title: [Purification and characterization of recombinant Escherichia coli dextransucrase].
abstract: 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.
authors.affiliation: Department of Pharmaceutical Engineering, Hefei University of Technology, Hefei 230009, China. cactus0926@ah163.com
authors.forename: Wang
authors.initials: W
authors.lastname: Yajie
authors.forename: Zhang
authors.initials: Z
authors.lastname: Hongbin
authors.forename: Hu
authors.initials: H
authors.lastname: Xueqin
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chunbao
authors.forename: Hu
authors.initials: H
authors.lastname: Youjia
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase