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Title:   Author  item
                                            Cameron, Randall

                                                         - *Randy*item
                                            Savary, Brettitem
                                            Hotchkiss, Arlanditem
                                            Fishman, Marshallitem
                                            Chau, Hoa

                                                         - *Rose*item
                                            BAKER, ROBERT - USDA, RETIREDitem
                                            GROHMANN, KAREL - USDA, RETIRED
  Submitted to: Journal of Agricultural and Food Chemistry
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 12/19/2002
                                    Publication  Date: 2/21/2003
                                    Citation: CAMERON, R.G., SAVARY, B.J., HOTCHKISS, A.T., FISHMAN, M.L., CHAU, H.K., BAKER, R.A., GROHMANN, K. SEPARATION AND CHARACTERIZATION OF A SALT-DEPENDENT PECTIN METHYLESTERASE  FROM CITRUS SINENSIS VAR. VALENCIA FRUIT TISSUE. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2003. v. 51(7). p. 2070-2075.Interpretive Summary: The appealing, flavorful cloud that characterizes most citrus juices is dependent on unmodified pectin.  If not properly treated, an enzyme naturally found in citrus juices will modify the pectin and cause the juice cloud to fall to the bottom of the container.  Pectin also is extracted from citrus fruit peel and used as a gelling agent for jams,  jellies and other confectionaries.  It is also used to stabilize acidic proteins present in dairy products such as yogurt.  Many of the functional properties of pectin depend on its chemical structure.  The enzyme in citrus juices that affects it's structure and properties is pectin methylesterase and its function is the removal of methyl groups from the pectin molecule.  There are four different forms of this enzyme in citrus fruit.  The one we have characterized is one that did not destabilize the juice cloud.  The other three forms of the enzyme did  cause the cloud to fall out of suspension.  We were able to show that this pectin methylesterase requires salt for activity, is not very active under acidic conditions and is unable to alter pectin structure such that the pectin falls out of solution. We also showed that this enzyme can produce stretches within a pectin molecule that are not methylated at neutral pH's, that may improve its function in some industrial applications.
                                Technical Abstract: A salt-dependent pectin methylesterase (PME) has been isolated from sweet orange (Citrus sinensis var. Valencia) fruit rag tissue.  It was separated from other PME's in this tissue by a combination of DEAE, heparin, CM-sepharose and gel filtration chromatography.  It is a Type II PME (salt dependent) and exhibits optimal activity between 0.1 - 0.2 M  NaCl at pH 7.5.  It has a pH optimum from 7.5 - 8.5 at 200 mM NaCl and 8.5 - 9.5 at 50 mM NaCl.  Endpoint DE values of 43.7% and 39.9% were obtained at pH 4.5 and 7.5 respectively.  The enzyme has an apparent molecular mass of 32.4 kDa by gel filtration chromatography and 33.5 kDa by denaturing electrophoresis.  It has a pI of 10.1 and exhibits only one activity band after IEF.  The enzyme has a Km of 0.0487 mg/mL**-1 and Vmax of 4.2378 nkatal/mg**-1 protein with 59% DE pectin at pH 7.5, 0.2 M NaCl.  De-blocking the N-terminus revealed a partial peptide composed of  SVTPNV.  The calcium-sensitive pectin ratio (CSPR) increased from 0.0450 +/ 0.0105 to 0.8290 +/ 0.0332 after de-esterification with Peak 4 PME suggesting a blockwise mode of action at pH 7.0.


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