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Titolo:
Rapid evolution of reversible denaturation and elevated melting temperature in a microbial haloalkane dehalogenase
Autore:
Gray, KA; Richardson, TH; Kretz, K; Short, JM; Bartnek, F; Knowles, R; Kan, L; Swanson, PE; Robertson, DE;
Indirizzi:
Diversa Corp, San Diego, CA 92121 USA Diversa Corp San Diego CA USA 92121Diversa Corp, San Diego, CA 92121 USA Dow Chem Co USA, Biotechnol Capabil, San Diego, CA 92121 USA Dow Chem Co USA San Diego CA USA 92121 l Capabil, San Diego, CA 92121 USA
Titolo Testata:
ADVANCED SYNTHESIS & CATALYSIS
fascicolo: 6-7, volume: 343, anno: 2001,
pagine: 607 - 617
SICI:
1615-4150(200108)343:6-7<607:REORDA>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
ALPHA-HELIX STABILITY; ACID SIDE-CHAINS; X-RAY STRUCTURE; CRYSTAL-STRUCTURE; DIRECTED EVOLUTION; HALIDE-BINDING; ENZYME; PEPTIDES; SPECIFICITY; MUTATIONS;
Keywords:
calorimetry; dehalogenation; enzyme catalysis; halogens; molecular evolution;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
45
Recensione:
Indirizzi per estratti:
Indirizzo: Gray, KA Diversa Corp, 4955 Directors Pl, San Diego, CA 92121 USA Diversa Corp 4955 Directors Pl San Diego CA USA 92121 A 92121 USA
Citazione:
K.A. Gray et al., "Rapid evolution of reversible denaturation and elevated melting temperature in a microbial haloalkane dehalogenase", ADV SYNTH C, 343(6-7), 2001, pp. 607-617

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 acommercially viable process. A strategy to increase enzyme performance wasundertaken to increase the reaction temperature, however it was determinedthat 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) = It minutes at 55 degreesC) 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 acombination of all eight single-site mutations (Dhla8) increased the half-life by another factor of ten. Thus, the final Was protein was 30,000 timesmore 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 significantincrease in rate with no deactivation at 55 degreesC. Differential scanning calorimetry demonstrated that mutational combinations in both Dhla5 and Dhla8 led to an 8 degreesC increase in T-m and substantiated that partial reversibility (Dhla5) and full reversibility of Dhla8. Thermal denaturation of Was was fully reversible upon scanning up to 90 degreesC. Bioreactor studies showed that improved thermal stability of Dhla5 and Was correlated qualitatively with increased productivity when haloalkane hydrolysis was conducted using immobilized forms of these evolved enzymes under high temperatureconditions.

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Documento generato il 15/07/20 alle ore 14:11:48