An increase in the transglycosylation activity of Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.
The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.
Matsui
I
I
National Chemical Laboratory for Industry, Ibaraki, Japan.
Ishikawa
K
K
Miyairi
S
S
Fukui
S
S
Honda
K
K
D006031:Glycosylation; D006868:Hydrolysis; D016296:Mutagenesis; D012442:Saccharomycopsis / Q000201:enzymology*; D013329:Structure-Activity Relationship; D000516:alpha-Amylases / Q000235:genetics / Q000502:physiology*
D000516:alpha-Amylases