title: Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation.
abstract: Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98kDa 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 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 beta-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 98kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K(m,app)=0.16+/-0.02 mg/mL and k(cat,app)=79+/-10s(-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,app)=488+/-23mL/(mgs) 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 microM, respectively.
authors.affiliation: Department of Systems Biology, Søltofts Plads, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Malene Bech
authors.initials: MB
authors.lastname: Vester-Christensen
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.forename: Henrik
authors.initials: H
authors.lastname: Naested
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D016018:Least-Squares Analysis; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014170:Transformation, Genetic
chemical_list: D003505:Cyclodextrins; D005936:Glucans; D011994:Recombinant Proteins; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase