ISOLATION OF AN L-RHAMNOSE ISOMERASE-CONSTITUTIVE MUTANT OF PSEUDOMONAS SP. STRAIN LL172 - PURIFICATION AND CHARACTERIZATION OF THE ENZYME

A soil bacterium, strain 172a, which inductively produced L-rhamnose i somerase (L-rhamnose ketolisomerase, EC 5.3.1.14) and could not grow o n L-lyxose acquired the ability to grow on L-lyxose following cultivat ion in a mineral salts medium containing L-lyxose as the sole carbon s ource for about 4 d. This L-lgxose-utilizing mutant (LL172) was isolat ed and found to produce L-rhamnose isomerase constitutively. Based on various bacteriological characteristics, the parent strain was identif ied as Pseudomonas sp. and the mutant was confirmed to be derived from the parent strain. L-Rhamnose isomerase was purified from extracts of Pseudomonas sp. LL172 by polyethylene glycol precipitation, anion exc hange chromatography on DEAE-Toyopearl 650M and gel filtration on Seph adex G-150. The enzyme was found to be homogeneous by polyacrylamide g el electrophoresis. The apparent molecular weight of the enzyme was es timated to be 150,000 by gel filtration on Sephadex G-150. Based on so dium dodecyl sulfate (SDS) gel electrophoresis, the enzyme is most lik ely composed of 4 identical subunits of molecular weight of approximat ely 42,000. The enzyme was optimally active at pH 9.0 and was stable i n the pH range of 5.0-11.0. The optimum temperature for activity was 6 0 degrees C (10 min, pH 9.0) and the enzyme was stable up to 60 degree s C (10 min, pH 9.0). The isoelectric point of the enzyme was estimate d to be 5.1. The substrate specificity of the enzyme was broad and the enzyme required manganese ions for maximum activity. The K-m for L-rh amnose isomerase was 55 mM and the V-max was 182.6 U/mg. The equilibri um ratio between L-rhamnose and L-rhamnulose was 55:45.