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 - Naested, Henrik
AU - Svensson, Birte
PY - 2010/1
Y1 - 2010/1
N2 - Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-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 α-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 α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (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 Km,app = 0.16 ± 0.02 mg/mL and kcat,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, kcat,app/Km,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with Kd of 27.2, 0.70, and 34.7 μM, respectively.
AB - Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-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 α-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 α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (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 Km,app = 0.16 ± 0.02 mg/mL and kcat,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, kcat,app/Km,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with Kd of 27.2, 0.70, and 34.7 μM, respectively.
KW - Amino Acid Sequence
KW - Base Sequence
KW - Bioreactors
KW - Biotechnology
KW - Cloning, Molecular
KW - Cyclodextrins
KW - Electrophoresis, Polyacrylamide Gel
KW - Fermentation
KW - Glucans
KW - Glycoside Hydrolases
KW - Hordeum
KW - Least-Squares Analysis
KW - Molecular Sequence Data
KW - Pichia
KW - Plasmids
KW - Recombinant Proteins
KW - Subcellular Fractions
KW - Surface Plasmon Resonance
KW - Transformation, Genetic
U2 - 10.1016/j.pep.2009.08.016
DO - 10.1016/j.pep.2009.08.016
M3 - Journal article
C2 - 19733243
SN - 1046-5928
VL - 69
SP - 112
EP - 119
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -