RESUMEN
Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.
Asunto(s)
Bacillus/química , Bacillus/clasificación , Bacillus/enzimología , Bacillus/genética , Bacillus/crecimiento & desarrollo , Bacillus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Estabilidad de Enzimas/química , Estabilidad de Enzimas/clasificación , Estabilidad de Enzimas/enzimología , Estabilidad de Enzimas/genética , Estabilidad de Enzimas/crecimiento & desarrollo , Estabilidad de Enzimas/metabolismo , Variación Genética/química , Variación Genética/clasificación , Variación Genética/enzimología , Variación Genética/genética , Variación Genética/crecimiento & desarrollo , Variación Genética/metabolismo , Genotipo/química , Genotipo/clasificación , Genotipo/enzimología , Genotipo/genética , Genotipo/crecimiento & desarrollo , Genotipo/metabolismo , Calor/química , Calor/clasificación , Calor/enzimología , Calor/genética , Calor/crecimiento & desarrollo , Calor/metabolismo , Concentración de Iones de Hidrógeno/química , Concentración de Iones de Hidrógeno/clasificación , Concentración de Iones de Hidrógeno/enzimología , Concentración de Iones de Hidrógeno/genética , Concentración de Iones de Hidrógeno/crecimiento & desarrollo , Concentración de Iones de Hidrógeno/metabolismo , Lipasa/química , Lipasa/clasificación , Lipasa/enzimología , Lipasa/genética , Lipasa/crecimiento & desarrollo , Lipasa/metabolismo , Filogenia/química , Filogenia/clasificación , Filogenia/enzimología , Filogenia/genética , Filogenia/crecimiento & desarrollo , Filogenia/metabolismoRESUMEN
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).