Single step liquefaction and saccharification of corn starch using an acidophilic, calcium independent and hyperthermophilic pullulanase

摘要

A novel thermoacidophilic pullulanase (Tk-PUL) from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is described here that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions. The gene encoding Tk-PUL was cloned and expressed in E. coli cells. The purified recombinant enzyme possesses the following properties: shows both pullulanase and α-amylase activities, displays highest activity at 95-100° C., active over a broad pH range (3.0-8.5) with optimum working pH 3.5, stable for several hours at 90° C. and displays a half-life of 45 minutes at 100° C., activity and stability are independent of calcium and other metal ions, and hydrolyzes maltotriose. Moreover, recombinant Tk-PUL can be used for single step liquefaction and saccharification of corn starch (without any α-amylase or β-amylase) at pH 4.2 in the absence of calcium.