Purified acid-stable alpha-amylase from fungal origin

#The present invention relates to a purified acid-stable fungal alpha-amylase and to a process for obtaining said purified alpha-amylase. Furthermore the use of the purified alpha-amylase in free and immobilized form for obtaining specific glucose syrups is demonstrated.

#Acid-stable alpha-amylases from fungal origin have been discovered a long time ago. For example, the acid-stable alpha-amylase from A. niger has been known for over 30 years (Y. Minoda, K. Yamada, Agr. Biol. Chem., 27(11), 806-811(1963)) and has been thoroughly characterised by Minoda, Yamada and co-workers. The same authors showed the stabilisation of the enzyme activity in the presence of Ca.sup.2+. The presence of an acid-unstable alpha-amylase in the Aspergillus niger preparations was disclosed by the same authors. The acid-stable alpha-amylase enzyme has a pH optimum between 3 and 4 and the optimal temperature is in the range of 70 to 75.degree. C.

#Several procedures have been developed for obtaining acid-stable alpha-amylase in purified form. One method (Minoda and Yamada, cited above) uses fractional precipitation with ammonium sulfate, rivanol and acetone to obtain the enzyme in crystalline form. It was found that this crystalline alpha-amylase was contaminated with an acid-unstable alpha-amylase, whereas glucoamylase and transglucosidase were found to be removed. The acid-unstable alpha-amylase could subsequently be removed by treatment of the alpha-amylase mixture at acidic pH (pH 2.5) and 37.degree. C., followed by fractionation. A further purification was performed by recrystallisation with acetone and gelfiltration with Sephadex G-50.

#Determination of enzyme activities is by standard methods.

#Generally as provided by the enzyme supplier.

#Glucoamylase activity is determined by hydrolysis of para-nitrophenyl-alpha-D-glucopyranoside (K. A. Holm, Analyst, 3, 927-929 (1986)). Basically, the procedure is a s follows. An enzyme preparation is allowed to react under standard conditions (pH 4.2, 55.degree. C.) with para-nitrophenyl-alpha-D-glucopyranoside. In the presence of a glucoamylase this substrate is degraded to glucose and para-nitrophenolate which has a yellow color. The amount of para-nitrophenolate produced is determined colorimetrically. Enzyme activity is calculated from a standard curve expressing the relation between the concentration of para-nitrophenolate and the absorbance.