title: Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae.
abstract: The endoglucanase coded by celA (GenBank Access No. Y12512) from Paenibacillus barcinonensis, an enzyme with good characteristics for application on paper manufacture from agricultural fibers, was expressed in Saccharomyces cerevisiae by using different domains of the cell wall protein Pir4 as translational fusion partners, to achieve either secretion or cell wall retention of the recombinant enzyme. Given the presence of five potential N-glycosylation sites in the amino acid sequence coded by celA, the effect of glycosylation on the enzymatic activity of the recombinant enzyme was investigated by expressing the recombinant fusion proteins in both, standard and glycosylation-deficient strains of S. cerevisiae. Correct targeting of the recombinant fusion proteins was confirmed by Western immunoblot using Pir-specific antibodies, while enzymatic activity on carboxymethyl cellulose was demonstrated on plate assays, zymographic analysis and colorimetric assays. Hyperglycosylation of the enzyme when expressed in the standard strain of S. cerevisiae did not affect activity, and values of 1.2 U/ml were obtained in growth medium supernatants in ordinary batch cultures after 24 h. These values compare quite favorably with those described for other recombinant endoglucanases expressed in S. cerevisiae. This is one of the few reports describing the expression of Bacillus cellulases in S. cerevisiae, since yeast expressed recombinant cellulases have been mostly of fungal origin. It is also the first report of the yeast expression of this particular endoglucanase.
authors.affiliation: Unidad de Microbiología, Facultad de Farmacia, Universidad De Valencia, Avda. Vicente Andrés Estelles s/n, 46100, Burjassot, Valencia, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Mormeneo
authors.forename: Fi Javier
authors.initials: FJ
authors.lastname: Pastor
authors.forename: Jesús
authors.initials: J
authors.lastname: Zueco
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D056507:Paenibacillus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C482291:CIS3 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C074775:CelA endoglucanase; D002480:Cellulase