title: Expression and enzymatic properties of rice (Oryza sativa L.) monolignol β-glucosidases.
abstract: Monolignol glucosides and their β-glucosidases are found in monocots, but their biological roles are unclear. Phylogenetic analysis of rice (Oryza sativa L.) glycoside hydrolase family GH1 β-glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol β-glucosidases. An optimized Os4BGlu16 cDNA and cloned Os4BGlu18 cDNA were used to express fusion proteins with His6 tags in Pichia pastoris and Escherichia coli, respectively. The secreted Os4BGlu16 fusion protein was purified from media by immobilized metal affinity chromatography (IMAC), while Os4BGlu18 was extracted from E. coli cells and purified by anion exchange chromatography, hydrophobic interaction chromatography and IMAC. Os4BGlu16 and Os4BGlu18 hydrolyzed the monolignol glucosides coniferin (kcat/KM, 21.6mM(-1)s(-1) for Os4BGlu16 and for Os4BGlu18) and syringin (kcat/KM, 22.8mM(-1)s(-1) for Os4BGlu16 and 24.0mM(-1)s(-1) for Os4BGlu18) with much higher catalytic efficiencies than other substrates. In quantitative RT-PCR, highest Os4BGlu14 mRNA levels were detected in endosperm, embryo, lemma, panicle and pollen. Os4BGlu16 was detected highest in leaf from 4 to 10 weeks, endosperm and lemma, while Os4BGlu18 mRNA was most abundant in vegetative stage from 1 week to 4 weeks, pollen and lemma. These data suggest a role for Os4BGlu16 and Os4BGlu18 monolignol β-glucosidases in both vegetative and reproductive rice tissues.
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Supaporn
authors.initials: S
authors.lastname: Baiya
authors.affiliation: Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center for Scientific and Technological Equipment, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Hua
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Watsamon
authors.initials: W
authors.lastname: Ekkhara
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand. Electronic address: cairns@sut.ac.th.
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D000595:Amino Acid Sequence; D044602:Cellulases / Q000235:genetics / Q000378:metabolism*; D002934:Cinnamates / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli; D035264:Flowers / Q000201:enzymology; D017343:Genes, Plant; D005960:Glucosides / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D010666:Phenylpropionates / Q000378:metabolism*; D010802:Phylogeny; D010843:Pichia; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018514:Plant Structures / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002934:Cinnamates; D018076:DNA, Complementary; D005960:Glucosides; D010666:Phenylpropionates; D010940:Plant Proteins; D012333:RNA, Messenger; D008031:Lignin; D044602:Cellulases; D001617:beta-Glucosidase; C028305:syringin; C016316:coniferin
keywords: Expression; Glycosides; Monolignols; Rice (Oryza sativa L.); β-Glucosidase