ABSTRACT
As more bioactivities of oligosaccharides have been elucidated, researches on biosynthesis of oligosaccharides have drawn more concerns in Glycobiology. A lot of enzymatic methods for the synthesis of oligosacchrides have been developed employing recombinant E. coli expressed glycosyltranferase or synthase of nucleotide-sugar. This review focuses on the recent progress in the production of oligosaccharides using bacteria especially by genetically engineered bacteria. The key point concering the oligosaccharides biosynthesis in recombinant E. coli, such as enzyme expression, NDP-sugar provision and biosynthesis pathway, was discussed.
Subject(s)
Bacteria , Genetics , Bacterial Proteins , Genetics , Metabolism , Carbohydrate Sequence , Escherichia coli , Genetics , Metabolism , Genetic Engineering , Methods , Glycosyltransferases , Genetics , Metabolism , Industrial Microbiology , Methods , Molecular Sequence Data , OligosaccharidesABSTRACT
Cyclodextrin glucanotransferase, the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Although many related enzyme properties are well known, the crucial factors in product specificity determination remain to be answered. Here, the recent research progresses of cyclodextrin glucanotransferase, especially those about the evolution of product specificity, were reviewed, and the scientific problems were discussed.
Subject(s)
Archaeal Proteins , Genetics , Metabolism , Bacillus , Genetics , Bacterial Proteins , Genetics , Metabolism , Biocatalysis , Cyclodextrins , Metabolism , Evolution, Molecular , Glucosyltransferases , Classification , Genetics , Metabolism , Mutation , Thermoanaerobacterium , Genetics , ThermococcusABSTRACT
In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.