Enhanced Production Of Streptokinase By Chemical Mutagenesis Of Streptococcus agalactiae EBL-20

Abstract Streptokinase (SK) is an enzyme that is used for the treatment of cardiovascular diseases. The current study focused on the enhanced production of SK by inducing mutation in Streptococcus agalactiae EBL-20 and optimization of medium components and culture conditions for the maximum growth of mutant derived strain. S. agalactiae EBL-32 was selected as a potent mutant after exposure of S. agalactiae EBL-20 to EMS for 180 minutes. SK activity obtained from mutant derived strain was found to be 1.6 fold higher as compared to the activity achieved by wild strain. Nutritional requirements of the mutated strain were optimized by single factor analysis method suggesting glucose as the optimum carbon source; yeast extract and peptone as a suitable nitrogen sources and corn steep liquor (CSL) as an appropriate substrate for the maximum SK production. The culture conditions determined by response surface methodology (RSM) suggested that a temperature value of 37.5⁰C and pH 7 of the fermentation medium with 2.50 mL inoculum size for 36 hours of incubation was optimum for maximum yield of SK. Hence the optimization studies resulted into 1.92 fold increase in the yield of SK suggesting the new isolate suitable for commercial scale production of SK.

Enhanced alkaline cellulases production by the thermohalophilic Aspergillus terreus AUMC 10138 mutated by physical and chemical mutagens using corn stover as substrate

Abstract A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70 percent increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Efeito residual do extrato acetato de etila de Trichilia pallida Swartz ( Meliaceae ) para lagartas de diferentes idades de Spodoptera frugiperda ( J. E. Smith, 1797 ) ( Lepidoptera: Noctuidae ) / Residual effect of ethyl acetate extract of Trichilia pallida Swartz ( Meliaceae ) for Spodoptera frugiperda ( J. E. Smith, 1797 ) ( Lepidoptera: Noctuidae ) larvae of diferents ages

Inseticidas de origem vegetal mostram limitada persistência em condições ambientais já que são degradados pela temperatura, por raios ultravioletas e por outros fatores ambientais. Com o objetivo de avaliar o efeito residual do extrato acetato de etila de Trichilia pallida, plantas de milho cultivadas em casa de vegetação foram pulverizadas com o referido extrato na concentração de 2 por cento, e as suas folhas utilizadas para alimentação de lagartas de Spodoptera frugiperda, em laboratório. As folhas foram oferecidas às lagartas aos 1, 3 e 7 dias após a pulverização, correspondendo cada época a um tratamento. Foram utilizados dois grupos de lagartas: recém-eclodidas e com 10 dias de idade. As folhas foram trocadas diariamente e, 10 dias após o início do experimento, foram avaliados a mortalidade, a largura da cápsula cefálica e o peso das lagartas. Verificou-se que, nas três épocas, o extrato reduziu a sobrevivência e prolongou o desenvolvimento larval. A intensidade destes efeitos foi maior quanto menor o intervalo entre a aplicação do extrato e o início do fornecimento das folhas aos insetos. Lagartas alimentadas com folhas tratadas com o extrato desde a eclosão foram mais afetadas que aquelas alimentadas a partir dos 10 dias.

RAPD analysis of herbicide-resistant Brasilian rice lines produced via mutagenesis

Over the last two decades, mutational techniques have become one of the most important tools available to progressive rice- breeding programs. In a mutation-breeding program initiated in 1999 at the Instituto Agronômico of Campinas, SP, Brazil, a rice line, IAC103, was selected for mutational studies with gamma radiation and ethyl methyl sulfonate mutagenesis, with the aim of developing a herbicide-resistant crop. After mutagenesis, surviving plants were exposed to glufosinate to check for herbicide resistance, which was examined up to the second generation. A detailed RAPD analysis was made of the resistant plants. Eighty Operon technology primers were tested and 10 were selected for a detailed study of RAPD markers that could tag herbicide resistance genes. Resistant and susceptible lines produced variation in the RAPD patterns and certain bands were found only in certain lines. These results suggest genetic ligation that will be confirmed through a genetic segregation study

Mutagen sensitivity of an insecticide-resistant strain of Drosophila melanogaster to ethyl methanssulfonate: comparison with a standard strain

Duas cepas de Drosophila melanogaster, uma resistente a vários inseticidas (MRA) e a outra sensível (BK), foram analisadas quanto à mutabilidade face ao conhecido agente alquilante metanosulfato de etila(EMS). Os resultados relativos à induçäo de letales recessivos ligados ao sexo indicam que, embora a cepa padräo seja mais sensível aos efeitos mutagênicos do EMS, com uma taxa de mutaçäo induzida que é aproximadamente o dobro do valor obtido para a cepa MRA, ambas as cepas säo suficientemente sensíveis, apresentando um incremento da mutabilidade em células germinais masculinas pós-meióticas dependente de dose

Ethyl methanesulfonate-induce mutations in Phaseoulus vulgaris L.

Os alcalóides furoquinoleínico esquimianina e benzofenantridínico celeritrina, extraídos de uma espécie da família Rutaceae foram testados quanto ao aspecto tóxico-genético através do cromoteste-SOS. Nos testes realizados na ausência de metabolizaçäo, ambos alcalóides näo mostraram atividade genotóxica, sendo que a esquimianina apresentou um efeito citotóxico nas concentraçöes mais elevadas. Na presença de mistura de ativaçäo metabólica, a esquimianina mostrou-se genotóxica sendo que este efeito foi mais acentuado quando se empregou fraçäo microssomal induzida com Aroclor 1254 em relaçäo aquela induzida com 3-metilcolantreno

Combined effect of ethylmethane sulfonate and 60CO gamma radiation on the induction of lethal mutations in Drosophila melanogaster

Duas linhagens de Drosophila melanogaster foram tratadas com doses independentes e conjuntas de etilmetanosulfonato (EMS) e radiaçäo gama de 60CO. As linhagens Columbia (CO3) e Riverside, Califórnia (RC1) foram tratadas com três doses de EMS (0.003; 0.006 e 0.012 M), ou com quatro doses de radiaçäo (2,5; 5; 10 e 15 KR) ou, ainda, com três doses conjuntas dos dois mutagênicos (0.002M + 10 KR; 0.003 + 5 KR e 0.004M + 2.5 KR). Foram estudadas diferenças de sensibilidade entre as duas linhagens, quanto à induçäo de letais recessivos ligados ao sexo, e determinadas as relaçöes existentes entre altas doses de mutagênico e freqüência da mutaçäo induzida. A relaçäo entre dose de EMS e produçäo de letais se distribuiu dentro de uma curva parabólica. Tal comportamento decorreu ou da eliminaçäo seletiva das células mais sensíveis sob o ponto de vista da mutabilidade, ou da saturaçäo na capacidade de absorçäo do mutagênico, quando administrado em altas doses. Na dose de 0.006 M de EMS, a percentagem de letais obtida indicou a existência na linhagem CO3, de resistência à açäo deste mutagênico. No tratamento conjunto com EMS e radiaçäo, a linhagem CO3 apresentou um efeito sinergístico, enquanto a RC1 mostrou um efeito aditivo. Dados anteriores sugerem que a linhagem CO3 apresenta um mecanismo de reparo mais eficiente, já que o tratamento conjunto parece inibir as enzimas de reparo, a linhagem CO3 é mais atingida por este tipo de inibiçäo