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

A novel thermostable type I pullulanase gene (named pul GSM ) is cloned from Geobacillus stearothermophilus DSMZ456. The purified Pul GSM 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 Pul GSM . Through site‐directed mutagenesis, three mutants (carrying the mutations L211C, E306I and R471E named Pul GSM ‐M211, Pul GSM ‐M306 and Pul GSM ‐M471) are generated and characterized in detail. As showing the best enzymatic properties, Pul GSM ‐M471 is directly immobilized on epoxy‐functionalized supports to obtain the immobilized enzyme lx‐Pul GSM ‐M471. The temperature tolerance results show an enhanced T 1/2 of 6.5 h (mutated Pul GSM ‐M471) and 8.5 h (mutated and immobilized lx‐Pul GSM ‐M471) at 70 °C in comparison to the wild type (4 h). Compared to the commercial pullulanase from Bacillus acidopullulyticus (PDB:2WAN), Pul GSM or Pul GSM ‐M471 can be used directly in maize starch saccharification process without adjusting the pH, which reduces cost and improves efficiency.