Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted alpha-d-glucopyranosyl units and 29 mol% 1,6-disubstituted alpha-d-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted alpha-d-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted alpha-d-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted alpha-d-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted alpha-d-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.