PRODUCTION OF RECOMBINANT HIGH pI-BARLEY α-GLUCOSIDASE

α-glucosidase of glycoside hydrolase family 31 plays in barley seeds a crucial role in embryonic development. The concerted action of α-glucosidase, β-amylase, α-amylase, and limit dextrinase is responsible for supply of glucose in the germinating seed as energy source for the embryo and growing plantlet [1].

Recently, expression and characterization of the recombinant full length, fully functional barley high pI α-glucosidase in Pichia pastoris has been achieved. To enable production of recombinant protein in mg amounts, a transformant harbouring a clone encoding the N-terminally hexa histidine tagged recombinant form of the enzyme was propagated using a high cell-density fermentation procedure.

This system resulted in successful expression under the highly sensitive methanol utilization phase conducting the fermentation process using a BiostatB 5 L reactor. The recombinant high pI α-glucosidase was purified in a yield of 42 mg from the culture (3.5 liter) by a four step purification procedure.

Remarkably, the purified enzyme exhibited slightly higher molecular mass of 100 kDa in SDS-PAGE than 92 kDa calculated from the primary structure. This difference in apparent molecular mass suggested glycosylation of the recombinant α-glucosidase. The enzyme activity was highly stable during the 5 day long fermentation.

Characterisation of the enzymatic properties confirmed the specific activity actually to be superior to that of the native enzyme purified from malt [2]. The kinetic parameters Km, Vmax and kcat for hydrolysis of maltose were 1.7 mM, 139 nM s-1 and 85 s-1 respectively. The presented data illustrate the first successful production of enzymatically active full length recombinant high pI barley α-glucosidase [2].

Further characterisation of the enzyme specificity is ongoing and positions for mutational analysis are identified as guided by first three-dimensional structure solved of a member of GH 31 [3] and multiple sequence alignment. This project is funded under the 5th Framework Programme of the European Commission, Contract Reference QLRT-2000-02400, "New Products from Starch-Derived 1,5-Anhydro-D-Fructose (NEPSA)" [1] MacGregor, A.W.; Sissons, M.J. (1994) Journal of Cereal Science 19, 161-169. [2] Frandsen, T.P.; Lok, F.; Mirgorodskaya, E. (2000) Plant Physiology 123, 275-286. [3] Lovering, A.L.; Lee, S.S.; Kim Y.W.; Withers SG, Strynadka, N.C. (2004) J.

Biol. Chem. [Epub ahead of print]