title: Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation.
abstract: We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. 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 using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.
authors: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA.
authors: Prasanna
authors: P
authors: Vasu
authors: Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA; College of Agriculture and Technology, Arkansas State University, Jonesboro, AR, USA. Electronic address: bsavary@astate.edu.
authors: Brett J
authors: BJ
authors: Savary
authors: US Horticultural Research Laboratory, Agricultural Research Service, US Department of Agriculture, Ft. Pierce, FL, USA.
authors: Randall G
authors: RG
authors: Cameron
mesh_terms: D029441:Carica / Q000737:chemistry*; D002845:Chromatography / Q000379:methods*; D005638:Fruit / Q000737:chemistry*; D013058:Mass Spectrometry; D010206:Papain / Q000737:chemistry*; D010368:Pectins / Q000737:chemistry*
chemical_list: D010368:Pectins; D010206:Papain
keywords: Biochemical characterization; Carica papaya; Chromatography purification; Fruit latex; Liquipanol; MALDI-TOF-MS; Pectin methylesterase