d-lactate-selective amperometric biosensor based on the cell debris of the recombinant yeast Hansenula polymorpha.
A d-lactate-selective biosensor has been developed using cells' debris of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha, overproducing d-lactate: cytochrome c-oxidoreductase (EC 1.1.2.4, d-lactate dehydrogenase (cytochrome), DlDH). The H. polymorpha DlDH-producer was constructed in two steps. First, the gene CYB2 was deleted on the background of the С-105 (gcr1 catX) strain of H. polymorpha impaired in glucose repression and devoid of catalase activity to avoid specific l-lactate-cytochrome c oxidoreductase activity. Second, the homologous gene DLD1 coding for DlDH was overexpressed under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration in the Δcyb2 strain. The selected recombinant strain possesses 6-fold increased DlDH activity as compared to the initial strain. The cells debris was used as a biorecognition element of a biosensor, since DlDH is strongly bound to mitochondrial membranes. The cells' debris, prepared by mechanic disintegration of recombinant cells, was immobilized on a graphite working electrode in an electrochemically generated layer using an Os-complex modified cathodic electrodeposition polymer. Cytochrome c was used as additional native electron mediator to improve electron transfer from reduced DlDH to the working electrode. The constructed d-lactate-selective biosensors are characterized by a high sensitivity (46.3-61.6 A M(-1)m(-2)), high selectivity and sufficient storage stability.
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine.
Oleh V
OV
Smutok
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine. Electronic address: dmytruk@cellbiol.lviv.ua.
Kostyantyn V
KV
Dmytruk
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine.
Maria I
MI
Karkovska
Anal. Chem.-Elektroanalytik & Sensorik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
Wolfgang
W
Schuhmann
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine; Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Sokolowska Street 26, 36-100 Kolbuszowa, Poland.
Mykhailo V
MV
Gonchar
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine; Department of Biotechnology and Microbiology, University of Rzeszow, Zelwerowicza 4, 35-601 Rzeszow, Poland.
Andriy A
AA
Sibirny
D015374:Biosensing Techniques; D045304:Cytochromes c / Q000737:chemistry; D055664:Electrochemical Techniques; D004566:Electrodes; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D007773:Lactates / Q000032:analysis*; D010088:Oxidoreductases / Q000737:chemistry; D010843:Pichia / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D007773:Lactates; D011994:Recombinant Proteins; D045304:Cytochromes c; D010088:Oxidoreductases
Amperometric biosensor; Cells׳ debris; Recombinant yeast; d-lactate; d-lactate: cytochrome c-oxidoreductase