title: Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization.
abstract: Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a 'smooth' region and a 'hairy' region. The 'smooth' region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The 'hairy' region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40% the viscosity of pectin with a degree of esterification ≥85%. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8% esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
authors.forename: Claudio
authors.initials: C
authors.lastname: Pérez-Fuentes
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
authors.forename: María
authors.initials: M
authors.lastname: Cristina Ravanal
authors.affiliation: Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile. Electronic address: jeyzaguirre@unab.cl.
authors.forename: Jaime
authors.initials: J
authors.lastname: Eyzaguirre
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011133:Polysaccharide-Lyases; C027349:pectin lyase
keywords: Acidic pectin lyases; Biotechnological applications of pectin lyase; Gene sequencing; Homogalacturonan degradation; Polysaccharide lyase family 1