| abstract |
Described is a method for improving the properties of a cellulolytic enzyme by amino acid substitution, deletion or insertion, the method comprising the steps of: n a. constructing a multiple alignment of at least two amino acid sequences known to have three-dimensional structures similar to endoglucanase V (EGV) from Humicola insolens known from Protein Data Bank entry 4ENG; n b. constructing a homology-built three-dimensional structure of the cellulolytic enzyme based on the structure of the EGV; n c. identifying amino acid residue positions present in a distance from the substrate binding cleft of not more than 5 Å; n d. identifying surface-exposed amino acid residues of the enzyme; n e. identifying all charged or potentially charged amino acid residue positions of the enzyme; n f. choosing one or more positions wherein the amino acid residue is to be substituted, deleted or where an insertion is to be provided; and n g. carrying out the substitution, deletion or insertion by using conventional protein engineering techniques. Also described are cellulase variants obtained by this method. |