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.
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.
Naested
Henrik
H
Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
Kramhøft
Birte
B
Lok
Finn
F
Bojsen
Kirsten
K
Yu
Shukun
S
Svensson
Birte
B
D002846:Chromatography, Affinity; D017931:DNA Primers; D005285:Fermentation; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014462:Ultrafiltration; D000520:alpha-Glucosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; D000520:alpha-Glucosidases