RESUMEN
L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 µg/mL) and HeLa cells (IC50, 8.3 µg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 µg/mL) and RAW 264.7 cell (IC50, 59.3 µg/mL).
Asunto(s)
Antineoplásicos/química , Glutaminasa/química , Streptomyces/enzimología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Biocatálisis , Proliferación Celular/efectos de los fármacos , Estabilidad de Enzimas , Glutaminasa/metabolismo , Glutaminasa/farmacología , Glutamina/química , Glutamina/metabolismo , Células HeLa , Células Hep G2 , Humanos , Cinética , Streptomyces/química , Especificidad por SustratoRESUMEN
Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).