title: Structural characterization of the thermally tolerant pectin methylesterase purified from citrus sinensis fruit and its gene sequence.
abstract: Despite the longstanding importance of the thermally tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, the unequivocal identification of the protein and its corresponding gene has remained elusive. TT-PME was purified from sweet orange [ Citrus sinensis (L.) Osbeck] finisher pulp (8.0 mg/1.3 kg tissue) with an improved purification scheme that provided 20-fold increased enzyme yield over previous results. Structural characterization of electrophoretically pure TT-PME by MALDI-TOF MS determined molecular masses of approximately 47900 and 53000 Da for two principal glycoisoforms. De novo sequences generated from tryptic peptides by MALDI-TOF/TOF MS matched multiple anonymous Citrus EST cDNA accessions. The complete tt-pme cDNA (1710 base pair) was cloned from a fruit mRNA library using RT- and RLM-RACE PCR. Citrus TT-PME is a novel isoform that showed higher sequence identity with the multiply glycosylated kiwifruit PME than to previously described Citrus thermally labile PME isoforms.
authors: Eastern Regional Research Center, Agricultural Research Service , U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, United States.
authors: Brett J
authors: BJ
authors: Savary
authors: Prasanna
authors: P
authors: Vasu
authors: Randall G
authors: RG
authors: Cameron
authors: T Gregory
authors: TG
authors: McCollum
authors: Alberto
authors: A
authors: Nuñez
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D032084:Citrus sinensis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase