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 degrees C similar to that of wild-type GA. Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80 degrees 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 degrees 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 degrees C.