Expression and biochemical characterization of a Bacillus subtilis catalase in Pichia pastoris X-33.

Catalase, which catalyzes the decomposition of H2O2 to H2O and O2, is widely used to reduce H2O2 in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 ± 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 °C and pH 11.0. Its Km for hydrogen peroxide was 10.9 ± 0.5 mM, and its kcat/Km was 5788.1 ± 25.6 s-1 mM-1. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.

Effects of PI3K inhibitor NVP-BKM120 on acquired resistance to gefitinib of human lung adenocarcinoma H1975 cells.

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 µmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 µmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 µmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 µmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 µmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 µmol/L)-treated group or between DMSO-treated control group and gefitinib (1 µmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 µmol/L), and NVP-BKM120 (1 µmol/L) or NVP-BKM120 (1 µmol/L) plus gefitinib (1 µmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 µmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.