Cloning, sequencing and expression of a dextransucrase gene (dexYG) from Leuconostoc mesenteroides.
The gene dexYG encoding the dextransucrase from an industrial strain of Leuconostoc mesenteroides 0326 was isolated by PCR. The nucleotide sequence of the dexYG gene consists of an open reading frame (ORF) of 4,584 bp, coding for a 1,527 aa protein with a Mr of 170 kDa. The results were analysed by a BLAST similarity search of the GenBank database, which revealed the amino acid sequence was similiar to dsrD derived from L. mesenteroides Lcc4. The dexYG gene was subcloned into the plasmid pET28a(+) and was expressed in E. coli BL21 (DE3) by IPTG induction. The pH value was one of the main reasons which caused the degradation of enzyme activity in the later stage of induction. The highest activity was reached 36 U/ml after 5 h induction in medium at pH 6.0. Biotransformation yield of the enzyme reached 65% and the molecular weight of transformed dextran was more than 68 kDa in 2 h.
Zhang
Hongbin
H
State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai, People's Republic of China. zhb5678@163.com
Hu
Youjia
Y
Zhu
Chunbao
C
Zhu
Baoquan
B
Wang
Yajie
Y
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D005964:Glucosyltransferases; C020025:dextransucrase