Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae.
The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.
Kimura
I
I
Kagawa Prefectural Fermentation and Food Experimental Station, 1351-1 Nouma, Uchinomi-cho, Shouzu-gun, Kagawa 761-4421, Japan.
Yoshioka
N
N
Kimura
Y
Y
Tajima
S
S