Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation.
Protein Expr Purif
; 69(1): 112-9, 2010 Jan.
Article
em En
| MEDLINE
| ID: mdl-19733243
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
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Hordeum
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Biotecnologia
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Fermentação
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Glicosídeo Hidrolases
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Dinamarca