Structural Characterization of the Thermally Tolerant Pectin Methylesterase Purified from Citrus sinensis Fruit and Its Gene Sequence

• Published in: Journal of agricultural and food chemistry Vol. 61; no. 51; pp. 12711 - 12719
• Main Authors: Savary, Brett J, Vasu, Prasanna, Cameron, Randall G, McCollum, T. Gregory, Nuñez, Alberto
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 26-12-2013
• Subjects: Amino Acid Sequence; Biological and medical sciences; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - isolation & purification; Carboxylic Ester Hydrolases - metabolism; citrus juices; Citrus sinensis; Citrus sinensis - chemistry; Citrus sinensis - enzymology; Citrus sinensis - genetics; complementary DNA; Enzyme Stability; expressed sequence tags; Fruit - enzymology; Fruit - genetics; Fundamental and applied biological sciences. Psychology; genes; glycosylation; Hydrogen-Ion Concentration; kiwifruit; messenger RNA; Molecular Sequence Data; Molecular Weight; nucleotide sequences; oranges; pectinesterase; peptides; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; polymerase chain reaction; product quality; pulp; sequence analysis; fruit cell wall enzyme; MALDI-TOF MS; orange juice cloud stability; Citrus sinensis; purification; thermostable pectinesterase; PCR cloning; Rutaceae; Esterases; Matrix assisted laser desorption ionization; Cell wall; Thermal stability; Characterization; Dicotyledones; Angiospermae; Pectinesterase; Purification; Fruit; Nucleotide sequence; Enzyme; Tolerance; Carboxylic ester hydrolases; Polymerase chain reaction; Orange juice; Hydrolases; Spermatophyta
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf403914u

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.