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.

Oxidative profiles of endurance horses racing different distances / Perfil oxidativo em cavalos de enduro competindo em diferentes distâncias

Increased oxidative stress during prolonged endurance exercises may result in muscle damage, fatigue and decreased performance. An adequate stress response during training is critical to obtain improved results and high animal welfare standards. The aim of this study was to evaluate the red blood cell haemolysate concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and catalase (CAT) and the plasma concentrations of malondialdehyde (MDA) from endurance horses in different distances at high speed in a tropical climate. Fifteen horses were tested; five at 160km (18.54 - 17.16km/h race speed), five at 120km (21.53 - 17km/h race speed) and five at 80km (20.06 - 18.01km/h race speed). Blood samples were collected at rest, immediately after and three hours after the horses left the final vet check and three, seven and fourteen days after the race. No significant increases (P > 0.05) in the levels of SOD, GPx, GSH, CAT or MDA were observed for any of the times or distances examined. Based on these observations, we conclude that reactive oxygen species (ROS) formation during exercise evokes specific adaptations, such as increased antioxidant/oxidative damage-repairing enzyme activity, increased resistance to oxidative stress and lower levels of oxidative damage...

Aumento do estresse oxidativo durante o exercício prolongado pode resultar em fadiga muscular, lesões e diminuição do desempenho. Uma adequada resposta a esse estresse durante o treinamento é fundamental para a obtenção de melhores resultados e bem-estar dos animais. O objetivo deste estudo foi avaliar a concentração de superóxido dismutase (SOD), glutationa peroxidase (GPx), glutationa reduzida (GSH) e catalase (CAT) no hemolisado sanguíneo e malondialdeído (MDA) plasmático em cavalos de enduro correndo em diferentes distâncias, com alta média de velocidade, em clima tropical. Quinze cavalos foram testados, cinco em 160km (18.54-17.16km/h), cinco em 120km (21.53-17km/h) e cinco em 80km (20.06-18.01km/h). Amostras de sangue foram coletadas em repouso, imediatamente e três horas depois que os cavalos passaram pela inspeção veterinária final e três, sete e 14 dias após a corrida. Não houve aumentos significativos (P>0,05) dos níveis de SOD, GPx, GSH, CAT ou MDA em nenhum tempo nem distâncias analisadas. Com base nessas observações, pode-se concluir que as espécies reativas de oxigênio (ROS) formadas durante o exercício provocam adaptações específicas, tais como atividade antioxidante aumentada da enzima, maior resistência ao estresse oxidativo e menores níveis de danos oxidativos...

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 percent NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98 percent sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 percent activity at 80 0C after 4 h and more than 70 percent activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 percent active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Cellulolytic potential of a novel strain of Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda gut

A cellulolytic bacterial strain, designated P118, isolated from the gut of the tropical fish Parotocinclus maculicauda was identified as belonging to the genus Paenibacillus based on phenotypic and chemotaxonomic characteristics and the 16S rRNA gene sequence. The novel strain was Gram-positive, spore-forming and rod-shaped. Catalase but not oxidase was produced. Carboxymethylcellulose was hydrolyzed but starch or gelatin was not. Acetoin production was negative whereas nitrate reduction and urease production were positive. Many carbohydrates served as carbon sources for growth. MK-7 was the predominant isoprenoid quinone. Anteiso-C15:0 (38.73 percent) and C16:0 (20.85 percent) were the dominant cellular fatty acids. Strain P118 was closely related to Paenibacillus amylolyticus NRRL NRS-290, P. pabuli HSCC 492, P. tundrae Ab10b, P. xylanexedens B22a, and P. tylopili MK2 with 98.3-98.8 percent 16S rRNA gene sequence similarity. The results presented here suggest that strain P118 represents a novel species of the genus Paenibacillus and it is a potential strain for further studies concerning its role in the production of industrially important products from cellulosic biomass.

Catalase activity of different Candida species after exposition to specific antiserum

Antisera were developed in rabbits after challenge with intracellular antigens of Candida albicans, C. tropicalis and C. parapsilosis. Microorganism catalase has been correlated with virulence, resistance to drugs and immunogenicity. The intracellular catalase is consistently present in strains of Candida and in this paper, the enzyme activity was analysed by PAGE after exposition to antisera. The catalases of C. albicans, C. parapsilosis and C. tropicalis were immunogenic and differed in their binding to specific antibodies raised in rabbits. Tests of cross-reactivity between different Candida species showed that when antiserum from C. albicans immunized rabbit was incubated with intracellular extracts of these three Candida species, the catalases activities were abolished. However, the antisera from C. parapsilosis or C. tropicalis immunized rabbits did not affect the catalase activity of C. albicans; the enzyme of C. albicans was inactivated only by the antiserum to the catalase of own C. albicans. The antiserum to the catalase of C. tropicalis was species-specific and did not cross-react with catalases of C. albicans and C. parapsilosis. The activities of Aspergillus niger and bovine catalases were not affected by the antiserum from any Candida immunized rabbits. This report is a preliminary study of specific antisera that react against intracellular catalase of Candida sp. and neutralize the enzymatic activity. Further study is necessary to develop species-specific antibody once differences in the susceptibility of the Candida species to commonly used antifungal drugs make identification to the species level important.

Antisoros foram desenvolvidos em animais em resposta à injeção de antígenos intracelulares de Candida albicans, C. tropicalis e C. parapsilosis. A presença de catalase nos microrganismos tem sido relacionada à virulência, resistência a drogas e imunogenicidade. A catalase intracelular está sempre presente nas cepas de leveduras do gênero Candida e neste trabalho, sua atividade enzimática foi analisada por PAGE, após exposição aos antisoros obtidos. Verificou-se que as catalases de C. albicans, C. tropicalis e C. parapsilosis foram imunogênicas e apresentaram diferenças quanto à sua ligação aos anticorpos específicos produzidos nos coelhos. Testes de reação cruzada mostraram que quando o antisoro obtido do animal imunizado com C. albicans foi incubado com os extratos intracelulares de cada uma das três espécies, a atividade da catalase intracelular foi neutralizada em todos os casos. Entretanto, os antisoros obtidos da imunização com C. parapsilosis e C. tropicalis não afetaram a atividade da catalase de C. albicans; a catalase de C. albicans foi neutralizada somente pelo antisoro específico. O antisoro anti-C. tropicalis foi espécie-específico e não reagiu com as catalases de C. albicans e C. parapsilosis. As atividades das catalases bovina e de Aspergillus niger não foram afetadas por nenhum dos antisoros produzidos contra Candida sp. Este trabalho é uma análise preliminar de antisoros específicos reagentes com a catalase intracelular de Candida sp., capazes de neutralizar a atividade enzimática. Novos estudos são importantes para o desenvolvimento de anticorpo espécie-específico, uma vez que diferentes espécies de Candida apresentam diferente susceptibilidade aos antifúngicos de uso corrente.

Extraction and purification of a catalase from Candida albicans

Candida albicans yeast cells H317 were grown to mid-log phase, mechanically disrupted and the resulting crude extract clarified by centrifugation. This catalase rich fraction (1.26 x 10(-4) units/ml) was fractionated by liquid phase isoelectric focusing in a pH gradient ranging from 3 to 10 using the Rotofor Isoelectric Focusing Preparative Cell. After isoelectric separation, fractions containing catalase activity were focused between pH 6.7 and 9.3. Active fractions were pooled and re-focused. After the second fractionation, catalase activity increased to 1.52 x 10(-2) units/ml and was restricted to fractions ranging from pH 7.6 to 8.8. To this point a 121 fold purification was achieved. Native polyacrylamide gel electrophoresis analysis of active fractions revealed a band migrating between 272,000 and 132,000 daltons which showed catalase activity. Purification of C. albicans catalase will allow us to evaluate its potential role in protecting this opportunistic pathogen from products of the oxidative burst. Antibodies generated against the catalase provide means for the evaluation of neutralizing fungal defenses against products of the oxidative burst during phagocytosis

Purification of rat intestinal cinnabarinate synthase and its possible role in hyperplasia of the small intestine in diabetic rats.

Streptozotocin diabetic rats fed ad libitum exhibited hyperplasia of the small intestine. As compared to the control animals, the intestine of experimental animals grew in weight, length and total RNA and DNA contents. Intestinal cinnabarinate synthase activity in diabetic rats was however significantly lower. Developmental studies in albino rats indicated that, attainment of the terminal and highest activity of the enzyme tends to correspond with cessation of further increase in RNA and DNA contents of the intestine, thereby suggesting a possible relationship between cinnabarinate synthase and the hyperplastic changes observed. It was also observed that some properties of this enzyme, such as Km and Vmax are modified in diabetic condition. The enzyme was purified to apparent homogeneity and some of its kinetic and other properties were studied.

Purification and properties of goat liver catalase: two pH optima.

Goat liver catalase (EC 1.11.1.6) has been purified to homogeneity using the techniques of ammonium sulfate fractionation, DEAE-cellulose chromatography and gel-filtration through Ultrogel AcA-34 involving two alternating steps of column chromatography. The homogeneity of the purified enzyme was tested by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. The enzyme is a tetramer having a subunit molecular weight of 58,000 +/- 3000, contains six sulfhydryl groups per mole of the enzyme and shows pH optima at pH 6.8 and 7.7. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indoleacetic acid, cysteine, formaldehyde and sodium azide inhibit the enzyme non-competitively with Ki values of 4 +/- 1, 2.5 +/- 0.8, 6 +/- 1.5, 0.48 +/- 0.15 and 0.0013 +/- 0.0003 mM, respectively. Sulfhydryl group binding agents as well as thiol reagents inhibit the enzyme activity.