Reasoning Enantioselectivity and Kinetics of Seleno-Subtilisin from the Subtilisin Template

Novel Biocatalysts by Chemical Modification of Known Enzymes: Cross-Linked Microcrystals of the Semisynthetic Peroxidase Seleno-Subtilisin

“…The ee of 1 was determined by HPLC on a Daicel-Chiralcel OB-H. Contrary to previous studies [8,9] the blank reaction was negligible using CLC-seleno-subtilisin. Kinetic resolution of racemic hydroperoxide 1 or 2 was carried out accordingly in preparative 1-mmol scale yielding typically 42 % of the enantiomerically enriched hydroperoxides (for stereoanalytics and enantioselectivity cf.…”

“…As a result of the introduction of selenium into the protease subtilisin, the semisynthetic seleno-subtilisin revealed glutathione peroxidase activity, [7] and catalyzed the kinetic resolution of several racemic hydroperoxides [8,9] (Scheme 2).…”

“…Substrate-and stereoselectivity are comparable due to identical binding sites. [9,14] However, synthetic application of semisynthetic enzymes has to date been limited to 10-mmol scale and the chemically modified enzyme was not reusable. In contrast, CLC-selenosubtilisin is insoluble in water and organic solvents and easily recovered after synthesis by centrifugation or filtration.…”

“…Kinetic resolution of racemic hydroperoxide 1 or 2 was carried out accordingly in preparative 1-mmol scale yielding typically 42 % of the enantiomerically enriched hydroperoxides (for stereoanalytics and enantioselectivity cf. [8,9] Ralf Giernoth, Patrick Huebler, and Joachim Bargon* PHIP-NMR spectroscopy (PHIP parahydrogen induced polarization) is an analytical method that provides profound insight into the mechanisms of homogeneously catalyzed hydrogenations, presupposed that the two parahydrogen nuclei remain J-coupled in the product molecule.…”

“…By utilizing a well-known protein framework, details about substrate selectivity or affinity may be transferred and predicted from the template to the semisynthetic enzyme. [9] Experimental Section Subtilisin Carlsberg (Novo Nordisk, 54 % (w/w) active protein) was prepurified by fractionated acetone precipitation and crystallized in 12 % Na 2 SO 4 in 30 mm acetate/NaOH buffer (pH 5.5; 16 8C). [11] Treatment of subtilisin crystals with 1.5 % glutardialdhyde (45 min) in 30 mm KH 2 PO 4 / NaOH (pH 7.5) containing 13 % Na 2 SO 4 resulted in cross-linked crystals (CLC) of subtilisin (25 % overall yield at gram-scale).…”