Saturation Mutagenesis of Toluene ortho -Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

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Abstract
Directed evolution of toluene ortho -monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells ( V max of 9.3 versus 4.5 nmol · min −1 · mg of protein −1 and unchanged K m ), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol · min −1 · mg of protein −1 [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o -cresol (50%), m -cresol (25%), and p -cresol (25%) are formed, in contrast to the 98% o -cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography ( V max of 2.61 versus 0.95 nmol · min −1 · mg of protein −1 and unchanged K m ) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol · min −1 · mg of protein −1 ). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol · min −1 · mg of protein −1 ). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o -cresol (28%), m -cresol (18%), and p -cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.