Cloning and characterization of industrially important alpha-galactosidase genes from the human pathogen aspergillus fumigatus

Söyler, U. Betül
In this study, molecular cloning studies were performed on the a-galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable a-galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results were obtained from these screenings. However, the DNA probes used were shown to be homologous to the a-galactosidase gene (agl1) of Trichoderma reesei. After the completion of the genome project of A. fumigatus, regions with homology to a-galactosidase genes were searched on the genome of A. fumigatus. PCR-based cloning studies were performed by designing specific primers for these regions. Two a-galactosidase genes, namely aglA and aglB were amplified with these specific primers, sequenced, and ligated to vector pUC19. The recombinant plasmid was then used to transform E. coli XL1 Blue MRF̕ cells. aglB gene consists of an open reading frame of 1684 bp containing six introns. The gene encodes a protein of 447 amino acids with a signal sequence of 22 amino acids and four N-glycosylation sites. aglA gene has an open reading frame of 1599 bp without introns. The gene encodes a protein of 532 amino acids with a putative signal sequence of 21 amino acids and four putative N-glycosylation sites. Cloning of a-galactosidase genes represents a first step towards the high level expression of these enzymes in a GRAS host.