L-Arabinose isomerase and D-xylose isomerase from Lactobacillus reuteri: characterization, coexpression in the food grade host Lactobacillus plantarum, and application in the conversion of D-galactose and D-glucose.
The L-arabinose isomerase (L-AI) and the D-xylose isomerase (D-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. L-AI displayed maximum activity at 65 °C and pH 6.0, whereas D-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the L-AI- and D-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum . The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified L-AI converted D-galactose to D-tagatose with a maximum conversion rate of 35%, and the D-XI isomerized D-glucose to D-fructose with a maximum conversion rate of 48% at 60 °C.
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Science Vienna , Muthgasse 18, 1190 Vienna, Austria.
Petra
P
Staudigl
Dietmar
D
Haltrich
Clemens K
CK
Peterbauer
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005690:Galactose / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D048191:Lactiplantibacillus plantarum / Q000235:genetics* / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C023298:xylose isomerase; D005947:Glucose; D005690:Galactose