Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli.
The dextransucrase gene dsrX from Leuconostoc mesenteroides CGMCC 1.544 was cloned into the vector pET-28a(+) and expressed as a N-terminal His(6)-tag fusion protein of 167.57 kDa in Escherichia coli BL21(DE3). DsrX with the high volumetric activity of 8.8 U ml(-1) culture and the specific activity of 97.37 U mg(-1) crude enzyme extracts was measured in the optimized recombinant expression system. The resultant expression level of the fusion protein amounted to 24.5% of the total cell proteins. The results of affinity chromatography and western blotting indicated that the three sensitive sites of proteolysis existed in the N-terminal catalytic domain of DsrX. Both the recombinant and native enzyme activity were slightly activated by 1 mmol l(-1) Mn(2+) and strongly inhibited by 1 mmol l(-1) Cu(2+) or Al(3+), and their optimum pH values were 5.4. The optimum temperature of the recombinant enzyme for dextran synthesis was 30 degrees C, which was 10 degrees C less than that of the native one. The transglucosylation products of two enzymes were studied by using thin layer chromatography and high-performance anion exchange chromatography. It could be concluded that the better sample-pretreatment temperature in SDS-PAGE was 37 degrees C, which significantly improved the detection of thermal instable enzyme than that of 100 degrees C.
Yalin
Yang
Y
Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie Street, Haidian District, Beijing 100081, PR China.
Jin
Luo
L
Jianhua
Wang
W
Da
Teng
T
Zigang
Tian
T
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase