ВИДІЛЕННЯ, ОЧИЩЕННЯ, СТАБІЛІЗАЦІЯ ТА ХАРАКТЕРИСТИКА ФЛАВОЦИТОХРОМУ b2 ІЗ КЛІТИН НАДПРОДУЦЕНТА OGATAEA POLYMORРHA «tr1» (gcr1 catX CYB2)

Abstract

A new method of isolation, purification, and stabilization of L-lactate: cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b 2 , FC b 2 ) from the cells of recombinant yeast Ogataea ( Hansenula ) polymorpha “tr1” ( gcr1 catX CYB2 ) has been developed and physico-chemical characterisation of purified preparations has been done. The strain is characterized by impairment of glucose catabolic repression and a five-fold overproduction of FC b 2 , compared to a parent wild-type strain. The effect of several detergents on the yield of membrane-incorporated FC b 2 was studied and the enzyme isolation procedure has been optimized. A new method of affinity chromatography for purification of FC b 2 , based on the use of cytochrome c as a natural ligand, immobilized on aminopropyl silochrome, has been developed. The efficiency of the developed affinity chromatography was compared to ion exchange chromatography on DEAE-Toyopearl 650M cellulose. The specific activity of FC b 2 preparations for both types of chromatography was about 10 U·mg -1 and enzyme yield was in the range of 75–95 %. The purity of the purified FC b 2 was evaluated by SDS-PAAG electrophoresis under denaturing conditions. The effect of a number of stabilizing agents and storage conditions has been investigated in order to provide the maximum activity of the FC b 2 . The best stabilizing effect was observed using 70 % ammonium sulfate and by storage of the preparation at -20 °C. Some physico-chemical parameters of the purified enzyme from O. polymorpha “tr1” cells have been studied (molecular mass and spectral characteristics of oxidized and reduced enzyme forms).