Isolation of an L-Rhamnose Isomerase-Constitutive Mutant of Pseudomonas sp. Strain LL172 : Purification and Characterization of the Enzyme

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A soli 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 stalts medium containing L-lyxose as the sole carbon for about 4 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 apprent molecular weight of the enzyme was estimated to be 150,000 by gel filtration on Sephadex G-150. Based on sodium dedecyl 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℃ (10 min, pH 9.0) and the enzyme was stable up to 60℃ (10 min, pH9.0). The isoelectric point of the enzyme was estimated to be 5.1. The substraye 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.
社団法人日本生物工学会の論文
1997-10-25

Isolation of an L-Rhamnose Isomerase-Constitutive Mutant of Pseudomonas sp. Strain LL172 : Purification and Characterization of the Enzyme

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