PROTEIN ENGINEERING OF GLUCOAMYLASE TO INCREASE pH OPTIMUM, SUBSTRATE SPECIFICITY AND THERMOSTABILITY

摘要

A fungal glucoamylase including a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair. The mutation provides increased thermal stability and reduced isomaltose formation to the enzyme. A fungal glucoamylase including a 311-314Loop mutation wherein reduced isomaltose formation is provided by the mutation is also provided. A fungal glucoamylase including a mutation Ser411Ala wherein increased pH optimum and reduced isomaltose formation is provided by the mutation is also provided. Combinations of the mutations in engineered glucoamylases are also provided as are combinations with other glucoamylase mutations that provide increased thermal stability, increased pH optimum and reduced isomaltose formation for cumulative improvements in the engineered glucoamylases. Also provided is a fungal glucoamylase including a mutation of Ser30Pro coupled with at least two stabilizing mutations forming a disulfide bond between the two stabilizing members. A fungal glucoamylase including a Ser30Pro/Gly137Ala/311-314 Loop is provided. A fungal glucoamylase including a mutation Ser30Pro/Glu137Ala/Ser411Ala is also provided.