Substrate specificity of a galactose 6-phosphate isomerase from Lactococcus lactis that produces d-allose from d-psicose.
We purified recombinant galactose 6-phosphate isomerase (LacAB) from Lactococcus lactis using HiTrap Q HP and Phenyl-Sepharose columns. The purified LacAB had a final specific activity of 1.79units/mg to produce d-allose. The molecular mass of native galactose 6-phosphate isomerase was estimated at 135.5kDa using Sephacryl S-300 gel filtration, and the enzyme exists as a hetero-octamer of LacA and LacB subunits. The activity of galactose 6-phosphate isomerase was maximal at pH 7.0 and 30 degrees C, and enzyme activity was independent of metal ions. When 100g/L of d-psicose was used as the substrate, 25g/L of d-allose and 13g/L of d-altrose were simultaneously produced at pH 7.0 and 30 degrees C after 12h of incubation. The enzyme had broad specificity for various aldoses and ketoses. The interconversion of sugars with the same configuration except at the C2 position was driven by using a large amount of enzyme in extended reactions. The interconversion occurred via two isomerization reactions, i.e., the interconversion of d-allose<-->d-psicose<-->d-altrose, and d-allose to d-psicose reaction was faster than d-altrose to d-psicose reaction.
Park
Ha-Young
HY
Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
Park
Chang-Su
CS
Kim
Hye-Jung
HJ
Oh
Deok-Kun
DK
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001709:Biotechnology; D002412:Cations; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005632:Fructose / Q000378:metabolism*; D005798:Genes, Bacterial; D005947:Glucose / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007661:Ketoses / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008956:Models, Chemical; D008970:Molecular Weight; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D017931:DNA Primers; D004269:DNA, Bacterial; D007661:Ketoses; C003243:psicose; D005632:Fructose; C002055:allose; D019747:Aldose-Ketose Isomerases; C023084:galactose-6-phosphate isomerase; D005947:Glucose