Genome mining for new α-Amylase and Glucoamylase encoding sequences and high level expression of a Glucoamylase from Talaromyces stipitatus for potential raw starch hydrolysis

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DOIResolve DOI: http://doi.org/10.1007/s12010-013-0460-3
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TypeArticle
Journal titleApplied Biochemistry and Biotechnology
ISSN0273-2289
Volume172
Issue1
Pages7386; # of pages: 14
SubjectFungal genomes; Amylase; Glucoamylase; Cold starch hydrolysis; Genome mining; Triticale; Potato starch; Pichia pastoris; Biocatalysts; Biorefinery
AbstractMining fungal genomes for glucoamylase and α-amylase encoding sequences led to the selection of 23 candidates, two of which (designated TSgam-2 and NFamy-2) were advanced to testing for cooked or raw starch hydrolysis. TSgam-2 is a 66-kDa glucoamylase recombinantly produced in Pichia pastoris and originally derived for Talaromyces stipitatus. When harvested in a 20-L bioreactor at high cell density (OD600 > 200), the secreted TSgam-2 enzyme activity from P. pastoris strain GS115 reached 800 U/mL. In a 6-L working volume of a 10-L fermentation, the TSgam-2 protein yield was estimated to be ∼8 g with a specific activity of 360 U/mg. In contrast, the highest activity of NFamy-2, a 70-kDa α-amylase originally derived from Neosartorya fischeri, and expressed in P. pastoris KM71 only reached 8 U/mL. Both proteins were purified and characterized in terms of pH and temperature optima, kinetic parameters, and thermostability. TSgam-2 was more thermostable than NFamy-2 with a respective half-life (t1/2) of >300 min at 55 °C and >200 min at 40 °C. The kinetic parameters for raw starch adsorption of TSgam-2 and NFamy-2 were also determined. A combination of NFamy-2 and TSgam-2 hydrolyzed cooked potato and triticale starch into glucose with yields, 71-87 %, that are competitive with commercially available α-amylases. In the hydrolysis of raw starch, the best hydrolysis condition was seen with a sequential addition of 40 U of a thermostable Bacillus globigii amylase (BgAmy)/g starch at 80 °C for 16 h, and 40 U TSgam-2/g starch at 45 °C for 24 h. The glucose released was 8.7 g/10 g of triticale starch and 7.9 g/10 g of potato starch, representing 95 and 86 % of starch degradation rate, respectively. © 2013 Her Majesty the Queen in Right of Canada.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); Aquatic and Crop Resource Development
Peer reviewedYes
NRC number55600
NPARC number21269873
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Record identifierc86908ed-5c34-4e93-8e1d-a84f9eb53361
Record created2013-12-13
Record modified2016-05-09
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