英文文献汇报培训资料.pptVIP

细胞色素P450酶系 孙婉菊 2014年12月17日 Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2 1.CYP106A2 from Bacillus megaterium ATCC 13368, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. 2.We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. 3.An enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis。 文献汇报 Cloning pBAR Twin: bovine AdR、Adx pACYC FHH28: CYP106A2 pBAR Tripel: bovine AdR、AdX、LbADH Selectivity of CYP106A2 With growing cells as a biocatalyst, a limited selectivity of both Prog and Tes hydroxylation was observed. No side product formation could be observed when resting cells were used as biocatalysts. Comparison of growing and resting cells for A) progesterone and B) testosterone conversion Substrate solubility and transport into the cell Highest activity was observed in presence of even 2m (15.4 vol%) propan-2-ol. During the second reaction cycle only 21% of Prog could be converted in the presence of propan-2-ol. Without this cosolvent 53% 15b-Prog was formed; This indicates the limited applicability of propan-2-ol as a cosolvent in this system. Influence of propan-2-ol on the activity (A) and stability (B) of the whole-cell biocatalyst 21% 53% Substrate solubility and transport into the cell biotransformation with“membrane-free” crude cell extract (CCE) and the whole-cell Fig. Conversion of progesterone by using crude cell extract of E. coli expressing CYP106A2, AdR, Adx and LbADH as biocatalyst The initial reaction rate increased : 0.4 U15?-Prog gwcw-1 to 9.6 U15?-Prog gwcw-1 glyophilisate-1 in cases of CCE. A loss in selectivity compared to resting cells. 20% side products are formed. Cofactor demand When propan-2-ol was added, the amount of NADPH drastically increased