OPTIMIZATION OF SYNTHESIS AND DEVELOPMENT OF PURIFICATION SCHEME FOR D-LACTATE : CYTOCHROM C OXIDOREDUCTASE FROM RECOMBINANT YEAST OGATAEA (HANSENULA) POLYMORРHA “tr6”

Еhe optimization of conditions for synthesis of D-lactate : cytochrome c oxidoreduc­tase (EC 1.1.2.4; DLDH) by recombinant yeast strain Ogataea (Hansenula) polymorpha “tr6” (gcr1 catX Δcyb2/DLD1) was performed. The strain is characterized by impairement of glucose catabolic repression, deletion of the CYB2 gene, responsible for the synthesis of L-lactate: cytochrome c oxidoreductase, and six-fold overproduction of DLDH. Moreover, an isolation and purification of the target enzyme using ion exchange chromatography was proposed. The optimization of the cultural medium composition and conditions for the yeast cells' cultivation to obtain the biomass with the highest specific activity of DLDH have been performed. Biomass of the cells O. polymorpha “tr6” is the highest in a medium with 1 % glycerol and 0.5 % racemic lactate, while the best specific activity of DLDH and the lowest non-specific ferricyanide reductase activity are observed in a medium with 1 % ethanol and 0.5% racemic lactate. The influence of some ions of divalent metals on the activity of DLDH in permeabilized cells and induction of the enzyme' synthesis in the cells of O. polymorpha “tr6” were investigated. It has been shown that the addition of Zn2+ ions to the cultural medium significantly increases the activity of DLDH in the cell-free extracts of the yeast cells. The isolation of the membrane-bound DLDH was carried out using methods of physical destruction of the cells. DLDH purification was performed on the DEAE-Toyopearl ion-exchange cellulose 650 M. The yield of the enzyme after chromatographic purification was about 50 %. The highest specific enzyme activity in purified fractions with a 7-fold purification degree was reached up to 1.1 U·mg-1. Some of the physicochemical properties of the purified preparation of DLDH of recombinant O. polymorpha yeast strain “tr6” have been investigated.