Directed evolution of Aspergillus niger glucoamylase to increase thermostability.
Using directed evolution and site-directed mutagenesis, we have isolated a highly thermostable variant of Aspergillus niger glucoamylase (GA), designated CR2-1. CR2-1 includes the previously described mutations Asn20Cys and Ala27Cys (forming a new disulfide bond), Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala, His391Tyr and Ser436Pro. In addition, CR2-1 includes several new putative thermostable mutations, Val59Ala, Val88Ile, Ser211Pro, Asp293Ala, Thr390Ser, Tyr402Phe and Glu408Lys, identified by directed evolution. CR2-1 GA has a catalytic efficiency (k(cat)/K(m)) at 35°C and a specific activity at 50°C similar to that of wild-type GA. Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol(-1) compared with that of wild-type GA. Thus, CR2-1 is more thermostable (by 5 kJ mol(-1) at 80°C) than the most thermostable A. niger GA variant previously described, THS8. In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol(-1), respectively, at 80°C.
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.
Allison
A
McDaniel
Erica
E
Fuchs
Ying
Y
Liu
Clark
C
Ford
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D005656:Fungal Proteins; D005087:Glucan 1,4-alpha-Glucosidase
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