title: Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity.
abstract: To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.
authors.affiliation: Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea.
authors.forename: So-Ra
authors.initials: SR
authors.lastname: Lee
authors.forename: Ah-Rum
authors.initials: AR
authors.lastname: Yi
authors.forename: Hong-Gyun
authors.initials: HG
authors.lastname: Lee
authors.forename: Myoung-Uoon
authors.initials: MU
authors.lastname: Jang
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Park
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Han
authors.forename: Tae-Jip
authors.initials: TJ
authors.lastname: Kim
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification*; D005964:Glucosyltransferases / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D013997:Time Factors
chemical_list: D003470:Culture Media; D005964:Glucosyltransferases; C020025:dextransucrase