title: Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan.
abstract: In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.
authors.affiliation: INRA, Unité de Recherche Biopolymères, Interactions, Assemblages, BP 71627, 44316 Nantes Cedex 03, France.
authors.forename: J
authors.initials: J
authors.lastname: Normand
authors.forename: E
authors.initials: E
authors.lastname: Bonnin
authors.forename: P
authors.initials: P
authors.lastname: Delavault
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D010368:Pectins; D006026:Glycoside Hydrolases; C067959:rhamnogalacturonase A