Cloning, characterization, and functional analysis of the EPG1-2 gene: a new allele coding for an endopolygalacturonase in Kluyveromyces marxianus.
A new allele, the EPG1-2 gene, which codes for an endopolygalacturonase in Kluyveromyces marxianus CECT1043, has been cloned. The gene has 1086 bp and the protein 362 amino acids, one more than the previously described Epg1p. Epg1-2p shows a high degree of similarity with the polygalacturonases of fungi and yeasts. The sequences common to all of the polygalacturonases of prokaryotes, fungi, and higher plants are also conserved in Epg1-2p. The EPG1-2 gene has been expressed in Pichia pastoris , and, when fused with the signal peptide of the alpha-factor of Saccharomyces cerevisiae , the protein is properly secreted into the media. The recombinant enzyme does not appear to be fully glycosylated by P. pastoris or not glycosylated in the same manner as in K. marxianus, but it maintains the same optimum temperature (55 degrees C) and pH (4.5) and the same stability at different temperatures and pH values as the native enzyme, also showing the same hydrolytic behavior. The recombinant strain produces 200-fold more enzyme than the wild-type strain of K. marxianus, making it a yeast of potential industrial interest for the production of endopolygalacturonase for the food industry.
Sieiro
Carmen
C
Department of Functional Biology and Health Sciences, Microbiology Area, Faculty of Biology, University of Vigo, Vigo, Spain. mcsieiro@uvigo.es
Sestelo
Ana Belén F
AB
Villa
Tomás G
TG
D000483:Alleles; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006031:Glycosylation; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D011096:Polygalacturonase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D011096:Polygalacturonase