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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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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.

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Acknowledgments

This work was supported by funding through the Canadian Triticale Biorefinery Initiative (CTBI) of the Agricultural Bioproducts Innovation Program of Agriculture and Agri-Food Canada (AAFC) and the National Bioproducts Program (NBP) of NRCC-AFCC-NRCan. Special thanks go to Francois Eudes and Connie Phillips for their encouragement and support. We thank Stephane Deschamp for his expert help in analytical analyses. This work is dedicated to the memory of Mo (Hamid Mohamed), a visionary and driving force behind the creation of the Canadian Biomass Innovation Network (CBIN) of which P.C.K. Lau is most grateful to have served.

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Authors and Affiliations

  1. Aquatic and Crop Resource Development, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, H4P 2R2, Canada

    Zhizhuang Xiao, Meiqun Wu, Stephan Grosse, Manon Beauchemin, Michelle Lévesque & Peter C. K. Lau

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  1. Zhizhuang Xiao
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  2. Meiqun Wu
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  3. Stephan Grosse
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  4. Manon Beauchemin
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  5. Michelle Lévesque
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  6. Peter C. K. Lau
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Correspondence to Peter C. K. Lau.

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Xiao, Z., Wu, M., Grosse, S. et al. 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. Appl Biochem Biotechnol 172, 73–86 (2014). https://doi.org/10.1007/s12010-013-0460-3

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