Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 101, Issue 6, Pages 2357-2369Publisher
SPRINGER
DOI: 10.1007/s00253-016-8025-3
Keywords
Geobacillus thermoleovorans; Recombinant amylopullulanase; Pichia pastoris; Wheat bran; Starch saccharification; Maltooligosaccharides
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Funding
- Indian Council of Medical Research (ICMR), Government of India, New Delhi [2013-ECDI-80/809]
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The 3'-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apu Delta C) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apu Delta C) was higher under GAP promoter (36.2 U ml(-1), alpha-amylase; 33.5 U ml(-1), pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml(-1)). The heavily glycosylated Gt-apu Delta C from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.
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