One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

A purificação de uma etapa e caracterização de uma xilanase livre de celulase de uma linhagem recentemente isolada alcalofílicos e moderadamente termofílico de Bacillus subtilis ASH. Xilanase foi purificada à homogeneidade de 10,5 vezes, com ~ por cento de recuperação 43 através de cromatografia de troca iônica através de CM- Sephadex C -50. A enzima purificada revelou uma única banda no gel SDS-PAGE com uma massa molecular de 23 kDa. Ele mostrou um pH ótimo de 7,0 e manteve-se estável na faixa de pH 6,0-9,0 . A temperatura ótima para atividade da enzima foi 55 º C. A xilanase purificada não perder nenhuma atividade até 45 º C , no entanto, manteve 80 por cento e 51 por cento de sua atividade após pré-incubação a 55 º C e 60 º C , respectivamente. A enzima obedecido Michaelis- Menton cinética para xilano de madeira de bétula com aparente km 3,33 mg / ml e Vmax 100 UI / ml. A enzima foi fortemente inibida por Hg2 +, Cu2 + , enquanto reforçada por Co2 + e Mn2 +. A enzima purificada pode ser armazenado a 4 º C por seis semanas sem nenhuma perda de atividade catalítica. A purificação mais rápido e econômico da xilanase livre de celulase de B. subtilis ASH por um passo-a processo juntamente com a sua estabilidade sensível a alta temperatura e pH alcalino torna potencialmente eficazes para aplicações industriais.

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash.

The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 °C. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg(2+)and Cu(2+)while enhanced by Co(2+) and Mn(2+). The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 ºC. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.