Polymerase and hydrolase activities of Zymomonas mobilis levansucrase separately modulated by in vitro mutagenesis and elevated temperature

A 1.7 kb gene encoding levansucrase of Zymomonas mobilis was cloned in Esch erichia coli. The gene product is able to hydrolyze sucrose and to synthesi ze levan. The production of levansucrase in recombinant E. coil strain was maximum when it was grown in M63 medium with glycerol (MY) rather than in L B medium. Maximum sucrase activity of wild type and mutant was observed at 50 and 40 degreesC, respectively, at pH 5. The levansucrase mutant was isol ated by in vitro mutagenesis using hydroxylamine. By mutation, the levan-fo rming activity was abolished but the sucrase activity of the wild type stra in was retained. The sucrase activity of the mutant was confirmed by native gel electrophoresis and zymogram analysis. This uncoupling of the sucrase activity from the levan-forming activity suggests a change in the protein c onformation due to mutation and elevated temperature. Thus, it is proposed that the active site for the sucrase and levan-forming activities could be different. (C) 2001 Elsevier Science Ltd. All rights reserved.