Mutations to alter Aspergillus awamori glucoamylase selectivity. III. Asn20-->Cys/Ala27-->Cys, Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Lys108-->Met, Gly137-->Ala, 311-314 Loop, Tyr312-->Trp and Ser436-->Pro.
Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild-type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.
Liu
H L
HL
Department of Chemical Engineering, Iowa State University, Ames 50011, USA.
Coutinho
P M
PM
Ford
C
C
Reilly
P J
PJ
D001230:Aspergillus / Q000201:enzymology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011134:Polysaccharides; D011994:Recombinant Proteins; D007534:Isomaltose; D008320:Maltose; C008315:maltodextrin; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose