Bioseparation of Alpha-Amylase by Forming Insoluble Complexes with Polyacrylate From a Culture of Aspergillus Oryzae Grown in Agricultural Wastes

PORFIRI, MA. CECIIA; BRAIA, MAURICIO; LOUREIRO, DANA; FARRUGGIA, BEATRIZ; ROMANINI, DIANA

Puerto Vallarta

Congreso; 16th International Conference on BioPartitioning and Purification, BPP; 2011

Alpha-amylases are members of family 13 in the classification of glycoside hydrolases (according to Henrissat [1]). The enzyme synthesized from Aspergillus oryzae is an extracellular endo-acting hydrolase that gives large oligosaccharides as products of starch degradation because of scission of internal alpha-1,4-linkages and its three dimensional structure has been well investigated by X-Ray Crystallography. Precipitation of insoluble complexes between this protein and polyacrylic acid (PAA) of different molecular weights was previously studied as a strategy of alpha-amylase concentration and purification [2]. In it we conclude that the enzyme form insoluble complexes with polyacrylic acid at pH under 5.00 (PAA 100,000 Da) and 4.00 (PAA 240,000 Da) with a molar ratio PAA/alpha-amylase 1/52 and 1/ 154, respectively. Taking into account all these results and verifying that PAA did not negatively affect enzyme activity we designed a methodology of protein separation by precipitating the enzyme with excess of polyelectrolyte at pH 3.00 and redissolving it at pH 6.00 (around mayor stability of the enzyme). The yields obtained showed high tendency of the enzyme to precipitate with a 73.79% and 64.26% for PAA 240,000 Da and 100,000 Da, respectively, envisioning a potential method for alpha-amylase concentration and purification [2]. The aim of this work was isolation of alpha-amylase from a culture of Aspergillus oryzae grown at the expenses of residues of wheat processing as starch source. To obtain the enzyme a fungal stain of A. Oryzae NRRL 695 was propagated in Potato-Glucose-Agar medium at 30ºC. The plates were grown for five days and spores were harvested by adding 15 ml of distilled water. These were counted by using the Thomas cell to inoculate an appropriate volume of the master spore suspension in a minimum medium containing wheat´s residues as sole carbohydrates source. This crop was kept on rotary shaker for four days at 30ºC, after which it was filtrated and supernatant used as alpha-amylase source. Electrophoresis in SDS-Page of this sample showed predominant band around 55kD corresponding to alpha-amylase. Precipitation of alpha-amylase by forming insoluble complexes with PAA of both molecular weights from this culture filtrated yielded a 73.52% and 70.35% with PAA 100,000 Da and 240,000 Da, respectively. In this work we also study the effect of this polyelectrolytes on the stability of the enzyme at the pH chosen for precipitation (3.00), as well as the stability of the precipitates formed after applied the methodology designed for concentration. The polymers showed a stabilizing effect on the activity of the alpha-amylase at pH 3.00, intensified when the complex are in the precipitated form before been redissolved. Our results show that the precipitation with PAA may be considered as a suitable strategy for obtaining and concentrating alpha-amylase from Aspergillus oryzae grown in wastes.