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

作者：Savary Brett J^*; Vasu Prasanna; Cameron Randall G; McCollum T Gregory; Nunez Alberto

来源：Journal of Agricultural and Food Chemistry, 2013, 61(51): 12711-12719.

DOI：10.1021/jf403914u

• Citrus sinensis
• fruit cell wall enzyme
• orange juice cloud stability
• MALDI-TOF MS
• PCR cloning
• purification
• thermostable pectin esterase

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.

• 出版日期2013-12-25