Abstract
| Original language | English |
|---|---|
| Journal | Protein Expression and Purification |
| Volume | 69 |
| Issue number | 1 |
| Pages (from-to) | 112-119 |
| ISSN | 1046-5928 |
| DOIs | |
| Publication status | Published - 2010 |
Keywords
- Pullulan hydrolysis
- High cell-density fermentation
- Cyclodextrin affinity
- Pichia pastoris
- Barley limit dextrinase
Cite this
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Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation. / Vester-Christensen, Malene Bech; Abou Hachem, Maher; Næsted, Henrik; Svensson, Birte.
In: Protein Expression and Purification, Vol. 69, No. 1, 2010, p. 112-119.Research output: Contribution to journal › Journal article › Research › peer-review
TY - JOUR
T1 - Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation
AU - Vester-Christensen, Malene Bech
AU - Abou Hachem, Maher
AU - Næsted, Henrik
AU - Svensson, Birte
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Pullulan hydrolysis
KW - High cell-density fermentation
KW - Cyclodextrin affinity
KW - Pichia pastoris
KW - Barley limit dextrinase
U2 - 10.1016/j.pep.2009.08.016
DO - 10.1016/j.pep.2009.08.016
M3 - Journal article
C2 - 19733243
VL - 69
SP - 112
EP - 119
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -