Data Availability StatementAll data generated or analyzed during this study are
Data Availability StatementAll data generated or analyzed during this study are included in this published article. was significantly enhanced from 3.2 to 158?U/L. Under the optimized condition, the enzyme was produced on a large-scale, and highly indicated cholesterol oxidase was purified from cell lysate by column nickel affinity chromatography. Km and Vmax ideals of the purified enzyme for cholesterol were estimated using LineweaverCBurk storyline. Further, the optimum pH and optimum temp for the enzyme activity were also determined. We statement a straightforward and AMD3100 manufacturer easy protocol for cholesterol oxidase production AMD3100 manufacturer which can be performed in any laboratory. sp. SA-COO (ChOA) secretory production has been proved inside a Mouse monoclonal to LAMB1 host-vector system (Murooka et al. 1986). Also, the gene has been cloned and sequenced (Ishizaki et al. 1989). Nomura AMD3100 manufacturer et al. successfully indicated the gene in (Nomura et al. 1995). Further, the thermal stability of the ChOA was improved in another study (Nishiya et al. 1997). Recombinant ChOA production in a large amount facilitates its biochemical characterization and its use in industrial processes. To this end, in the current study, we have taken a straightforward and effective approach to maximize ChOA production by optimizing the culture and induction parameters in shaking flasks. Materials and methods Strains, materials, and culture media host strains were obtained from Novagen (Madison, WI, USA). Synthesis of plasmid pET24b-was ordered to Bio Basic Inc. (ON, Canada). Ni-CAM HC Resin, isopropyl–d-thiogalactopyranoside (IPTG), kanamycin and chloramphenicol were purchased from Sigma-Aldrich (MO, USA). All other chemicals were prepared from Merck chemical company (Darmstadt, Germany). The following liquid media were used: LuriaCBertani (LB, 10?g/L peptone, 5?g/L yeast extract, 5?g/L NaCl, Merck), Super Broth (SB, 32?g/L peptone, 20?g/L yeast extract and 5?g/L NaCl, Merck), Terrific Broth (TB, 12?g/L AMD3100 manufacturer peptone, 24?g/L yeast extract, 8?g/L glycerol, 17?mM KH2PO4 and 72?mM K2HPO4, Merck). Optimization of recombinant ChOA expression Expression of ChOA in different hosts Initially, three different strains capability for the production of recombinant ChOA were assessed under our routine laboratory conditions. At first, gene (GenBank accession number M31939) was designed into pET24b(+) expression plasmid between plasmid was transformed into chemically competent cells of host strains. We used 50?g/mL kanamycin in the solid and liquid medium of each of the three strains and additional 25? g/mL chloramphenicol in the case of and harboring pET24-plasmid was made in 3?mL of LB media. Then, 10?mL of three different medium types including LB, TB, and SB were inoculated with a pre-culture with the ratio of 1 1:100. When OD600nm reached 0.6, the cultures were induced with 0.5?mM IPTG and incubated at 37?C, 160?rpm for 6?h. The cultures were harvested and the pellet was resuspended in 0.5?mL of PBS buffer. After sonication, the cell lysate was centrifuged at 13,000cells containing pET24-were grown overnight in LB media. Fresh culture (4 flasks) containing 10?mL?TB media was inoculated (1:100) and incubated at 37?C, 160?rpm. When the OD600nm of cultures reached 0.3, 0.6, 1.2 and 1.8, induction was made with 0.5?mM IPTG. Each culture was incubated for 6?h at 37?C, 160?rpm. The harvested cells were resuspended in 0.5?mL of buffer (PBS, pH 7) and disrupted by sonication, then centrifuged at 13,000harboring gene. The induction was done at OD600nm???0.6 by adding IPTG in a final concentration of 0.25?mM. After the induction, the flasks were incubated at 15?C, 25?C, and 37?C on a rotary shaker with a speed of 160?rpm. In order to determine the optimal post-induction incubation time, 2?mL of culture media from each flask was withdrawn at different time (6, 8, 16, and 24?h) intervals. The collected samples were centrifuged and pellets were resuspended in the.