[Purification and characterization of recombinant Escherichia coli dextransucrase]. 2008

Wang Yajie, and Zhang Hongbin, and Hu Xueqin, and Zhu Chunbao, and Hu Youjia
Department of Pharmaceutical Engineering, Hefei University of Technology, Hefei 230009, China. cactus0926@ah163.com

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D007934 Leuconostoc A genus of gram-positive, facultatively anaerobic bacteria whose growth is dependent on the presence of a fermentable carbohydrate. It is nonpathogenic to plants and animals, including humans. Leukonostoc
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005964 Glucosyltransferases Enzymes that catalyze the transfer of glucose from a nucleoside diphosphate glucose to an acceptor molecule which is frequently another carbohydrate. EC 2.4.1.-. Glucosyltransferase
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial

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