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Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Author:
Li, Youran, Zhang, Liang, Ding, Zhongyang, Shi, Guiyang
Source:
Process Biochemistry 2013 v.48 pp. 1303-1310
ISSN:
1359-5113
Subject:
polypeptides, fermentation, Komagataella pastoris, fermenters, methanol, open reading frames, pullulanase, yeasts, starch, amino acid sequences, gene expression, amino acids, genes, maltose, pH, molecular weight, isoamylase, Bacillus lentus
Abstract:
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 90kDa. 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 318U/mL extracellular isoamylase activity after 72h in a 10L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90kDa, 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.
Agid:
1140253