Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite.

OBJECTIVES: To find a simple enzymatic strategy for the efficient synthesis of the expensive 5'-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate. RESULTS: The practical synthetic strategy for the 5'-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5'-OH omeprazole sulfide with a high conversion yield (85-90 %). CONCLUSIONS: A highly efficient synthesis of 5'-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli.

The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifactors) such as 2(4-1) fractional factorial, 2(3) full factorial, and 2(2) rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO4, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges characterized by %T-20 (proportion of peptide to the total cell protein) were observed, narrowed down, and further investigated to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources, MgSO4, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55-0.75 with 0.7 mM isopropyl-beta-D-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical optimization allowed the production of T-20 to be increased more than twofold (from 6 to 14%) within a shorter induction duration (from 6 to 4 h) at the shake-flask scale.

Statistical medium formulation and process modeling by mixture design of experiment for peptide overexpression in recombinant Escherichia coli.

The medium formulation and robust process modeling for anti-HIV peptide (T-20) production by recombinant Escherichia coli overexpression were studied by employing a crossed experimental design. The crossed design, a mixture design combined with process factor (induction duration), was used to find the optimal medium formulation and process time. The optimal settings for three major components (7.75 mL of NPK sources, 5.5 mL of glucose, and 11.75 mL of MgSO4) characterized by %T-20 (14.45%), the proportion of peptide to the total protein, were observed in a total of 100 mL of medium inducted at an optical density of 0.67 with 0.7 mM isopropyl-beta-D-thiogalactopyranoside) for a 3-h induction duration at shake-flask scale. These conditions were further investigated to find robust process conditions (8.2 mL of NPK sources, 5.6 mL of glucose, and 11.3 mL of MgSO4, and a 3.5-h induction duration time) for T-20 production (13.9%) by applying propagation of error.