title: Development of a continuous bioconversion system using a thermophilic whole-cell biocatalyst.
abstract: The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.
authors: Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors: Pham Huynh
authors: PH
authors: Ninh
authors: Kohsuke
authors: K
authors: Honda
authors: Yukako
authors: Y
authors: Yokohigashi
authors: Kenji
authors: K
authors: Okano
authors: Takeshi
authors: T
authors: Omasa
authors: Hisao
authors: H
authors: Ohtake
mesh_terms: D001709:Biotechnology / Q000379:methods; D001711:Biotransformation; D004798:Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D005404:Fixatives / Q000378:metabolism; D005649:Fumarate Hydratase / Q000235:genetics* / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D005976:Glutaral / Q000378:metabolism; D006358:Hot Temperature; D008293:Malates / Q000378:metabolism; D060847:Metabolic Engineering; D008854:Microscopy, Electron; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D004798:Enzymes; D005404:Fixatives; D005650:Fumarates; D008293:Malates; D011994:Recombinant Proteins; C030298:malic acid; D005649:Fumarate Hydratase; D005976:Glutaral