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Rapid Evolution of Reversible Denaturation and Elevated Melting Temperature in a Microbial Haloalkane Dehalogenase
Author(s) -
Gray Kevin A.,
Richardson Toby H.,
Kretz Keith,
Short Jay M.,
Bartnek Flash,
Knowles Ryan,
Kan Lynn,
Swanson Paul E.,
Robertson Dan E.
Publication year - 2001
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/1615-4169(200108)343:6/7<607::aid-adsc607>3.0.co;2-m
Subject(s) - thermostability , chemistry , saturated mutagenesis , dehalogenase , active site , directed evolution , biochemistry , enzyme , enzyme kinetics , rhodococcus rhodochrous , denaturation (fissile materials) , mutagenesis , mutant , systematic evolution of ligands by exponential enrichment , protein engineering , rhodococcus , gene , rna , nuclear chemistry
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 (t 1/2 = 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‐lifeby 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 T m 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 hightemperature conditions.