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

Este estudo demonstra como a Beta-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 10-3 mol.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.

In this study, it was demonstrated that Beta-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 30ºC 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.

AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India

AmpC â-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC â-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC â -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57 percent) strains were resistant to 3GC, among which 63(47 percent) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7 percent) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9 percent (51/63) of screen positive isolates were detected by Amp C disc test and 93.6 percent (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9 percent) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.

Epidemiology and antimicrobial resistance of staphylococci isolated from different infectious diseases

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5 percent), amikacin (2.3 percent) and gatifloxacin (3.8 percent). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for â-lactamase production where, 61.7 percent of S. aureus and 42.9 percent of CoNS were positive for â-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the â-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.

Producción y caracterización parcial de beta-galactosidasa de Kluyveromyces lactis propagada en suero de leche desproteinizado / Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey

The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83 per cent, total solids 6.17 per cent, protein 0.44 per cent, lactose 4.85 per cent, acidity 0.43 per cent and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8 per cent. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

ß- galactosidasa de alta pureza obtenida por cromatografía de interacción hidrofóbica / High purity ß galactosidase obtained by hidrophobic interaction chromatography

Se presenta un procedimiento de obtención de ß-galactosidasa recombinante en Escherichia coli con una pureza superior al 95 % basado en dos pasos de purificación y solo uno de ellos cromatográficos. Se combinaron en el trabajo la precipitación salina y la cromatografía de interacción hidrofóbica en Fractogel TSK butilo 650 M, alcanzándose en esta última una alta resolución mediante el ajuste efectivo de los parámetros determinantes en esta operación

Purificación de ß-galactosidasa recombinante y su evaluación como enzima marcadora en un sistema inmunoenzimático con anticuerpos monoclonales / Purification of recombinant ß-galactosidase and its evaluation as label enzyme in an immunoenzymatic assay with monoclonal antibodies

La enzima ß-galactosidasa producida por vía recombinante en E. Coli fue purificada por cromatografía de intercambio iónico y filtración por gel, lográndose una preparación de enzima de más del 95 % de pureza. Su capacidad como enzima marcadora en inmunoensayo enzimático sobre fase sólida (ELISA), fue probada comparándose con peroxidasa, la cual ha sido ampliamente usada en estos ensayos. Se realizaron conjugados con anticuerpo monoclonal anti-rotavirus para un sistema de detección de rotavirus, utilizando el agente heterobifuncional SPDP. Se obtuvieron niveles similares de sensibilidad para los conjugados realizados con peroxidasa y ß-galactosidasa; igualmente no se observaron diferencias significativas en el parámetro unión específica/unión no específica. En el presente trabajo mostramos los resultados obtenidos en la purificación de la enzima, su seguimiento por actividad enzimática y su uso como enzima marcadora en inmunoensayo enzimático

Inmovilización covalente de ß-galactosidasa de A. fonsecaeus sobre sílice aminada / Covalent immobilization on aminated silica of ß-galactosidase from A. fonsecaeus

La ß-galactosidasa semipurificada del A. fonsecaus fue inmovilizada covalentemente sobre sílice aminada activada con glutaraldehído. Se estudió la influencia del área específica del soporte, de la concentración de proteínas y del PH de inmovilización sobre los rendimientos del proceso. Se demostró que el área específica más efectiva fue de 122 m2 (aproximadamente 250A) y que la concentración de proteínas y el PH óptimo de inmovilización fueron de 1,6 g/l y 8,6 respectivamente. La enzima inmovilizada tiene una zona de PH óptimo para la hidrólisis del orto-nitrofenil ß-D-galactopiranósido (ONPG) semejante a la enzima libre. Los resultados obtenidos utilizando la ß-galactosidasa purificada de A. oryzae son comparables a los resultados obtenidos con los preparados semipurificados de ß-galactosidasa de A. fonsecaus. Este estudio crea las premisas para el empleo de la ß-galactosidasa de esta nueva fuente microbiana, en la inmovilización covalente sobre otros soportes utilizables en procesos industriales, para los que las propiedades de esta enzima sean relevantes

Inmovilización covalente de ß-galactosidasa de A. fonsecaeus sobre derivados celulósicos del maíz: comparación con la ß-galactosidasa del A. oryzae / Covalent immobilization on corn cellulose derivative of ß-galactosidase from A. fonsecaeus: comparison with ß-galactosidase from A. oryzae

La ß-galactosidasa del A. fonsecaeus con actividad específica entre 19-24 U/mg, a concentraciones de 1,6-3,3 g/l y PH 8,6 fue inmovilizada covalentemente sobre derivados celulósicos del maíz (DCM), con altos rendimientos (667 U/g de DCM). El mayor porcentaje de enzima inmovilizada y los valores superiores de actividad enzimática por gramo de soporte, se obtuvieron con el empleo de DCM de 0,8 mm. Fueron determinadas algunas características cinéticas de la ß-galactosidasa inmovilizada de A. fonsecaeus y A. oryzae en reactor antidifusional utilizando ortonitrofenil-ß-D-galactopiranósido (ONPG) y lactosa como sustrato. La enzima de A. fonsecaeus presenta valores de Km aparente inferiores a la de A. oryzae en forma inmovilizada, contrario a lo que ocurre en forma libre. La hidrólisis continua de soluciones de lactosa en reactores de columna empacada con DCM ß-galactosidasa de ambos microorganismos, produjo resultados similares y comparables a los obtenidos en otros sistemas reportados. La enzima inmovilizada del A. fonsecaeus fue muy estable; los tiempos de vida media a 35-C y 50-C, fueron de 240 y 80 días respectivamente. La enzima inmovilizada puede ser empleada para la hidrólisis continua de la lactosa del suero lácteo a 50-C y PH 4,5 con una productividad de 0,9 g de lactosa/g de DCM-enzima/hora.