Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme
Autor: | Torben Halkier, Maria Hockauf, Lene Venke Kofod, Lene Nonboe Andersen, Sakari Kauppinen, Stephan Christgau, Kurt Dörreich, Henrik Dalbøge |
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Jazyk: | angličtina |
Rok vydání: | 1996 |
Předmět: |
food.ingredient
DNA Complementary Pectin Molecular Sequence Data Carbohydrates Saccharomyces cerevisiae Biochemistry Esterase food Aspergillus oryzae Polysaccharides Amino Acid Sequence Cloning Molecular Molecular Biology Peptide sequence Chromatography High Pressure Liquid Glycoproteins biology Base Sequence Sequence Homology Amino Acid Aspergillus niger Aspergillus aculeatus Cell Biology biology.organism_classification Yeast Recombinant Proteins Kinetics Aspergillus Electrophoresis Polyacrylamide Gel Heterologous expression Carboxylic Ester Hydrolases Research Article |
Popis: | 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 °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. |
Databáze: | OpenAIRE |
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