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

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.Unusual carbohydrates, simply termed "rare sugars," are referred to as sugars that exist in an extremely scanty amount in nature; they include,tagatose, allitol, and others. Rare sugars have been proven to be of paramount significance not only in food industries but also in pharmaceutical and nutritional industries on account of their multipurpose applications, such as their use as reducedcalorie sweeteners, inhibitors of microbial growth, bulking agents, and memory enhancers (4,(22)(23)(24)(25). Yet the unavailability and scarcity of these uncommon sugars result in their high cost and limitations in usage as starting materials or research reagents. Consequently, there has been a dearth of literature reporting on the mass production of rare sugars. In recent years, however, microbial and enzymatic reactions have become increasingly significant and have played pivotal roles in the production of unusual sugars.We are mainly working on the production and/or mass production of various kinds of rare sugars by using microorganisms screened from a variety of ecological habitats and their enzymes. For example, Pseudomonas cichorii ST-24 and Acinetobacter sp. strain DL-28, which produce D-tagatose 3-epimerase and L-ribose isomerase, respectively, were isolated from soil and utilized for the preparation of a number of uncommon sugars (1,(16)(17)(18)(19)32). In order to improve the productivity of the sugars, the enzymes were later genetically engineered (14,15,26). Furthermore, members of our laboratory recently isolated a mutant s...

Enzymes for the biocatalytic production of rare sugars

“…Two isomerases that prefer deoxy-sugars as their substrates are L-fucose isomerase [21,47] and L-rhamnose isomerase [57,58]. These enzymes also tolerate other modifications at centers higher than C3 and can use epimeric or functionalized sugars as substrates.…”

The Structures of l-Rhamnose Isomerase from Pseudomonas stutzeri in Complexes with l-Rhamnose and d-Allose Provide Insights into Broad Substrate Specificity

“…5 L-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeri L-RhI; Swiss Prot accession number Q75WH8, 430 amino acid residues, 46,975 Da) 6 shows a broader substrate specificity than L-RhI from Escherichia coli (E. coli L-RhI; Swiss Prot accession number P32170, 419 amino acid residues, 47,199 Da) 2 , catalyzing the isomerization between various aldoses and ketoses, as well as between L-rhamnose and L-rhamnulose, in the presence of appropriate metal ions. [5][6][7] Leang et al reported that a recombinant His-tagged P. stutzeri L-RhI efficiently catalyzed the isomerization between L-rhamnose and L-rhamnulose, L-mannose and L-fructose, L-lyxose and L-xylulose, D-ribose and D-ribulose, and D-allose and D-psicose, as listed in Figure 1(a) with the enzyme activities. Since some of them are so-called "rare sugars" which exist in small amounts in nature, P. stutzeri L-RhI is exploited for industrial applications in rare sugar production.…”

Characterization of a recombinant thermostable l-rhamnose isomerase from Thermotoga maritima ATCC 43589 and its application in the production of l-lyxose and l-mannose

“…The optimum temperatures and half-lives of RhaAs from the mesophiles E. coli, P. stutzeri, and B. pallidus are 60, 60, and 65°C, respectively, and as 10 min at 50°C, 10 min at 60°C, and 60 min at 60°C, respectively (Badia et al 1991;Leang et al 2004c;Poonperm et al 2007b). However, RhaA from the hyperthermophile T. maritima examined in this study has an optimum of 85°C and a half-life of 773 h at 75°C, which is 773 times higher than that from B. pallidus RhaA at 60°C.…”