Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough.
Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K m of 8.6 mM and a V max of 20 μmol/mg/min. According to (1)H and (13)C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.
Université de Toulouse, Laboratoire de Biotechnologies Agroalimentaire et Environnementale, EA 4565, Institut Universitaire de Technologie, Université Paul Sabatier, 24, Rue d'Embaquès, 32000 Auch, France.
Myriam
M
Amari
Luisa Fernanda Gomez
LF
Arango
Valérie
V
Gabriel
Hervé
H
Robert
Sandrine
S
Morel
Claire
C
Moulis
Bruno
B
Gabriel
Magali
M
Remaud-Siméon
Catherine
C
Fontagné-Faucher
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005516:Food Microbiology; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D058836:Weissella / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D003911:Dextrans; D011994:Recombinant Proteins; D013395:Sucrose; D005964:Glucosyltransferases; C020025:dextransucrase