Pectinesterases purification

Tucker,G. A.,Robertson,N.G. and Grierson,D. (1982) Purification and changes in activities of tomato pectinesterase isoenzymes. Journal of the Science of Food and Agriculture. 33. 396-400. 

Macdonald, H.M. Evans, R. Spenser, W.J. 1993. Purification and properties of the major pectinesterases in lemon fruits (Citrus limon). J. Sci. Food Agric. 62 163-168. 

Seymour, T.A. Preston, J.F. Wicker, L. Lindsay, J.A. Marshall, M.R. 1991a. Purification and properties of pectinesterases of Marsh white grapefruit pulp. J. Agric. Food Chem. 39 1080-1085. 

Versteeg, C. 1979. Pectinesterases from the orange fruit - Their purification, general characteristic and juice cloud destabilizing properties. Ph.D. thesis. Agricultural Univ., The Netherlands. 

Versteeg, C. Rombouts, F.M. Pilnik, W. 1978. Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensm. Wiss. Technol. 11 267-274. 

Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel — filtration on Sefadex G — 75 was found to be 35,000. 

After purification of the enzyme preparation both at Sephadexes of various types and at Toyopearl HW—56 two fractions were obtained (fractions 1 — 2). One of them (fraction 1) possessed pectinesterase activity under the condition of absence of the activity of other pectolytic enzymes. ... 

The results obtained after purification of pectinesterase preparation using columns with various gels certified that the active fraction (fraction 1 has greater molecular weight) was not subjected to further separation on the tested gels. Components of fraction 1 increased an activity of elute. That can be explained by their acid properties. Pectinesterase activity was accumulated in fraction 1. Activity of the other components of pectolytic complex was not found in the other studied probes of fraction 1. 

Partial purification of pectinesterase preparation enlarged to some extent its specific activity. 

Much attention has now been devoted to the purification and characterization of tomato pectinesterase, apparently because of the high content thereof in the ripe fruits. 

By means of gel electrophoresis on cross-linked, hydrolyzed starch,99 with simultaneous checking for proteins, lipids, and pectinesterase activity, it was found, however, that the product isolated after the separation on CM-Sephadex C-50 constitutes but one of five multiple forms of tomato pectinesterase, and is the one present in preponderant proportion98 (see Fig. 4). The accompanying lipid and sugar components were separated from this pectinesterase form in the course of the purification procedure. After analysis of the hydro-lyzate of the final product for fatty acids, as well as for carbohydrate components, it was possible to exclude the possibility of a lipoprotein,30 as well as glycoprotein,100 character of this form of tomato pectinesterase. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

In a study of the pectinesterase from bananas,64,85,102 three pectinesterase fractions were obtained after respective extraction with water, 150 mM sodium chloride, and 150 mM sodium chloride of pH 7.5. The fractions obtained were further purified by fractional salting-out with ammonium sulfate, and chromatography on columns of DEAE- and CM-cellulose. A 50-fold purification was achieved, and the individual, purified fractions were characterized with respect to different effects of cations, inhibition by sucrose, and reaction kinetics. 

Relatively less attention has been devoted to the isolation of pectinesterase from oranges.35,36 Jansen and coworkers87 described a purification of pectinesterase by adsorption on the cell walls of oranges this adsorption is not specific, but serves to provide a 7.5-fold increase of specific activity. 

In the purification of pectinesterase from the fruits of Citrus nat-sudaidai,61 fractional salting-out with ammonium sulfate was followed by chromatography on a column of DEAE-cellulose and by separation of the active fraction on Sephadex G-100. A preparation (purified solution) having a specific activity 460-fold greater than that of the original extract was obtained. Its homogeneity was checked by disc electrophoresis, and its amino acid content was determined and fundamental, kinetic data were obtained. 

The method was used in a modified form for the purification of endo-D-galacturonanase from tomatoes.212 Elution from the column by use of a linear pH gradient led to the separation of pectinesterase and of three multiple molecular forms of endo-D-galacturonanase dif-... 

A purification procedure including all of the separation principles then known led to a 156-fold purification of exopectate lyase of Clostridium multifermentans, but pectinesterase could not be re-moved.51,105... 

MacDonnell, L. R. Jang, R. Jansen, E. F. Lineweaver, H. Specificity of pectinesterases from several sources with some notes on purification of orange pectinesterase. Arch. Biochem., 1950, 28, 260-273. 

Manabe, M. Purification and properties of Citrus natsudaidai pectinesterase. Agric. Biol. Chem., 1973, 37, 1487-1491. ... 

SakeUaris, G., Evangelopoulos, A. E. (1989). Production, purification and characterization of extracellular pectinesterase from Lactobacillus plantarum (str. BA 11). Biotechnology and Applied Biochemistry, 11, 503—507. 

A review of the occurrence, purification, properties, and modes of action of hemicellulases has dealt with L-arabinanases, D-galactanases, D-mannanase, and D-xylanases, while a comprehensive review of pectolytic enzymes has discussed their classification and the assay and properties of pectinesterases and poly-D-galacturonate hydrolases and lyases. ... 

Nucieotidases.—Since the 5 -nucleotidase from Dictyostelium discoideum interacted with immobilized concanavalin A, it appears to be a glycoprotein. Pectinesterases.—The occurrence, formation, assay, purification, and specificities of pectinesterases have been reviewed. Pectinesterase activity was detected both in healthy and diseased onions Allium cepa), but not in cultures of the invading bacterium (Pseudomonas cepacia) Both the pectinesterase and poly-D-galact-uronate lyase activities of Clostridium multifermentans are associated with a single complex. ... 

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