Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum

Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Molecular biotechnology 2015-06, Vol.57 (6), p.539-548
Hauptverfasser: Iwai, Marin, Yamada, Hiroyuki, Ikemoto, Takeshi, Matsumoto, Shotaro, Fujiwara, Daisuke, Takenaka, Shigeo, Sakamoto, Tatsuji
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-015-9847-4