RESUMO
A new fusion gene (Bgl-licMB), encoding ß-1,3-1,4-glucanase both from Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB), was constructed via end-to-end fusion and expressed in Escherichia coli to improve hydrolytic activity and thermostability of ß-1,3-1,4-glucanase. The results of enzymatic properties showed that the catalytic efficiency (K(cat)/K(m)) of the fusion enzyme for oat ß-glucan was 2.7 and 20-fold higher than that of the parental Bgl and licMB, respectively, and that the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bgl and licMB were both less than 30%. These properties make this particular ß-1,3-1,4-glucanase a good candidate for application in brewing and animal-feed industries.
Assuntos
Bacillus/enzimologia , Clostridium thermocellum/enzimologia , Endo-1,3(4)-beta-Glucanase/genética , Endo-1,3(4)-beta-Glucanase/metabolismo , Avena/química , Bacillus/genética , Clostridium thermocellum/genética , Endo-1,3(4)-beta-Glucanase/química , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Temperatura Alta , Hidrólise , Cinética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , beta-Glucanas/metabolismoRESUMO
In this study, carboxymethyl chitosan-graft-poly-(ε-caprolactone) copolymers (CMCS-g-PCL) were synthesized and used to encapsulate apatinib to prepare apatinib-loaded CMCS-g-PCL (CPA) micelles. CPA micelles' sizes were 100-150nm at pH 7.4 while aggregated to 300-350nm at pH 6.4, and the release rate at pH 6.4 was faster than pH 7.4, indicating CPA micelles have a pH-responsive activity. Furthermore, the release rate decreased with an increased grafting ratio of CMCS-g-PCL, which was shown by the results of release experiments from CPA-2 to CPA-10 micelles. A series of cell experiments demonstrated that blank micelles were non-toxic for human umbilical endothelial cells (HUVECs) below 0.125mg/ml, CPA micelles had significant inhibiting effect on HUVECs as IC50 was near 3.125µg/ml, and the drug effect could be adjusted by altering grafting ratio of CMCS-g-PCL. These results suggest that CPA micelles may be used as an effective drug delivery system for anti-angiogenesis cancer therapy.