Modulation of hydrolysis and transglycosylation activity of Thermus maltogenic amylase by combinatorial saturation mutagenesis.
The roles of conserved amino acid residues (Val329-Ala330- Asn331-Glu332), constituting an extra sugar-binding space (ESBS) of Thermus maltogenic amylase (ThMA), were investigated by combinatorial saturation mutagenesis. Various ThMA mutants were firstly screened on the basis of starch hydrolyzing activity and their enzymatic properties were characterized in detail. Most of the ThMA variants showed remarkable decreases in their hydrolyzing activity, but their specificity against various substrates could be altered by mutagenesis. Unexpectedly, mutant H-16 (Gly-Leu-Val-Tyr) showed almost identical hydrolyzing and transglycosylation activities to wild type, whereas K-33 (Ser-Gly-Asp-Glu) showed an extremely low transglycosylation activity. Interestingly, K-33 produced glucose, maltose, and acarviosine from acarbose, whereas ThMA hydrolyzed acarbose to only glucose and acarviosine-glucose, which proposes that the substrate specificity, or hydrolysis or transglycosylation activity of ThMA can be modulated by combinatorial mutations near the ESBS.
Oh
Su-Won
SW
Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Korea.
Jang
Myoung-Uoon
MU
Jeong
Chang-Ku
CK
Kang
Hye-Jeong
HJ
Park
Jung-Mi
JM
Kim
Tae-Jip
TJ