Rapid Evolution of Reversible Denaturation and Elevated Melting Temperature in a Microbial Haloalkane Dehalogenase

Abstract

Haloalkane dehalogenases have the potential for use in high‐value biocatalytic processes to convert haloalkanes into epoxides via intermediate haloalcohols. Initial bioreactor studies probing the hydrolysis of 1,2,3‐trichloropropane by immobilized wild‐type dehalogenase isolated from Rhodococcus rhodochrous 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 elevated temperatures. The accelerated laboratory evolution technique of Gene Site Saturation Mutagenesis (GSSM TM) was used to create a clonal enzyme library comprising all single site sequence variants of the Rhodococcus enzyme. Using high throughput screening techniques and rapid kinetics assays, this library was probed for improvements in thermostability and for the ability of the enzyme to undergo a fully reversible cycle of thermal denaturation‐renaturation. Eight single site mutants were discovered that had considerable effects on these aspects of the dehalogenase phenotype. Compared to the parental dehalogenase (t1/2 = 11 minutes at 55 °C) single site variants have half‐lives ranging from 300 minutes to 2700 minutes. Combinations of these mutations dramatically improved the half‐life demonstrating the enhancing effects of mutational additivity. Combining five of the mutations 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‐life by another factor of ten. Thus, the final Dhla8 protein was 30,000 times more stable than the parent molecule as measured by its ability to refold after denaturation at high temperature. Kinetic analysis showed that the improvement in thermal stability associated with Dhla5 did not negatively affect the rate of catalysis at ambient temperature, and allowed a significant increase in rate with no deactivation at 55 °C. Differential scanning calorimetry demonstrated that mutational combinations in both Dhla5 and Dhla8 led to an 8 °C increase in Tm and substantiated that partial reversibility (Dhla5) and full reversibility of Dhla8. Thermal denaturation of Dhla8 was fully reversible upon scanning up to 90 °C. Bioreactor studies showed that improved thermal stability of Dhla5 and Dhla8 correlated qualitatively with increased productivity when haloalkane hydrolysis was conducted using immobilized forms of these evolved enzymes under high temperature conditions.

Principles of Enzyme Optimization for the Rapid Creation of Industrial Biocatalysts

“…Completely arational methods that subject the entire gene to saturation mutagenesis at every site in the corresponding protein offer one way to explore the space of all single amino acid mutations, but they often require extensive molecular biology efforts to manufacture and manage the screening of hundreds of small libraries [30][31][32][33][34] or thousands of sequence-verified clones [35]. New developments for randomly accessing a wider array of mutations include random insertion and deletion mutagenesis (RID) [36] and trinucleotide exchange (TriNEx) protocols [37].…”

Directed evolution of enzymes for biocatalysis and the life sciences

“…Secondary characterization showed that residues Ala190 and Phe191 were mutational hotspots. The same technique was used to increase the thermal stability of a microbial haloalkane dehalogenase [22]. Subsequent recombination of the single site mutations improved the half-life at 55°C from 11 min for the parental enzyme to 29,000 min and the melting temperature of the final variant was increased 8°C.…”

Transformation and biodegradation of 1,2,3-trichloropropane (TCP)

“…By using error prone PCR and DNA shuffling, Bosma et al (2002) generated a DhaA mutant (e.g., a variant called DhaAM2 with the mutations C176Y and Y273F) that had three times higher catalytic efficiency ( k cat / K m   =  280 s −1  M −1 ) than wild-type enzyme. Similarly, Gray et al (2001) performed in vitro evolution studies which also yielded a mutant with a substitution at position 176 and a mutation close to the N terminus that showed higher activity with TCP as compared to wild-type, and further mutations enhanced the stability of the enzymes.…”