A MODIFIED ORTHO-CLEAVAGE PATHWAY IN PSEUDOMONAS-PUTIDA STRAIN-87 - PURIFICATION AND PROPERTIES OF DIENELACTONE HYDROLASE

Dienelactone hydrolase (DLH)(3) was purified to electrophoretic homoge neity from Pseudomonas putida strain 87 grown on 3-chlorobenzoate. The specific activity of the purified enzyme was 50.5 U/mg protein. The e nzyme has monomeric structure, 22 kD molecular mass, pH optimum 7.6, a nd is active within the temperature range from 20 to 45 degrees C. The enzyme activity is inhibited by p-chloromercuribenzoate (p-CMB), but not by EDTA; complete inhibition of the enzyme activity was achieved w ith two equivalents of p-CMB. The V-max values of DLH for trans-dienel actone and 2-chlorodienelactone were nearly identical (247.9 and 247 U /mg protein, respectively) and were twice higher than that for cis-die nelactone. However, the K-m value for 2-chlorodienelactone was twofold higher than that for trans-dienelactone (6.9 and 14.2 mu M, respectiv ely). Comparison of physicochemical and kinetic properties of the stud ied DLH and formerly characterized DLHs involved in degradation of chl orinated and fluorinated compounds in other microorganisms placed the DLH from P. putida 87 in the third group of enzymes, i.e., to the grou p from gram-negative bacteria with modified ortho-cleavage pathway die nelactone hydrolases.