[Construction and culture conditions of dextransucrase-secreting engineered strain].
OBJECTIVE
Dextransucrase was a glucosyltransferases catalyzing the transfer of D-glucopyranosyl units from sucrose to synthesize alpha-glucans or oligosaccharides.


METHODS
dexYG gene (GenBank Accession No. DQ345760), encoding the dextransucrase from Leuconstoc mesenteriodes 0326, was subcloned into expression plasmid pET28a(+). The recombinant plasmid was then transformed into E. coli BL21 (DE3). Kanamycin resistant transformants were selected and verified by restriction endonuclease assay.


RESULTS
Dextransucrase could be efficiently expressed in engineered strain BL21 (DE3)/pET28-dexYG by Isopropyl beta-D-thiogalactopyranoside (IPTG) induction, although the growth of E. coli host was inhibited during induction. Recombinant enzyme producing conditions such as induction time, IPTG concentration, incubation temperature, cell density (OD(600)) and pH value were studied. The optimum conditions for producing dextransucrase were as follows: incubation at 25 degrees C, 0.5 mmol/L Isopropyl beta-D-thiogalactopyranoside (IPTG) induction at cell density (OD(600)) of 1.0 for 5h, pH 6.0. Under these conditions, the recombinant dextransucrase activity was increased from 5.39U/mL to 35.62 U/mL. The highest activity under the optimal culture conditions after 5h induction in medium with pH 6.0 was 3.5 times as that of in Luria-Bertani medium without pH-adjustment. Moreover, the pH value was one of the main reasons that caused the degradation of enzyme in the later stage of induction.


CONCLUSIONS
These results showed that dextransucrase could be efficiently heterologous expressed in E. coli and a strong dextransucrase activity had been detected.
Zhang
Hongbin
H
Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China. zhb5678@163.com
Zhu
Chunbao
C
Hu
Youjia
Y
Zhu
Baoquan
B
Wang
Yajie
Y
D018533:Biomass; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007934:Leuconostoc / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D013696:Temperature
D007544:Isopropyl Thiogalactoside; D005964:Glucosyltransferases; C020025:dextransucrase