Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus.
A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.
Ghazi
Iraj
I
Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquimica, CSIC, Cantoblanco, 28049 Madrid, Spain.
Fernandez-Arrojo
Lucia
L
Garcia-Arellano
Humberto
H
Ferrer
Manuel
M
Ballesteros
Antonio
A
Plou
Francisco J
FJ
D001230:Aspergillus / Q000201:enzymology*; D005503:Food Additives / Q000737:chemistry; D006602:Hexosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D001426:Bacterial Proteins
D005503:Food Additives; D006602:Hexosyltransferases; D013395:Sucrose; C021451:inulosucrase; D001426:Bacterial Proteins