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.