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SECTION II. APPLIED BIOTECHNOLOGY IN THE PHARMACEUTICAL INDUSTRY... 331The organic layer was collected and concentrated using a rotary evaporator. The residue was dissolved in 2 mL of methanol for production analysis [5]. 2.5. Production analysis by HPLC and LC-MS The crude pradimicin A extracts of wild-type and mutant strains was analyzed by HPCL and LC-MS. For HPLC analysis of pradimicin A, the Agilent HPLC 1260II system equipped with a reverse-phase C18 column (Agilent, 5 %u03bcm, 250 %u00d7 4.6 mm) was used for analysis. A binary gradient was performed, consisting of solvent A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile), with a flow rate set at 1 mL/min.LC/MS analysis was performed using the ACQUITY UPLC/SYNAPT G2-S system (Waters Corporation). The liquid chromatography utilized a binary-linear gradient with solvent A (water containing 0.1% TFA) and solvent B (acetonitrile). The gradient increased solvent B from 0% to 100% over 12 minutes, at a flow rate of 0.3 mL/min. High-resolution electrospray ionization mass spectrometry (HRESIMS) was performed under conditions of 3 kV voltage, a gas flow rate of 600 L/h, and a gas temperature of 300 %u00b0C.3. RESULTS AND DISCUSSION3.1. Ribosome engineering of A. hibisca P172-2To enhance the production of pradimicin A in A. hibisca P172-2, Ribosome engineering of A. hibisca P172-2 was done by plating spores of wild-type strain on MS plates with streptomycin concentrations of 200 %u03bcg/mL. After 7 days incubated at 28%u00b0C, the mutant strains were selected. The colonies with more dark-red color were achieved. In the results, 20 colonies were collected. The survival rate is 0,1%. All these strains were incubated in production media to determine the pradimicin production.