Brett J. Savary

Gas chromatography–mass spectrometry method for determining the methanol and acetic acid contents of pectin using headspace solid-phase microextraction and stable isotope dilution

A simple, fast, and direct procedure was developed for the simultaneous determination of the methanol and acetic acid present as esters in the plant cell wall polysaccharide pectin. After base-hydrolysis of esters and acidification of pectin samples, headspace solid-phase microextraction (SPME) was performed using a Carboxen-PDMS fiber assembly. Methanol and acetic acid were separated by gas chromatography with a Chrompak PoraPlot Q capillary column and detected using electron impact mass spectrometry with selected ion monitoring. Stable deuterated isotopomers (d3-methanol and d3-acetic acid) were used as internal standards and for constructing calibration curves, providing accurate and absolute quantification of analytes. The methanol and acetic acid contents in 1 mg quantities of fruit and vegetable pectins were readily quantified by this procedure.

Structural Characteristics of Pumpkin Pectin Extracted by Microwave Heating

UNLABELLED To improve extraction yield of pumpkin pectin, microwave heating was adopted in this study. Using hot acid extraction, pumpkin pectin yield decreased from 5.7% to 1.0% as pH increased from pH 1.0 to 2.0. At pH 2.5, no pectin was recovered from pumpkin flesh powder. After a pretreatment at pH 1.0 and 25 °C for 1 h, pumpkin powder was microwave-extracted at 120 °C for 3 min resulting in 10.5% of pectin yield. However, premicrowave treatment at 60 °C for 20 min did not improve extraction yield. When microwave heating at 80 °C for 10 min was applied after premicrowave treatment, final pectin yield increased to 11.3%. When pH was adjusted to 2.0, the yield dropped to 7.7% under the same extraction conditions. Molecular shape and properties as well as chemical composition of pumpkin pectin were significantly affected depending on extraction methods. Galacturonic acid content (51% to 58%) of pumpkin pectin was lower than that detected in commercial acid-extracted citrus pectin, while higher content of neutral sugars and acetyl esters existed in pumpkin pectin structure. Molecular weight (M(w) ) and intrinsic viscosity (η(w) ) determined for microwave-extracted pumpkin pectins were substantially lower than acid-extracted pectin, whereas polydispersity was greater. However, microwave-extracted pectin at pH 2.0 had more than 5 times greater M(w) than did the pectin extracted at pH 1.0. The η(w) of microwave-extracted pectin produced at pH 2.0 was almost twice that of other microwave-extracted pectins, which were comparable to that of acid-extracted pectin. These results indicate that extraction yield of pumpkin pectin would be improved by microwave extraction and different pectin structure and properties can be obtained compared to acid extraction. PRACTICAL APPLICATION Pumpkin is a promising alternative source for pectin material. Pumpkin pectin has a unique chemical structure and physical properties, presumably providing different functional properties compared to conventional commercial pectin sources. Depending on the conditions to produce pumpkin pectin, diverse molecular structures can be obtained and utilized in various food applications.

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.

PERFUSION CHROMATOGRAPHY SEPARATION OF THE TOMATO FRUIT-SPECIFIC PECTIN METHYLESTERASE FROM A SEMIPURIFIED COMMERCIAL ENZYME PREPARATION

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a “ubiquitous-type” isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties. Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.

Identification of thermolabile pectin methylesterases from sweet orange fruit by peptide mass fingerprinting.

The multiple forms of the enzyme pectin methylesterase (PME) present in citrus fruit tissues vary in activity toward juice cloud-associated pectin substrates and, thus, in their impact on juice cloud stability and product quality. Because the proteins responsible for individual PME activities are rarely identified by structural properties or correlated to specific PME genes, matrix-assisted laser desorption-ionization with tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was investigated as a direct means to unequivocally identify the thermolabile (TL-) PME isoforms isolated from sweet orange [ Citrus sinensis (L.) Osbeck] fruit tissue. Affinity-purified TL-PME preparations were separated by SDS-PAGE prior to trypsin digestion and analyzed by MS for peptide mass fingerprinting. The two major PME isoforms accumulated in citrus fruit matched existing accessions in the SwissProt database. Although similar in size by SDS-PAGE, isoform-specific peptide ion signatures easily distinguished the two PMEs.

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13-hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13-hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo-product, 13-oxo-9(Z), 11(E)tridecadienoic acid (13-OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13-hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13-hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13-OTA along with the 13-ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13- and 9-hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH 4 ) 2 SO 4 precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.

Structural Characterization of the Thermally Tolerant Pectin Methylesterase Purified from Citrus sinensis Fruit and Its Gene Sequence

Despite the longstanding importance of the thermally tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, the unequivocal identification of the protein and its corresponding gene has remained elusive. TT-PME was purified from sweet orange [ Citrus sinensis (L.) Osbeck] finisher pulp (8.0 mg/1.3 kg tissue) with an improved purification scheme that provided 20-fold increased enzyme yield over previous results. Structural characterization of electrophoretically pure TT-PME by MALDI-TOF MS determined molecular masses of approximately 47900 and 53000 Da for two principal glycoisoforms. De novo sequences generated from tryptic peptides by MALDI-TOF/TOF MS matched multiple anonymous Citrus EST cDNA accessions. The complete tt-pme cDNA (1710 base pair) was cloned from a fruit mRNA library using RT- and RLM-RACE PCR. Citrus TT-PME is a novel isoform that showed higher sequence identity with the multiply glycosylated kiwifruit PME than to previously described Citrus thermally labile PME isoforms.