Thermostability improvement ofAspergillus awamoriglucoamylase via directed evolution of its gene located on episomal expression vector inPichia pastoris

Novel thermostable forms of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene with its localization on a new episomal expression vector pPEHα in Pichia pastoris cells. Of 3000 yeast transformants screened, six new thermostable GA mutants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asp9His/Ile82Phe, Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of every single substitution in the double mutants, we have constructed an appropriate single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg mutations caused an increased thermostability. While Leu7Met and Asp9His mutations decreased the thermal stability of GA, and Gln338Leu had little effect, the synergistic effect of double mutant forms Leu7Met/His391Tyr, Asp9His/Ile82Phe and Ser8Arg/Gln338Leu revealed the significant thermostability improvement as compared to wild type GA.