Bridging the Bridge − Stabilization of CalB against H2O2 and its Application in Epoxidation Reactions

M. Steinhagen, A. Gräbner, J. Meyer, A. E. W. Horst, A. Drews, D. Holtmann, M. B. Ansorge-Schumacher,

Chemo-enzymatic reactions combine the advantages of chemistry and biocatalysis. Chemical synthesis provides precursor molecules and low costs, whereas the application of enzymes provides selectivity. Additionally, the mild reaction conditions of enzymatic approaches allow the chemical application of intermediates and/or products that otherwise are not accessible. One example is the in situ production of peroxycarboxylic acids (peracids) by Candida antarctica lipase B (CalB). In contrast to the harsh conditions of chemical peracid synthesis, CalB catalyzed reactions run at low temperatures and without additives. Unfortunately, the enzyme is rapidly inactivated by the oxidative environment. Herein, we report on CalB stabilization by preventing disulfide cleavage after H2O2 exposure. Therefore, a bismaleimide functionalized linker was used to convert all the enzyme’s disulfide bridges to more stable thioether linkages. These bonds are still affected by hydrogen peroxide but will not open upon oxidation. A two- to fourfold excess of this linker was optimal to avoid enzyme oligomerization. At the same time, a 1.5 fold increase in half life time after exposure to hydrogen peroxide was achieved. To our knowledge, such an approach to intramolecular disulfide stabilization has never been reported before, but might be a general strategy for enzyme engineering. Furthermore, a carrier screening was performed, identifying different optimal carrier types for CalB immobilization. The combination of stabilization by disulfide conjugation and immobilization was expanded by the adoption of a thermostabilized double mutant. Finally, chemo-enzymatic epoxidations of alkenes were examined in batch experiments and under continuous process conditions. Activity loss was reduced by 50% and a 75% increase in long-term stability was achieved in comparison to the commercial preparation.

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