Designing of a novel dextransucrase efficient in acceptor reactions.
Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119 kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5' end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.
Department of Biochemistry, Gebze Institute of Technology, Muallimkoy, Gebze, Kocaeli, Turkey.
Mahmut
M
Parlak
Department of Genetics, Research Institute of Experimental Medicine, Istanbul University, Fatih, Istanbul, Turkey. Electronic address: dustek@istanbul.edu.tr.
Duran
D
Ustek
Department of Biochemistry, Gebze Institute of Technology, Muallimkoy, Gebze, Kocaeli, Turkey. Electronic address: tanriseven@gyte.edu.tr.
Aziz
A
Tanriseven
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D002122:Calcium Chloride / Q000737:chemistry; D020134:Catalytic Domain; D003911:Dextrans / Q000096:biosynthesis; D004800:Enzymes, Immobilized / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D003911:Dextrans; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C020025:dextransucrase; D005982:Glutathione Transferase; D002122:Calcium Chloride
Dextran; Dextransucrase; Enzyme activity; Enzyme biocatalysis; Leuconostoc mesenteroides; Recombinant DNA