Functional expression of horseradish peroxidase in E-coli by directed evolution

In an effort to develop a bacterial expression system for horseradish perox idase (HRP), we inserted the gene encoding HRP into the pET-22b(+) vector ( Novagen) as a fusion to the signal peptide PelB. A similar construct for cy tochrome c peroxidase (CcP) leads to high CcP activity in the supernatant. Expression of the wild-type HRP gene in the presence of isopropyl-beta-D-th iogalactopyranoside (IPTG) yielded no detectable activity against ABTS (azi nobis(ethylbenzthiazoline sulfonate)). However, weak peroxidase activity wa s detected in the supernatant in the absence of IPTG. The HRP gene was subj ected to directed evolution: random mutagenesis and gene recombination foll owed by screening in a 96-well microplate format. From 12 000 clones screen ed in the first generation, one was found that showed Iii-fold higher HRP a ctivity than wild-type, amounting to similar to 110 mu g of HRP/L, which is similar to that reported from laborious in vitro refolding. No further imp rovement was obtained in subsequent generations of directed evolution. This level of expression has nonetheless enabled us to carry out further direct ed evolution to render the enzyme more thermostable and more resistant towa rd inactivation by H2O2. These results show that directed evolution can ide ntify mutations that assist proteins to fold more efficiently in Escherichi a coli. This approach will greatly facilitate efforts to "fine-tune" those many enzymes that are promising industrial biocatalysts, but for which suit able bacterial or yeast expression systems are currently lacking.