Structural Characterization of the Thermally Tolerant Pectin Methylesterase Purified from Citrus sinensis Fruit and Its Gene Sequence
peer-reviewed · Journal of Agricultural and Food Chemistry · 2013
| Date | 2013-12-26 |
| Type | peer-reviewed |
| Venue | Journal of Agricultural and Food Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/jf403914u |
| Citations (OpenAlex) | 12 |
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.
Methods and tools
- Citrus thermally tolerant PME: Purifies thermally tolerant pectin methylesterase from orange and uses MALDI-TOF/TOF de novo sequences to match ESTs and clone its gene.