| abstract |
In one aspect of the present invention there is provided a method for preparing a variant lipid acyltransferase enzyme comprising expressing in a host organism a nucleotide sequence which has at least 90% identity with a nucleotide sequence encoding a parent lipid acyltransferase and comprises at least one modification (suitably at least two modifications) at a position(s) which corresponds in the encoded amino acid sequence to an amino acid(s) located in a) the canyon region of the enzyme (i.e. preferably amino acid residues 31, 27, 85, 86, 119, and 120); and/or b) insertion site 1 (i.e. amino acid residues 22-36) and/or c) insertion site 2 (i.e. amino acid residues 74-88), wherein the canyon region, insertion site 1 and/or insertion site 2 are defined as that region which when aligned based on primary or tertiary structure corresponds to the canyon region, insertion site 1 or insertion site 2 (or the corresponding amino acid residues taught above) of the enzyme shown herein as SEQ ID No. 16 or 6. Preferable modifications are one or more of the following L3 IQ, H, N, T, F, Y or C (preferably L31 Q); M27R, G, H, K, Y, D, N, V, C, Q, L, E, S or F (preferably M27V); V85H, R5 D or E; I86R,Y, S, V, I, As T, M, F, C or L (preferably I86S or A); Al 19T or I; Y120K or E; W122S, L or A (preferably W122L); E201R; Q245S; F235A or V; W232G or S; and/or A236G or E. |