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| Titel:
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Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation |
| Type:
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Journal articleJournal article |
| Person(er):
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Technical University of Denmark
Technical University of Denmark
Email:
Technical University of Denmark
Technical University of Denmark
Email:
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| Uddrag:
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Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase I promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LID (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K-m,K-app = 0.16 +/- 0.02 mg/mL and k(cat,app) = 79 +/- 10 s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively. |
| Publiceret:
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in journal: Protein Expression and Purification (ISSN: 1046-5928) (DOI: http://dx.doi.org/10.1016/j.pep.2009.08.016), vol: 69, issue: 1, pages: 112-119, 2010 |
| DOI:
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