ALPHA-D-GALACTOSIDASES HAVING CHANGED REGIOSPECIFICITY AND METHOD FOR THEIR PRODUCTION Russian patent published in 2014 - IPC C12N9/24 C12N15/56 

FIELD: biotechnologies.

SUBSTANCE: described is alpha-D-galactosidase having changed regiospecificity in relation to α1.4-bond and containing Pro402Asp or Phe328Ala mutation in corresponding position of amino acid sequence of initial wild-type enzyme, extracted from Thermoioga maritima MSB8 strain. Production method of the specified alpha-D-galactosidase consists in selection of positions for site-directed mutagenesis of amino acid sequence of wild-type enzyme as follows: a) zymophores having high homology of their amino acid sequence and space structure similarity, which crystal structure contains residue of D-galactose, are overlapped in space; based on performed overlapping of structures potential arrangement of B-galactose in active centre of α-galactosidase from Thermotoga maritima MSB8 is determined; b) potential arrangement of D-galactose in active centre of α-galactosidase is determinec by molecular dynamics methods, general amino acid residues of fermentative centre forming hydrogen bonds with D-galactose are determined, and potential arrangement of D-galactose relative to a pair of catalytic amino acid residues of ferment is found out; c) in silieo mutations of wild type α-galactosidase are performed, positions of amino acid residues for site-directed mutagenesis are selected, after positions selection one of amino acid residues selected at stage c) is replaced by another amino acid residue, namely Pro402Asp or Phe328Ala. Invention allows obtaining regiospecific alpha-D-galactosidases in relation to α1.4-bond.

EFFECT: improving compound properties.

2 cl, 3 dwg, 2 tbl, 1 ex