Technical Abstract: We purified a single stable pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation, which is isolated from Carica papaya (L.) fruit latex. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography with the kiwi PME inhibitor protein to obtain an electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1,200 U/mg. The molecular weight determined by MALDI-TOF MS was 35,135, and peptide mass fingerprinting by trypsin digestion provided a large set of peptide ions for identifying the protein. The CpL-PME required salt for activity; it showed a broad pH range of 6-9 and moderate thermostability (optimum ca. 70 degrees C); and it readily converted a methylated citrus pectin to high calcium sensitivity with -6% deesterfication. These properties support further research to apply CpL-PME to tailor pectin nanostructure.