Development and Characterization of Biotransformation System toward Helogenated Compounds with Yeast

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(1) Chlorinated ethanes and ethenes are a particularly problematic class of pollutants for which biological treatment methods are increasingly being sought.A recombinant Saccharomyces cerevisiae strain expressing rat liver cytochrome P450 1A2 oxidized tirchloroethylene and reduced pentachloroethane and hexachloroethane. With trichloroethylene and pentachloroethane, the initial rates of cell-free membrane fractions were an order of magnitude higher per unit enzyme than those obtained with intact yeast.Hexachloroethane was reduced at comparable rates by both membranes and yeast. Trichloroethylene was oxidized to 2,2,2-trichloroacetaldehyde and 2,2,2-trichloroethanol. Intact yeast produced proportionally more trichloroethanol which kinetic studies indicated arose from trichloroacetaldehyde. Pentachloroethane underwent ruductive dehalogenation to yield trichloroethylene, part of which was oxidized to trichloroacetaldehyde and trichloroethanol. Hexachloroethane underwent reductive dehalogen … More ation to yield a mixture of tetrachloroethylene and pentachloroethane.(2) We examined whether or not a recombinant Saccharomyces cerevisiae strain expressing rat liver cytochrome P450 1A2 (P450 1A2) mutant proteins efficiently catalyze dehalogenation of trichloroethylene, pentachloroethane, and hexachloroethane. It was found that mutations at a putative distal site of P450 1A2 enormously enhanced turnover numbers toward those substrates both under the aerobic or anaerobic conditions. For example, a Thr319Ala mutation at the putative distal site markedly enhanced the degradation rate of trichloroethylene and pentachloroethane by 2-and 2.7-fold.respectively, under the aerobic conditions. The Thr319Ala mutation also strongly facilitated the reaction toward hexachloroethane up to 13-and 4.5-fold both under the aerobic and anaerobic conditions, respectively. Role of Thr319 in the cataklytic functions toward those dhlorinated hydrocarbons are discussed in association with reaction meachanism. Less