title: Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme.
abstract: Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.
authors.affiliation: Screening Biotechnology, Bagsvaerd, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Christgau
authors.forename: L V
authors.initials: LV
authors.lastname: Kofod
authors.forename: T
authors.initials: T
authors.lastname: Halkier
authors.forename: L N
authors.initials: LN
authors.lastname: Andersen
authors.forename: M
authors.initials: M
authors.lastname: Hockauf
authors.forename: K
authors.initials: K
authors.lastname: Dörreich
authors.forename: H
authors.initials: H
authors.lastname: Dalbøge
authors.forename: S
authors.initials: S
authors.lastname: Kauppinen
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development; D017386:Sequence Homology, Amino Acid
chemical_list: D002241:Carbohydrates; D018076:DNA, Complementary; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase