Protein Engineering of Novel Thermostable Pullulanase from Geobacillus stearothermophilus and Starch Saccharification Application

Abstract

A novel thermostable type I pullulanase gene (named pulGSM) is cloned from Geobacillus stearothermophilus DSMZ456. The purified PulGSM shows optimal activity at pH 6.0 and 65 °C. Protein engineering method (site‐directed mutagenesis and enzyme immobilization) is used to further improve the thermostability and catalytic efficiency of PulGSM. Through site‐directed mutagenesis, three mutants (carrying the mutations L211C, E306I and R471E named PulGSM‐M211, PulGSM‐M306 and PulGSM‐M471) are generated and characterized in detail. As showing the best enzymatic properties, PulGSM‐M471 is directly immobilized on epoxy‐functionalized supports to obtain the immobilized enzyme lx‐PulGSM‐M471. The temperature tolerance results show an enhanced T1/2 of 6.5 h (mutated PulGSM‐M471) and 8.5 h (mutated and immobilized lx‐PulGSM‐M471) at 70 °C in comparison to the wild type (4 h). Compared to the commercial pullulanase from Bacillus acidopullulyticus (PDB:2WAN), PulGSM or PulGSM‐M471 can be used directly in maize starch saccharification process without adjusting the pH, which reduces cost and improves efficiency.