Starch-binding domain shuffling in Aspergillus niger glucoamylase.
Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N-terminus, catalytic domain + linker + starch-binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIDeltaL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII approximately GAII approximately GAI > RGAIDeltaL approximately RGAI approximately GAE. Insoluble starch hydrolysis rates were GAI > RGAIDeltaL > RGAI >> GAE approximately RGAII > GAII, while insoluble starch-binding capacities were GAI > RGAI > RGAIDeltaL > RGAII > GAII > GAE. These results indicate that: (i) moving the SBD to the N-terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.macerans cyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch.
Cornett
Catherine A G
CA
Department of Food Science and Human Nutrition, 2114 Sweeney Hall, Iowa State University, Ames, IA 50011-2230, USA.
Fang
Tsuei-Yun
TY
Reilly
Peter J
PJ
Ford
Clark
C
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*
D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase