title: Identification of a novel Penicillium chrysogenum rhamnogalacturonan rhamnohydrolase and the first report of a rhamnogalacturonan rhamnohydrolase gene.
abstract: Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of β-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: s_wake_zo_1616@me.com.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: General Research Laboratory, Ozeki Corporation, 4-9 Imazu Dezaike-cho, Nishinomiya-shi, Hyogo 663-8227, Japan. Electronic address: hiroyuki.yamada@ozeki.co.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamada
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: swc02023@edu.osakafu-u.ac.jp.
authors.forename: Yuika
authors.initials: Y
authors.lastname: Kunishige
authors.affiliation: Division of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan. Electronic address: takenaka@vet.osakafu-u.ac.jp.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Takenaka
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: mami@biochem.osakafu-u.ac.jp.
authors.forename: Masami
authors.initials: M
authors.lastname: Nakazawa
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: mueda@biochem.osakafu-u.ac.jp.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueda
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan. Electronic address: sakamoto@biochem.osakafu-u.ac.jp.
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Sakamoto
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010368:Pectins / Q000737:chemistry / Q000378:metabolism; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D010368:Pectins; D006026:Glycoside Hydrolases; C090358:rhamnogalacturonan rhamnohydrolase
keywords: Gene cloning; Glycoside hydrolase family 78; Penicillium chrysogenum; Rhamnogalacturonan rhamnohydrolase; α-l-Rhamnosidase