Rapid evolution of reversible denaturation and elevated melting temperature in a microbial haloalkane dehalogenase

Haloalkane dehalogenases have the potential for use in high-value biocataly tic processes to convert haloalkanes into epoxides via intermediate haloalc ohols. Initial bioreactor studies probing the hydrolysis of 1,2,3-trichloro propane by immobilized wild-type dehalogenase isolated from Rhodococcus rho dochrous demonstrated, however, that productivity was too low to realize a commercially viable process. A strategy to increase enzyme performance was undertaken to increase the reaction temperature, however it was determined that the wild-type enzyme was not stable for long periods of time at elevat ed temperatures. The accelerated laboratory evolution technique of Gene Sit e Saturation Mutagenesis (GSSM((TM))) was used to create a clonal enzyme li brary comprising all single site sequence variants of the Rhodococcus enzym e. Using high throughput screening techniques and rapid kinetics assays, th is library was probed for improvements in thermostability and for the abili ty of the enzyme to undergo a fully reversible cycle of thermal denaturatio n-renaturation. Eight single site mutants were discovered that had consider able effects on these aspects of the dehalogenase phenotype. Compared to th e parental dehalogenase (t(1/2) = It minutes at 55 degreesC) single site va riants have half-lives ranging from 300 minutes to 2700 minutes. Combinatio ns of these mutations dramatically improved the half-life demonstrating the enhancing effects of mutational additivity. Combining five of the mutation s into a single protein (Dhla5) improved the half-life to 29,000 min and a combination of all eight single-site mutations (Dhla8) increased the half-l ife by another factor of ten. Thus, the final Was protein was 30,000 times more stable than the parent molecule as measured by its ability to refold a fter denaturation at high temperature. Kinetic analysis showed that the imp rovement in thermal stability associated with Dhla5 did not negatively affe ct the rate of catalysis at ambient temperature, and allowed a significant increase in rate with no deactivation at 55 degreesC. Differential scanning calorimetry demonstrated that mutational combinations in both Dhla5 and Dh la8 led to an 8 degreesC increase in T-m and substantiated that partial rev ersibility (Dhla5) and full reversibility of Dhla8. Thermal denaturation of Was was fully reversible upon scanning up to 90 degreesC. Bioreactor studi es showed that improved thermal stability of Dhla5 and Was correlated quali tatively with increased productivity when haloalkane hydrolysis was conduct ed using immobilized forms of these evolved enzymes under high temperature conditions.