title: 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.
abstract: Mining 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.
authors.affiliation: Aquatic and Crop Resource Development, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, H4P 2R2, Canada.
authors.forename: Zhizhuang
authors.initials: Z
authors.lastname: Xiao
authors.forename: Meiqun
authors.initials: M
authors.lastname: Wu
authors.forename: Stephan
authors.initials: S
authors.lastname: Grosse
authors.forename: Manon
authors.initials: M
authors.lastname: Beauchemin
authors.forename: Michelle
authors.initials: M
authors.lastname: Lévesque
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
mesh_terms: D000327:Adsorption; D057225:Data Mining; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D016681:Genome, Fungal / Q000235:genetics*; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010843:Pichia / Q000235:genetics; D017421:Sequence Analysis; D013213:Starch / Q000378:metabolism*; D032901:Talaromyces / Q000201:enzymology / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008670:Metals; D013213:Starch; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase