Development and Characterization of Biotransformation System toward Helogenated Compounds with Yeast

(1)氯化乙烷和乙烯是一类特别有问题的污染物，人们越来越多地寻求生物处理方法。一株表达大鼠肝细胞色素P450 1A2的重组酿酒酵母氧化三氯乙烯和还原五氯乙烷和六氯乙烷。使用三氯乙烯和五氯乙烷，每单位酶的无细胞膜组分的初始速率比使用完整酵母获得的初始速率高一个数量级。六氯乙烷被膜和酵母以相当的速率还原。三氯乙烯被氧化为2,2,2-三氯乙醛和2,2,2-三氯乙醇。完整的酵母按比例产生更多的三氯乙醇，动力学研究表明这是由三氯乙醛产生的。五氯乙烷经生产脱卤制得三氯乙烯，其中一部分被氧化为三氯乙醛和三氯乙醇。六氯乙烷经过还原脱卤，得到四氯乙烷和五氯乙烷的混合物。(2)我们检测了一株表达大鼠肝细胞色素P450 1A2 （P450 1A2）突变蛋白的重组酵母是否能有效催化三氯乙烯、五氯乙烷和六氯乙烷的脱卤反应。研究发现，P450 1A2远端位点的突变极大地增加了在好氧或厌氧条件下对这些底物的周转数量。例如，假设的远端位点Thr319Ala突变显著提高了三氯乙烯和五氯乙烷的降解率，分别提高了2倍和2.7倍。分别在有氧条件下。Thr319Ala突变也强烈促进了对六氯乙烷的反应，在好氧和厌氧条件下分别提高了13倍和4.5倍。并结合反应机理讨论了Thr319在这些氯代烃催化作用中的作用。少

(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