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

Abstract

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.

The Role of α-Glucosidase in Germinating Barley Grains

“…The major forms of a-glucosidase found in germinating cereal grains are exo-acting enzymes belonging to glycoside hydrolase family 31 (Frandsen and Svensson, 1998;Naested et al, 2006). The conventional view is that these enzymes act on maltose and other short maltooligosaccharides produced by amylases and limit dextrinase, converting them to Glc (Dunn, 1974;Manners, 1974;Osman, 2002).…”

New Insight into Structure/Function Relationships in Plant .ALPHA.-Amylase Family GH13 Members

“…20,21) Very recently, however, successful heterologous production of barley LD was achieved by a strategy involving secretory expression of this large multi domain enzyme during high cell density fermentation of transformed Pichia pastoris. 22,33) A fed batch fermentation enabled efficient production of LD and 34 mg electrophoretically homogenous LD was purified from the culture supernatant from 2 L starting batch by affinity chromatography on β CD Sepharose and Sephadex G200 gel filtration resulting in a yield of 85%. The activity (kcat,app Km,app) was 3.5 fold higher than of LD purified from germinating barley seeds.…”

Purificação e caracterização bioquímica da α-glicosidase termoestável produzida por Thermoascus aurantiacus CBMAI 756

“…niger, M. javanicus. (HENRISSAT, 1991;HENRISSAT;BAIROCH, 1993;CHIBA, 1997;FRANDSEN;SVENSSON, 1998;NAESTED et al;.…”

“…A α-glicosidase nativa da cevada apresenta massa molar de 92 kDa, enquanto a α-glicosidase recombinate da cevada expressa em Pichia pastoris apresentou massa molar de 100 kDa (NAESTED et al, 2006).…”

“…Além disso, na literatura é relatado que as Į-glicosidases de Aspergillus pertencem à família 31 e atuam mais eficientemente sobre a maltose do que sobre dextrinas mais longas (NAESTED et al;FRANDSEN;SVENSSON, 1998;SUGIMOTO et al, 2003;OKUYAMA et al, 2005), o mesmo foi concluído para a Į-glicosidase purificada de T. aurantiacus CBMAI 756.…”