Patent ID: 7220563

Claim:
A method of producing a glucose-6 oxidase comprising the steps of: (a) constructing a library of variants of a parent galactose oxidase (GAO), wherein the parent galactose oxidase has the amino acid sequence of SEQ ID NO:2, where said variant consists of an amino acid substitution at residue Q406 of SEQ ID NO:2 and one or two additional amino acid substitutions; and (b) selecting any galactose oxidase variants having increased glucose-6 oxidation activity as compared to the parent galactose oxidase, wherein said one or two additional amino acid substitutions are positioned at residues no more that 15 Å from a metal ion in the catalytic center of the parent galactose oxidase, based on 3-dimensinal structure of the amino acid sequence of SEQ ID NO:2, and wherein said one or two additional amino acid substitutions are at residues selected from the group consisting of S10, M70, P136, I169, V170, P171, A172,A173, A174, M185, W186, S187, S188, Y189, R190, N191, D192, A193, F194, G195, G1961 S197, I201, T202, L203, H224, D225, M226, F227, C228, P229, G230, I231, V241, T242, G243, G244, N245, D264, A247, R270, G271, Y272, Q273, S274, S275, G287, G288, W290, $291, G292, G293, V294, K297, D324, Q326, G327, L328, Y329, R330, S331, D332, N333, H334, A335, W336, P350, A381, M382, C383, G384, N385, S402, D404, Y405, T440, F441, H442, T443, G457, R460, G461, I462, P463, F464, E465, D466, V492, R493, V494, Y495, H496, S497, 1498, S499, G510, G511; G512, G513, L514, C515, G516, D517, C518, T520, N521, H522, N535, T578, A579, T580, H581, T582, V583, and A584 of SEQ ID NO:2.