The SRT4 gene encoding levansucrase (EC () was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the gene from the promoter in resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria and respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the levansucrase with those of the levansucrase suggested that one of these motifs may be involved in the specificity of the synthesized product. Disruption of the gene in the genome of resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of


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