Pentachlorophenol Hydroxylase: Analysis of Catalytic Abilities and Evolution of a Better Enzyme

摘要

Sphingobium chlorophenolicum is a Gram-negative soil bacterium that can mineralize pentachlorophenol (PCP), although degradation is slow and the bacterium cannot tolerate high levels of PCP. The rate of degradation is limited by the first enzyme in the pathway, PCP hydroxylase, which is a very poor catalyst. We have used DNA shuffling to produce mutant enzymes with modestly improved catalytic activities. We discovered that the reason for our limited success was that our selection was based upon publications claiming that the product formed by PCP hydroxylase is tetrachlorohydroquinone (TCHQ). Since TCHQ is less toxic than PCP, cells expressing better enzymes should be able to grow in the presence of higher levels of PCP. However, we found that the product formed from PCP is actually tetrachlorobenzoquinone (TCBQ), which is more toxic than PCP. We also discovered a previously unrecognized enzyme that converts TCBQ to TCHQ. Our new knowledge about the pathway will allow us to design a more effective selection for improved PCP hydroxylase enzymes. Finally, we have used genome shuffling to generate strains of S. chlorophenolicum that have greatly increased tolerance to the toxicity of PCP and have significantly improved rates of degradation.