title: Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions.
abstract: We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Elvi
authors.initials: E
authors.lastname: Restiawaty
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.forename: Kenji
authors.initials: K
authors.lastname: Okano
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000378:metabolism; D005991:Glycerol Kinase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007218:Industrial Microbiology; D050576:Membrane Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000378:metabolism; D013997:Time Factors
chemical_list: D050576:Membrane Fusion Proteins; D011122:Polyphosphates; D005991:Glycerol Kinase; D005990:Glycerol