title: Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum.
abstract: 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.
authors: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
authors: Marin
authors: M
authors: Iwai
authors: Hiroyuki
authors: H
authors: Yamada
authors: Takeshi
authors: T
authors: Ikemoto
authors: Shotaro
authors: S
authors: Matsumoto
authors: Daisuke
authors: D
authors: Fujiwara
authors: Shigeo
authors: S
authors: Takenaka
authors: Tatsuji
authors: T
authors: Sakamoto
mesh_terms: D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D010408:Penicillium chrysogenum / Q000201:enzymology*; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011133:Polysaccharide-Lyases