Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
34623 | Process Biochemistry | 2013 | 8 Pages |
•A novel isoamylase gene was cloned.•The gene was constitutively expressed at high level.•The enzyme was purified and characterized.
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 recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90 kDa, with its optimal activity at 70 °C, pH 6.5 and was quite stable between 30 °C and 70 °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.