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Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.
Naested, Henrik; Kramhøft, Birte; Lok, Finn; Bojsen, Kirsten; Yu, Shukun; Svensson, Birte.
Afiliação
  • Naested H; Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
Protein Expr Purif ; 46(1): 56-63, 2006 Mar.
Article em En | MEDLINE | ID: mdl-16343940
Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Pichia / Hordeum / Alfa-Glucosidases Idioma: En Revista: Protein Expr Purif Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2006 Tipo de documento: Article País de afiliação: Dinamarca País de publicação: Estados Unidos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Pichia / Hordeum / Alfa-Glucosidases Idioma: En Revista: Protein Expr Purif Assunto da revista: BIOLOGIA MOLECULAR Ano de publicação: 2006 Tipo de documento: Article País de afiliação: Dinamarca País de publicação: Estados Unidos