The thesis is devoted to construction of yeast strains overproducers of L- and
D-lactate-selective oxidoreductases, purification of the enzymes, their combination with nanosized materials, and development on the base of obtained bionanomolecules new spectrophotometric and biosensor approaches for analysis of L- and D-lactate, as well as bioreactors for enzymatic conversion of racemic lactic acid to pure enantiomers.
Using molecular genetics approaches, recombinant strain of yeast Ogataea polymorpha: "tr1" (gcr1 catX CYB2) – overproducer of L-lactate: ferricitohrome c-oxidoreductase (flavocytochrome b2, FC b2), characterized by an eight-fold increase in the specific activity of the enzyme, has been constructed. A recombinant producer of D-lactate: ferricitohrom c-oxidoreductase (DLDH) – O. polymorpha "tr6" (gcr1 catX CYB2Δ/DLD1), whose specific activity of DLDN is six-fold greater comparing with the parental strains, has been constructed also. Due to deletion of the CYB2 gene, the strain is not synthesizes FC b2. The conditions for the maximal enzymes synthesis for both recombinant yeast strains have been optimized.
For the first time, a new method of purifying FC b2 from extracts of yeast strains O. polymorpha "tr1" by affinity chromatography on aminopropylsilocromes modified with cytochrome c in the role of a ligand has been developed, and preparations of the enzyme with a specific activity of 10 U·mg-1 have been obtained. The scheme of purification of the membrane enzyme DLDN from the cells of O. polymorpha "tr6" has been developed and a purified enzyme preparation with a specific activity of 1.1 U·mg-1 has been obtained. For both enzyme preparations, a physico-chemical and enzyme characteristic has been performed.
The possibility of using the yeast producers of L- and D-lactate-specific oxidoreductases for production of pure D-enantiomer from racemic lactic acid and remediation of D-lactate from the model samples has been proved.
For the first time a new enzymatic-photometric method of quantitative analysis of L-lactate based on recombinant FC b2 and "Prussian blue" has been reported. The method of reusing of enzyme immobilized on ferromagnetic microparticles has been developed. It is shown that enzyme microparticles can be removed from the reaction solution by magnetic field and reused at least six times without loss of quality of L-lactate analysis.
A new photometric method of quantitative analysis of D-lactate based on the use of cell and subcellular fractions of O. polymorpha "tr6" and the formation of formazan as a final colored product has been developed.
The nanocarriers for immobilization of the enzymes based on gold nanoparticles were synthesized. A new method of gold nanoparticles formation on the surface of the working planar electrode in situ was developed. The physico-chemical and structural characteristics of the nanomaterials were performed by means scanning electron microscopy, X-ray spectral analysis, atomic force microscopy and transmitted electron microscopy.
On the basis of permeabilized cells O. polymorpha "tr1" - overproduced FC b2 new microbial amperometric biosensors with improved bioanalytical characteristics have been constructed. The construction of new microbial ampereometric biosensors with improved bioanalytical characteristics based on permeabilized FC b2-overproductive cells of O. polymorpha "tr1" enriched with an enzyme bound gold nanoparticles have been done. For the first time it has been shown that the enrichment of yeast cells of FC b2, due to the overexpression of the corresponding gene and the addition of immobilized enzyme gold nanoparticles into cells, significantly improves the main operational characteristics of the biosensor (sensitivity, detection limit, storage stability, etc.).
A new L-lactate-selective enzymatic mediatorless biosensor of the "third generation" based on purified recombinant FC b2 and gold nanoparticles has been developed.
New microbial biosensors for D-lactate with the use of cell debris, subcellular fractions enriched with mitochondria, as well, the cells of recombinant strain
O. polymorpha «tr6» have been developed and characterized. Due to the deletion of the CYB2 gene encoding L-lactate-selective flavocytochrome b2, microbial biosensors are highly selective to the D- lactate enantiomer.
The developed bioanalytical approaches based on recombinant cells, cell debris, subcellular fractions enriched with mitochondria, as well, purified enzyme preparations were used for quantitative analysis of L- and D-lactate in the real samples of human liquids, food products and transfusion pharmaceuticals. Due to its high sensitivity and selectivity, as well as reliability and ease of use, the developed enzymatic and microbial approaches of quantitative analysis of lactate can be using practically in clinical diagnosis, sports medicine and food technology.
