Biochemical studies on LevJ, a fructanase from Actinomyces naeslundii T14V.
The Actinomyces naeslundii T14V gene levJ encodes a sucrase with fructanase activity and may be responsible for the fructanase activity observed bound to the surface of A. naeslundii T14V cells. A large proportion of LevJ expressed in Escherichia coli was translocated to the periplasm, and translocation and enzymatic activity were not affected by deletion of a putative cell-wall anchor sequence. The pH optimum of the enzyme was found to be between 5.5 and 6.5 whether the substrate was sucrose or inulin, although inulinase activity was more sensitive than sucrose activity to perturbation of the pH from the optimum. The relation between LevJ inulinase activity and pH was similar to that of A. naeslundii whole cells. LevJ exhibited standard saturation kinetics with sucrose, and the K(m) was calculated to be 89 mM, but it was not possible to calculate a K(m) for inulin. Evidence for inhibition of inulinase activity but not sucrase activity by high concentrations of inulin was obtained.
Norman
J M
JM
Centre for Molecular Biotechnology, School of Life Science, Queensland University of Technology, Australia.
Giffard
P M
PM
D000190:Actinomyces / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001669:Biochemical Phenomena; D001671:Biochemistry; D002473:Cell Wall / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000378:metabolism; D013393:Sucrase / Q000737:chemistry / Q000235:genetics; D013395:Sucrose / Q000378:metabolism; D014178:Translocation, Genetic
D001426:Bacterial Proteins; D013395:Sucrose; D007444:Inulin; D006026:Glycoside Hydrolases; D013393:Sucrase; C021512:levanase; C023086:inulinase