ИСТИНА

A characteristic feature of the PA is a complicated and multi-stage processing, in the issue of which active heterodimer of PA is generated. Maturation of PA is the main problem in obtaining recombinant enzyme. In addition, the efficiency and speed of processing is highly dependent on the input mutations. Therefore, at present the task of creating a new producer strains and new designs that would allow obtain the active enzyme in a simpler way, is very urgent. The one possible way of solving the problem is to obtain the so-called permuted enzyme, whose N-terminus of α subunit is connected to the C-terminal β-subunit via a short spacer. This will avoid the complicated procedure of processing of the enzyme and will facilitate the expression of the enzyme in the bacteria E.coli. The goal of our work was to obtain permuted PA from Alcaligenes faecalis. The work involves the following steps: analysis of the structure of the enzyme and simulation of possible linkers; synthesis of plasmids with gene of permuted enzyme; expression of permuted AfPA in E.coli cells; optimization of cultivation; isolation and study properties of permuted AfPA. As of today we suggested several polylinkers by computer simulation, and received two permutants. We have studied the conditions of their expression, depending on the culture medium, and it was shown for one permutant that, firstly, its catalytic properties similar to wild-type enzyme, and secondly, it is approximately 12 times more thermal stable than wild-type enzyme.