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Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.

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Relation http://ir.cftri.com/14687/
https://doi.org/10.1016/j.enzmictec.2020.109653
 
Title Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia
pastoris and its efficient recovery for synthesis of isomaltooligosaccharides.
 
Creator Sandeep, Kumar
Sarma, Mutturi
 
Subject 01 Carbohydrates
04 Yeast
 
Description A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger
strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained
intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction
digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated
for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L
bioreactor. Specific growth rate of 0.1 h− 1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible
enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the
periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was
purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three
bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained
enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can
be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in
cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.
 
Date 2020
 
Type Article
PeerReviewed
 
Format pdf
 
Language en
 
Identifier http://ir.cftri.com/14687/1/Enzyme%20and%20Microbial%20Technology%20141%20%282020%29%20109653.pdf
Sandeep, Kumar and Sarma, Mutturi (2020) Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides. Enzyme and Microbial Technology, 141. p. 109653. ISSN 0141-0229