Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.
An enzyme with alpha-galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of alpha-galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a lambda EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa alpha-galactosidase is absent from a strain with a disruption of the aglA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa alpha-galactosidase A represents a minor extracellular alpha-galactosidase activity in A. niger.
den Herder
I F
IF
TNO Medical Biological Laboratory, AA Rijswijk, The Netherlands.
Rosell
A M
AM
van Zuilen
C M
CM
Punt
P J
PJ
van den Hondel
C A
CA
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D015345:Oligonucleotide Probes; D000519:alpha-Galactosidase