Influence of Different Substrates on the Production of a Mutant Thermostable Glucoamylase in Submerged Fermentation
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Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70 °C, with a 3.0 KJ mol−1 increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6 U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3 U/mL, followed by cassava starch and corn starch with 5.9 and 3.0 U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose–Sepharose affinity chromatography; the estimated molecular mass was 100 kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65 °C.
KeywordsMutant glucoamylase Thermostable enzyme Production Purification Corn starch Potato starch Cassava starch Thermo-inactivation
The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq) for their financial support. We are also gratefull to Dr. Clark Ford from Iowa State University for his teachings in this area and for donating our firts mutants.
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