Molecular cloning and expression in Escherichia coli of an exo-levanase gene from the endophytic bacterium Gluconacetobacter diazotrophicus SRT4.
Gluconacetobacter diazotrophicus produces levan from sucrose by a secreted levansucrase (LsdA). A levanase-encoding gene ( lsdB), starting 51 bp downstream of the lsdA gene, was cloned from strain SRT4. The lsdB gene (1605 bp) encodes a protein (calculated molecular mass 58.4 kDa) containing a putative 36-amino-acid signal peptide at the N-terminus. The deduced amino acid sequence shares 34%, 33%, 32%, and 29% identities with levanases from Actinomyces naeslundii, Bacillus subtilis, Paenibacillus polymyxa, and Bacteroides fragilis, respectively. The lsdB expression in Escherichia coli under the control of the T7 RNA polymerase promoter resulted in an active enzyme which hydrolyzed levan, inulin, 1-kestose, raffinose, and sucrose, but not melezitose. Levanase activity was maximal at pH 6.0 and 30 degrees C, and it was not inhibited by the metal ion chelator EDTA or the denaturing agents dithiothreitol and beta-mercaptoethanol. The recombinant LsdB showed a fourfold higher rate of hydrolysis on levan compared to inulin, and the reaction on both substrates resulted in the successive liberation of the terminal fructosyl residues without formation of intermediate oligofructans, indicating a non-specific exo-levanase activity.
Menéndez
Carmen
C
Centre for Genetic Engineering and Biotechnology, Plant Division, Plant-Microbe Interactions Laboratory, P.O. Box 6162, Havana 10600, Cuba.
Hernández
Lázaro
L
Selman
Guillermo
G
Mendoza
Milady F
MF
Hevia
Pedro
P
Sotolongo
Mailin
M
Arrieta
Juan G
JG
D016947:Acetobacteraceae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D043324:beta-Fructofuranosidase
D001426:Bacterial Proteins; D007477:Ions; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; C021512:levanase