title: Permeabilized cells of flavocytochrome b2 over-producing recombinant yeast Hansenula polymorpha as biological recognition element in amperometric lactate biosensors.
abstract: A L-lactate-selective microbial biosensor was developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing L-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, flavocytochrome b(2), FC b(2)). The construction of FC b(2)-producers by over-expression of the gene CYB2 H. polymorpha encoding FC b(2) is described. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration was transformed to the recipient strain H. polymorpha C-105 (gcr1 catX) impaired in glucose repression and devoid of catalase activity. The permeabilized cells were either immobilized on the graphite working electrode by physical entrapment of the cell suspension by means of a dialysis membrane or by integration of the cells in an electrochemically generated layer using a cathodic electrodeposition polymer. Phenazine methosulphate was used as a free-diffusing redox mediator. It was assumed that the mediator reacts with mitochondrial FC b(2) after entering the cells in the presence of L-lactate. The biosensor based on recombinant yeast cells exhibited a higher K(M)(app) value and hence expanded linear range toward L-lactate as compared to a similar sensor based on the initial cells of H. polymorpha C-105.
authors: Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine. smutok@cellbioll.lviv.ua
authors: Oleh
authors: O
authors: Smutok
authors: Kostyantyn
authors: K
authors: Dmytruk
authors: Mykhailo
authors: M
authors: Gonchar
authors: Andriy
authors: A
authors: Sibirny
authors: Wolfgang
authors: W
authors: Schuhmann
mesh_terms: D015374:Biosensing Techniques; D004563:Electrochemistry; D005822:Genetic Vectors; D007425:Intracellular Membranes / Q000201:enzymology; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics; D019344:Lactic Acid / Q000032:analysis* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics; D010539:Permeability; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D010957:Plasmids
chemical_list: D024101:Mitochondrial Proteins; D019344:Lactic Acid; D042844:L-Lactate Dehydrogenase (Cytochrome)