[Purification and characterization of recombinant Escherichia coli dextransucrase].
OBJECTIVE
To purify and characterize recombinant dextransucrase expressed in engineered strain BL21 (DE3)/pET28-dexYG.


METHODS
The dextransucrase gene (dexYG) was expressed in engineered strain after IPTG induction and the crude enzyme was obtained by sonication. We purified the recombinant dextransucrase by using ammonium sulfate precipitation and metal chelate affinity chromatography on a Ni-NTA column. Then we characterized catalytic kinetic parameter of purified enzyme.


RESULTS
This purification protocol resulted in a 11.4-fold purification with a yield of 37.5%. The molecular weight of dextransucrase measured by SDS-PAGE was 170 kDa ,which was similar to the enzyme from Leuconostoc mesenteroides. The enzyme had an optimum temperature between 25 and 30 degrees C and an optimum pH of 5.4. It was relatively stable in the range of pH 5.0 to 7.0, but the stability declined rapidly as soon as the temperature rose over 35 degrees C.The enzyme activity remained 59% after stored for 4 days at room temperature (25 degrees C), and lost 50% activity after stored for seven weeks at 4 degrees C. Ca2+ of 0.5 mmol/L could strongly activate the enzyme, Mg2+ of 1 mmol/L had little effect, Cu2+ and SDS could greatly inhibit the enzyme.


CONCLUSIONS
These results may provide an important basis for industrial applications of the recombinant dextransucrase.
Yajie
Wang
W
Department of Pharmaceutical Engineering, Hefei University of Technology, Hefei 230009, China. cactus0926@ah163.com
Hongbin
Zhang
Z
Xueqin
Hu
H
Chunbao
Zhu
Z
Youjia
Hu
H
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D007934:Leuconostoc / Q000737:chemistry / Q000201:enzymology*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase