Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori.
Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69 +/- kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the beta-(2-->1)-fructan (inulin) and beta-(2-->6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants K(m) and V(max) in the hydrolysis of inulin were 0.003 +/- 0.0001 mM and 175 +/- 5 micromol.min(-1).mg(-1). The same parameters in the hydrolysis of levan were 2.08 +/- 0.04 mg/ml and 1.2 +/- 0.02 micromol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P2(1)2(1)2(1) with cell parameters a=64.726 A (1A=0.1 nm), b=82.041 A and c=136.075 A. Crystals diffracted beyond 1.54 A, and useful X-ray data were collected to a resolution of 1.73 A.
Arand
Michael
M
Institute of Toxicology, University of Mainz, Mainz, Germany.
Golubev
Alexander M
AM
Neto
J R Brandao
JR
Polikarpov
Igor
I
Wattiez
R
R
Korneeva
Olga S
OS
Eneyskaya
Elena V
EV
Kulminskaya
Anna A
AA
Shabalin
Konstantin A
KA
Shishliannikov
Sergei M
SM
Chepurnaya
Olga V
OV
Neustroev
Kirill N
KN
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D018076:DNA, Complementary; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018076:DNA, Complementary; D006026:Glycoside Hydrolases; C023086:inulinase