β-galactosidase immobilization on controlled pore silica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.

Yacon (Polymnia sanchifolia) extract as a substrate to produce inulinase by Kluyveromyces marxianus var. bulgaricus

The inulinase production by yeast K marxianus var. bulgaricus growing in yacon extract was investigated. The microorganism showed good development in yacon, higher enzymatic activities were achieved at 30% and 40% (v/v) of extract. The cultivation temperature (20, 25, 30, 35, 40 degreesC) neither influenced the growth or the enzymatic activity. The optimum cultivation pH was 3.5. The highest activity was observed at 60 degreesC and pH 4.0. At temperature of 55 C and 60 C occurred sharp decrease in the enzyme activity. (C) 2004 Elsevier Ltd. All rights reserved.

Effects of culture conditions on the production of inulinase by Kluyveromyces marxianus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 degrees C for the free and 60 degrees C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kj/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver-Burk plots and the V-max and K-m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%. (c) 2008 Elsevier Ltd. All rights reserved.

Effect of Conditioning on the Production of Inulinase by Kluyveromyces marxianus var. bulgaricus through Fed-Batch Fermentation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of nitrogen source and sucrose concentration on inulinase production by Kluyveromyces marxianus in fed-batch fermentation

This work studied the influence of nitrogen source and sucrose concentration in the feeding medium for biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus. The results show that the best nitrogen source was a combination of 5 g/L of yeast extract and 10 g/L of peptone. Both cellular growth and enzymatic activity increased with sucrose concentration in the feeding medium (from 200 to 500 g/L). When the sucrose concentration reached 600 g/L, both cellular growth and enzymatic activity decreased.

Location of the β-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not β-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-β-D-galactopyranoside (ONPG), indicating that β-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that β-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncouplercarbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of β-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. © 1996 Kluwer Academic Publishers.

Estudo da bioconversão da lactose do soro de leite em bioetanol pela evedura Kluyveromyces marxianus 229

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Estudo da hemoglobina como adjuvante na infecção experimental de eqüinos, via intraperitoneal, com Escherichia coli e/ou Bacteroides fragilis

Pós-graduação em Medicina Veterinária - FCAV

Effect of divalent metal ions on the activity and stability of β-galactosidase isolated from Kluyveromyces lactis

In this study, it was demonstrated that β-galactosidase can be deactivated and reactivated with EDTA and divalent metal ions. The enzyme was deactivated after 20 minutes in EDTA solution. Maximal deactivation at the lowest EDTA concentration (10-3 mol.L-1) occurred in the presence of Tris-HCl buffer (pH 7.0). The enzyme recovered 50% of its initial activity after 10 minutes at Mg2+concentrations higher than 0.1 mmol.L-1. Experimental concentrations of 0.1 mmol.L-1 Mn2+ and 1.0 mmol.L-1 Co2+ were sufficient to reactivate the enzyme to around 300% of the control activity for the Mn2+ ion and nearly 100% for the Co2+ ion. The enzyme gradually lost its activity when the Co2+ concentration was 10-2 mol.L-1. Ni2+ and Zn2+ were unable to restore the catalytic activity. Km app and Vmax app were 1.95 ± 0.05 mmol.L-1 and 5.40 ± 0.86x10-2 mmol.min-1.mg-1, with o-NPG as substrate. Optimal temperature and pH were 34oC and 7.5. The half-life (t1/2) at 30°C was 17.5 min for the holoenzyme and 11.0 min for the apoenzyme. With respect to pH variation, the apoenzyme proved to be more sensitive than the holoenzyme. Keywords: β-galactosidase. Divalent metallic ions. Enzyme activity. Stability. RESUMO Efeito de íons metálicos divalentes na atividade e estabilidade da β-galactosidase isolada de Kluyveromyces lactis Este estudo demonstra como a β-galactosidase pode ser desativada e reativada usando EDTA e íons metálicos divalentes. A enzima foi desativada após 20 minutos na presença de EDTA. Desativação máxima para a menor concentração de EDTA (10-3 mol.L-1) ocorreu na presença do tampão Tris-HCl. A enzima recuperou 50% de sua atividade inicial após 10 minutos na presença de Mg2+ em concentrações superiores a 0,1mmol.L-1. Concentrações de 10-4 e 10-3mol.L-1 de Mn2+ e Co2+ foram suficientes para reativar a enzima em 300% comparado ao controle de íons Mn2+ e aproximadamente 100% para íons Co2+. A enzima perdeu gradualmente a sua atividade quando a concentração foi de 10-2 mol.L-1. Ni2+ e Zn2+ foram incapazes de restabelecer a atividade catalítica. Km app e Vmax app foram 1,95 ± 0,05 mmol.L-1 e 5,40 ± 0,86 x 10-2 mmol.min-1.mg-1. A temperatura e pH ótimos foram 34ºC e 7,5. A meia vida da holoenzima foi de 17,5 min a 30ºC e para a apoenzima foi de 11,0 min a 30ºC. Quanto à variação de pH, a apoenzima provou ser mais sensível que a holoenzima. Palavras-chave: β-galactosidase. Íons metálicos divalentes. Atividade enzimática. Estabilidade.

Experimental peritonitis in horses. Hematological and biochemistry aspects

Dezesseis eqüinos adultos foram distribuídos aleatoriamente em 4 grupos (GI, GII, GIII e GIV) constituídos por quatro animais, recebendo cada grupo o seguinte inóculo por via intraperitoneal: GI (100 X 10(7) unidades formadoras de colônia (UFC) de Escherichia coli diluídos em 500 ml de solução salina 0,9% estéril); GII (100 X 10(7) UFC de Bacteroides fragilis diluídos em 500 ml de solução salina 0,9% estéril); GIII (100 X 10(7) UFC de Escherichia coli associados a 100 X 10(7) UFC de Bacteroides fragilis diluídos em 500 ml de solução salina 0,9% estéril); GIV (testemunho - 500 ml de solução salina 0,9% estéril). Leucopenia ocorreu em todos os animais inoculados com bactérias, nas primeiras seis horas após as inoculações. Posteriormente a este período, verificou-se em alguns eqüinos inoculados leucocitose. Os eqüinos inoculados com culturas puras de E. coli ou B. fragilis apresentaram peritonites brandas e autolimitantes, enquanto os inoculados com a associação destas bactérias, apresentaram alterações laboratoriais de maior intensidade e duração.

Amniotic Fluid Interleukin-1 Beta and Interleukin-6, but not Interleukin-8 Correlate with Microbial Invasion of the Amniotic Cavity in Preterm Labor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clinical aspects of experimental peritonitis in horses

Dezesseis eqüinos adultos foram distribuídos aleatoriamente em 4 grupos (GI, GII, GIII e GIV), constituídos por quatro animais, recebendo cada grupo o seguinte inóculo por via intraperitoneal: GI (100 X 10(7) unidades formadoras de colônia (UFC) de Escherichia coli diluídos em 500ml de salina 0,9%); GII (100 X 10(7) UFC de Bacteroides fragilis diluídos em 500ml de salina 0,9%); GIII (100 X 10(7) UFC de E. coli associados a 100 X 10(7) UFC de B. fragilis diluídos em 500ml de salina 0,9%); GIV (testemunho - 500ml de salina 0,9%). Aumento da sensibilidade e tensão da parede abdominal, diarréia, diminuição dos sons intestinais e aumento da freqüência cardíaca foram os sinais mais freqüentemente observados nos eqüinos inoculados com cepas bacterianas. Eqüinos inoculados com culturas puras de E. coli ou B. fragilis apresentaram peritonites brandas e autolimitantes, enquanto que os inoculados com a associação dessas bactérias apresentaram sinais de maior intensidade e duração.

Experimental peritonitis in horses: peritoneal fluid composition

Dezesseis eqüinos adultos foram aleatoriamente divididos em quatro grupos de quatro animais que receberam inoculação intraperitoneal das seguintes suspenções: grupo I, 100×10(7) unidades formadoras de colônias (CFU) de E. coli diluídas em 500ml de solução salina a 0,9%; grupo II, 100×10(7) CFU de Bacteroides fragilis em 500ml de solução salina a 0,9%; grupo III, 100×10(7) CFU de E. coli combinados com 100×10(7) CFU de B. fragilis em 500ml de solução salina a 0,9%; grupo IV, 500ml de solução salina a 0,9%. Observou-se aumento significativo do número de leucócitos no líquido peritoneal quatro horas após as inoculações dos animais dos grupos I e II, e oito horas após as inoculações dos animais do grupo III. A contagem mais elevada foi de 516×10³ leucócitos/mm³. Aumentos significativos nas concentrações de fibrinogênio (1g/dl) e proteína total (9,1%) foram também observados. Eqüinos inoculados com culturas puras, tanto de E. coli quanto de B. fragilis, apresentaram peritonites mais brandas e autolimitantes, enquanto que eqüinos inoculados com associação das duas bactérias apresentaram alterações laboratoriais com maior intensidade e duração.