Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger.
Secretion of endo-1,5-alpha-L-arabinase A (ABN A) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing L-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA+ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-transformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.
Flipphi
M J
MJ
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Panneman
H
H
van der Veen
P
P
Visser
J
J
de Graaff
L H
LH
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data
C074451:Endo-arabinase; D006026:Glycoside Hydrolases