Cloning, Nucleotide Sequence, and Overexpression of the l -Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

Abstract

ABSTRACT The gene encoding l -rhamnose isomerase ( l -RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l -RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l -RhI from E. coli are conserved in that from P. stutzeri . The l -RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l -RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l -RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l -RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60°C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.