Using leaf strips as the recipiences, we efficiently transformed tobacco (Nicotiana tabacum) variety qinyan 95 by the Agrobacterium mediated method. After gradient selection with gentamycin, a number of transgenic plants were regenerated. Southern blot and RT-PCR analyses showed that the AANAT-HIOMT genes were integrated into the genome of the transgenic plants and the target genes could express at the level of RNA transcription. By RP-HPLC, we measured the melatonin contents in transgenic plants. The results showed that the melatonin level in YXu55 (containing the gentamycin-resistance gene, the AANAT gene and HIOMT gene) transgenic plants were much higher than those in pZP122 (control containing only the gentamycin-resistance gene) transgenic plants and nontransgenic plants. The content of melatonin in pZP122 transgenic plants was nearly the same as that in nontransgenic plants. Physiological determination of antioxidative characteristics demonstrated that 1) the capacity of total antioxidation, 2) the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and 3) the content of glutathione (GSH) were increased in YXu55 transgenic plants containing the AANAT-HIOMT genes as compared to the control plants (pZP122 or nontransgenic plants). At the same time, malonaldehyde (MDA) content did not appear remarkably difference between transgenic plants and nontransgenic plants. The above mentioned facts indicate enhancement of melatonin levels in YXu55 transgenic plants might help to reduce damage by oxidative stress.
Acetylserotonin O-Methyltransferase ; genetics ; Agrobacterium tumefaciens ; genetics ; Arylalkylamine N-Acetyltransferase ; genetics ; Catalase ; metabolism ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Melatonin ; biosynthesis ; Peroxidase ; metabolism ; Plants, Genetically Modified ; enzymology ; genetics ; Superoxide Dismutase ; metabolism ; Tobacco ; enzymology ; genetics ; Transduction, Genetic ; methods