Isolation of an l-rhamnose isomerase-constitutive mutant of Pseudomonas sp. strain LL172: Purification and characterization of the enzyme

Abstract

A soil bacterium, strain 172a, which inductively produced l-rhamnose isomerase ( l-rhamnose ketolisomerase, EC 5.3.1.14) and could not grow on l-lyxose acquired the ability to grow on l-lyxose following cultivation in a mineral salts medium containing l-lyxose as the sole carbon source for about 4 d. This l-lyxose-utilizing mutant (LL172) was isolated and found to produce l-rhamnose isomerase constitutively. Based on various bacteriological characteristics, the parent strain was identified 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 exchange chromatography on DEAE-Toyopearl 650M and gel filtration on Sephadex G-150. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The apparent molecular weight of the enzyme was estimated to be 150,000 by gel filtration on Sephadex G-150. Based on sodium dodecyl sulfate (SDS) gel electrophoresis, the enzyme is most likely composed of 4 identical subunits of molecular weight of approximately 42,000. The enzyme was optimally active at pH 9.0 and was stable in the pH range of 5.0–11.0. The optimum temperature for activity was 60°C (10 min, pH 9.0) and the enzyme was stable up to 60°C (10 min, pH 9.0). The isoelectric point of the enzyme was estimated 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-rhamnose isomerase was 55 mM and the V max was 182.6 U/mg. The equilibrium ratio between l-rhamnose and l-rhamnulose was 55:45.