Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme |
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Authors: | Fraga Vidal Reinaldo Moulis Claire Escalier Pierre Remaud-Siméon Magali Monsan Pierre |
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Institution: | (1) Cuban Research Institute on Sugarcane By-Products (ICIDCA), P.O. Box 4026, CH-10, CP:11000 Havana City, Cuba;(2) Universit? de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France;(3) CNRS, UMR5504, F-31400 Toulouse, France;(4) INRA, UMR792 Ing?nierie des Syst?mes Biologiques et des Proc?d?s, 31400 Toulouse, France;(5) Laboratoire d’Ing?nierie des Syst?mes Biologiques et des Proc?d?s, INSA DGBA, UMR CNRS 5504, UMR INRA 792, 135, avenue de Rangueil, F-31077 Toulouse Cedex 04, France |
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Abstract: | The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching
was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From
amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the
variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high
number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro,
and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in
the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase
ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was
obtained. |
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