Genetic and biochemical analysis of Aspergillus awamori glucoamylase thermostability

摘要: Aspergillus awamori glucoamylase (GA) is an enzyme involved in industrial com starch processing. Genetic and biochemical approaches were used to smdy the mechanisms governing GA thermostability. Three proline substimtion (Xaa-Pro) mutations constructed that predicted increase enzyme's stability by decreasing its conformational entropy of unfolding. When expressed ui Saccharomyces cerevisiae Ser30-Pro increased, Asp345-Pro did not alter Glu408-Pro greatly decreased as measured resistance irreversible thermoinactivation relative wildtype enzyme. The mutation was combined with other previously identified stabilizing examine whether combining such could cumulatively stabilize GA. SerSO-Pro stabilized when Asn20-Cys/Ala27-Cys mutations, which create a disulfide bond between positions 20 27. Similarly, Glyl37-Ala stabilized. mutant Asn20-Cys/Ala27-Cys/Ser30-Pro/Glyl37-Ala most stable variant increased activation energy for 4.4 kJ/moI at 65°C melting temperature (the 50% inactivated after 10 minutes) 3.9°C wild-type None mutants activity. Ser30-Pro/Glyl37-Ala presence 1.71 M glucose outperformed high-temperamre ix (65°C) saccharification DE maltodextrin. Using random mutagenesis, AspZSS-Asn expression recombinant S. grown elevated temperatures both Gly396-Ser genetic backgroimds. This may be due intraand/or extra-cellular proteolysis conferred Asp238-Asn amino acid substimtion. screening method identify Asp238-'Asn useful productiondeficient mutants.