Engineering horseradish peroxidase with enhanced enantioselectivity

There is an ever‐growing interest in stereoselective synthesis of enantiopure chiral compounds. Natural enzymes possess many appealing properties as asymmetric catalysts for organic synthesis. They are efficient, renewable, operate under mild conditions, and generate little waste. Here we report a new experimental platform based on yeast surface display and fluorescence‐activated cell sorting (FACS) that allows us to alter the enantioselectivity of a commercially important enzyme, horseradish peroxidase (HRP). HRP is a good choice for yeast surface display as it cannot be expressed in bacteria in a soluble form due to its four disulfide bonds and multiple glycosylations. Because wild‐type HRP only shows a marginal preference for L‐tyrosinol over D‐tyrosinol, we used these two substrates conjugated to fluorescent dyes to screen several libraries of HRP for enhanced enantioselectivity. These libraries contained approximately 2 x 10 6 unique sequences and were constructed by either subjecting the HRP gene to error‐prone PCR or by replacing five amino‐acid residues at, or adjacent to, the HRP active site with any of the standard 20 amino acid residues. We specifically focused on HRP‐catalyzed radical dimerization of two chiral phenolic substrates, tyrosinol supplied in solution and L‐tyrosine found naturally on yeast’s surface. By exploiting the aforementioned method, in just two rounds of evolution we both markedly increased (up to 40‐fold) and also reversed (up to 20‐fold) the enantioselelctivity of HRP. Funded by MIT‐DuPont Alliance and NIH grant R01‐GM66712.