Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90 kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318 U/mL extracellular isoamylase activity after 72 h in a 10 L fermenter. The recombinantenzyme was purified and characterized. It had an estimated molecular mass of 90 kDa, with its optimal activity at 70 degrees C, pH 6.5 and was quite stable between 30 degrees C and 70 degrees C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process. (C) 2013 Elsevier Ltd. All rights reserved.