title: Molecular cloning and characterization of active truncated dextransucrase from Leuconostoc mesenteroides B-1299CB4.
abstract: The open reading frame of dsrE563, a dextransucrase gene obtained from a constitutive mutant (CB4-BF563) of Leuconostoc mesenteroides B-1299, consists of 8,511 bp encoding 2,836 amino acid residues. DsrE563 contains two catalytic domains (CD1 and CD2). Two truncated derivative mutants DsrE563ΔCD2ΔGBD (DsrE563-1) and DsrE563ΔCD2ΔVR (DsrE563-2) of DsrE563 were constructed and expressed using the pRSETC vector in Escherichia coli. The derivatives DsrE563-1 (deletion of 1,620 amino acids from the C-terminus) and DsrE563-2 (deletion of 1,258 amino acids from the C-terminus and 349 amino acids from the N-terminus) were expressed as active enzymes. Both enzymes synthesized less-soluble dextran, mainly containing α-1,6 glucosidic linkage. The synthesized less-soluble dextran also had a branched α-1,3 linkage. DsrE563-2 showed 4.5-fold higher dextransucrase activity than that of DsrE563-1 and showed higher acceptor reaction efficiency than that of dextransucrase from L. mesenteroides 512 FMCM when various mono or disaccharides were used as acceptors. Thus, the glucan-binding domain was important for both enzyme expression and dextransucrase activity.
authors.affiliation: Department of Biotechnology and Bioengineering and Research Institute for Catalysis, Chonnam National University, Gwang-Ju, 500-757, Korea.
authors.forename: Hee-Kyoung
authors.initials: HK
authors.lastname: Kang
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Ko
authors.forename: Jong-Ho
authors.initials: JH
authors.lastname: Kim
authors.forename: Doman
authors.initials: D
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase