d-lactate-selective amperometric biosensor based on the cell debris of the recombinant yeast Hansenula polymorpha

• Published in: Talanta (Oxford) Vol. 125; pp. 227 - 232
• Main Authors: Smutok, Oleh V., Dmytruk, Kostyantyn V., Karkovska, Maria I., Schuhmann, Wolfgang, Gonchar, Mykhailo V., Sibirny, Andriy A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-07-2014
• Subjects: Amperometric biosensor; Biosensing Techniques; Cells׳ debris; Cytochromes c - chemistry; d-lactate; d-lactate: cytochrome c-oxidoreductase; Electrochemical Techniques; Electrodes; Electrons; Escherichia coli - metabolism; Hansenula polymorpha; Lactates - analysis; Oxidoreductases - chemistry; Pichia - chemistry; Plasmids - metabolism; Recombinant Proteins - chemistry; Recombinant yeast; Reproducibility of Results; Sensitivity and Specificity; Recombinant yeast; d-lactate; Amperometric biosensor; Cells׳ debris; d-lactate: cytochrome c-oxidoreductase
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2014.02.041

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 (46.3–61.6AM−1m−2), high selectivity and sufficient storage stability.
[Display omitted]
•A recombinant overproducer of d-lactate: cytochrome c-oxidoreductase was constructed.•The strain possesses 6-fold increase enzyme activity as compared to parental strain.•The cells׳ debris was used as a bioselective element of d-lactate biosensor.•Cytochrome c was used as additional native mediator to improve electron transfer.•The biosensor characterized by a high sensitivity, selectivity and storage stability.