Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue.
A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.
Cameron
Randall G
RG
Citrus and Subtropical Products Laboratory, South Atlantic Area, Agricultural Research Service, U.S. Department of Agriculture, 600 Avenue South, N.W., Winter Haven, Florida 33881, USA. rcameron@citrus.usda.gov
Savary
Brett J
BJ
Hotchkiss
Arland T
AT
Fishman
Marshall L
ML
Chau
Hoa K
HK
Baker
Robert A
RA
Grohmann
Karel
K
D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D032084:Citrus sinensis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010368:Pectins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*
D007527:Isoenzymes; D012965:Sodium Chloride; D010368:Pectins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase