A family 51 alpha-l-arabinofuranosidase from Penicillium purpurogenum: purification, properties and amino acid sequence.
The soft rot fungus Penicillium purpurogenum secretes a wide variety of xylanolytic enzymes to the medium, among them three alpha-l-arabinofuranosidases. This work refers to arabinofuranosidase 2 (ABF 2). This enzyme was purified to homogeneity and characterized; it is a glycosylated monomer with a molecular weight of 70 000 and an isoelectric point of 5.3. When assayed with p-nitrophenyl alpha-l-arabinofuranoside (pNPAra) the enzyme followed Michaelis-Menten kinetics with a K(M) of 0.098mm. The optimum pH is 5 and the optimal temperature 60 degrees C. ABF 2 showed weak activity on natural polymeric substrates, such as sugar beet arabinan, debranched arabinan, and arabinoxylan. These results, together with its low K(M) (pNPAra) and its activity towards short arabinooligosaccharides, suggest that the enzyme belongs to the exo alpha-l-arabinosyl hydrolases not active on polymers. The abf2 gene and its cDNA were sequenced, and the gene was found to possess seven introns. The mature protein is 618 amino acids long with a calculated molecular weight of 67 212. Amino acid sequence alignments show that the enzyme belongs to family 51 of the glycosyl hydrolases, although it differs in some properties from other enzymes of this family.
Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
Macarena
M
Fritz
María Cristina
MC
Ravanal
Christophe
C
Braet
Jaime
J
Eyzaguirre
D000595:Amino Acid Sequence; D001483:Base Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase