Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 18, 2004
Publication Date: February 18, 2005
Citation: Cameron, R.G., Savary, B.J., Hotchkiss, A.T., Fishman, M.L. 2005. Isolation, characterization and pectin modifying properties of a thermally tolerant pectin methylesterase from valencia oranges. Journal of Agricultural and Food Chemistry. 53:2255-2260. Interpretive Summary: Cloudiness in citrus juices is an appealing, flavorful characteristic and depends on the unchanged pectin that is present in the fruit and extracted with the juice. Unless the juice is pasteurized, enzymes present in the juice will modify the pectin, leading to settling of the cloud material and leaving behind an unappealing clear juice serum. Pectin also is extracted from citrus fruit peel and used as a gelling agent for jams, jellies and other confectionaries, and to stabilize some dairy products like yogurt. It is the physical structure of pectin that gives it these functional properties. Pectin methylesterase is an enzyme in citrus juices that alters the structural features of the pectin that leads to juice cloud loss and, also, allows gelling conditions to be controlled. There are four types of this enzyme in citrus juice and one is very resistant to heat. We have separated the heat resistant one from the other three and characterized its biochemical properties. We show that it does not need salt for activity but its activity increases in the presence of salt. This enzyme makes blocks in pectin without methyl groups giving it unique functional properties. It also is able to remove most of the methyl groups from pectin, which probably stimulates the loss of the juice cloud. Its properties differ sufficiently from the other pectin methylesterases in citrus so that it should be able to be activated while the other enzymes are inactivated.
Technical Abstract: A thermally tolerant, salt-independent pectin methylesterase (TT-PME) has been isolated from sweet orange (Citrus sinensis var. Valencia) fruit rag tissue. It was separated from other PMEs in this tissue by a combination of DEAE, heparin, and concanavalin A affinity chromatography. It is similar to a Type II PME (salt-independent) and exhibits optimal activity between 0.05 - 0.2 M NaCl at pH 7.5. It has a pH optimum from 5.5 - 8.5 at 200 mM NaCl but activity continued to increase up to pH 9.5 with 0 or 15 mM NaCl. An endpoint DE value of 21.1% and 11.7% were obtained at pH 4.5 and 7.5 respectively. The enzyme has an apparent molecular mass of 40.3 kDa by denaturing electrophoresis. It has a pI of 9.31 and exhibits only one activity band after IEF. The calcium sensitive pectin ratio increased from 0.045 ± 0.011 to 0.895 ± 0.011 after de-esterification with TT-PME suggesting a blockwise mode of action at pH 7.0. De-esterification of non-calcium-sensitive-pectin did not result in a reduction of the pectin molecular weight but did reduce the intrinsic viscosity to 5.03, suggesting a slightly lower charge density than produced by the two thermally labile PMEs (TL-PME) previously described. A lower charge density may indicate the production of a different distribution of demethylated blocks than produced by the TL-PMEs.