Production of enzymatically active recombinant full-length barley high pI α-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Recombinant barley high pI α-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 α-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 α-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 × s−1, and 85 s−1 using maltose as substrate. This work presents the first production of fully active recombinant α-glucosidase of glycoside hydrolase family 31 from higher plants.