Of pectinesterases

Pressey,R and Avants,J.K (1972) Multiple forms of pectinesterases in tomatoes. Phytochemistry. 11. 3139-3142. 

Cations Increase Activity and Enhance Permeation of Pectinesterase in Ultrafiltration... 

Evans, R. McHale, D. 1968. Multiple forms of pectinesterase in limes and oranges. Phytochemistry 17 1073-1075. 

Rouse, A.H. 1953. Distribution of pectinesterase and pectin in component parts of citrus fruits. Food Technol. 7 360-362. 

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. 

Properties of pectinesterase from Penicillium fellutanum Biourge and new developments in pectin applications... 

Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 °C. The enzyme was stable at pH 4 — 5 and 40 °C (pH 5) for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics =... 

Subject of the present message is study of the conditions for Penicillium fellutanum cultivation (that is a producer of pectinesterase) and analysis of some properties of pectinesterase enzymatic preparation. 

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. 

For study of substrate specificity pectin with various degrees of metoxilation (expressed as a percentage) were used beet substrate —37.8, apple substrate — 70, lemon—82. Specificity of pectinesterase action was analyzed under optimum temperature and acidity of the medium using beet, apple and lemon pectin according to the speed of methanol formation (M 10 min. ). 

Enzymatic preparation with predominant content of pectinesterase (obtained from Penicillium fellutanum culture liquid by isolation by acetone was purified. Primary enzymatic preparation was re — precipitated by three volumes of ethyl alcohol and centrifuged (6000 rev/min.) during 20 min. The obtained precipitate of partially purified pectinesterase preparation was dried in vacuum — desiccator. Sephadexes G — 50, G-75, G-lOO, G-200 "LKB" (Sweden) and Toyopearl HW-55 (Japan) were used for separation of enzymatic complex by gel — filtration. 

Molecular weight of the components of the enzymatic complex was determined using a Sephadex G —75 column after its calibration by dextrans with molecular weight equal to 10,000, 40,000 and 70,000 and rafinose with molecular weight of 504. Fractions were also analyzed by the disk —electrophoresis method in PAAG (7) using 7.5% polyacrilamide gel (pH 4.3). Activity of pectinesterase was determined by titrometric method [8]. The enzymatically released methanol analyzed by gas—liquid chromatography [9]. 

Acidity of the reaction mixes after incubation increased as the activity of the probe ro —se during determination of pectinesterase activity of the samples.1t was caused by the for—mation of carboxyl groups as a result of pectin ester bonds hydrolysis under pectinesterase ac—tion.That is why kinetic characteristics of substrate hydrolysis were measured according to the speed of pectin hydrolysis by continuously recorded titration of the free carboxyl groups (11). 

Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 °C. It is shown on Figure 1 that pectinesterase was stable at pH 4 — 5. At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 — 8%. At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. 

P. fellutanum 57699 is characterized by the predominant synthesis of pectinesterase. Enzymatic complex produced by the fungus is distinguished from known industrial producers for increased content of extracellular pectinesterase of pectolytical complex. 

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. 

Disk electrophoresis of pectinesterase enzyme preparation in polyacrilamide gel revealed 4 protein bands in toe fraction 1 after its gel — filtration through Toyopearl HW—55. One of the two obtained bands (a wider one) possessed pectinesterase activity, while toe second one did not reveal it. 

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

Thus, some features of pectinesterase from P. fellutanum were characterized as a result of the study. New preparation of the extracellular pectinesterase is recommended for production of lowmolecular pectin. 

The specificity of pectinesterase is not so marked with respect to the alcohol moiety of the ester, as it hydrolyzes ethyl esters of D-galacturonans at a rate 6-16% that of de-esterification of the methyl esters.36 Citrus natsudaidai pectinesterase de-esterifies the ethyl esters of pectic acid at a rate l/5th to l/7th of that for the methyl esters the propyl and allyl esters are attacked at l/20th to l/80th of the rate.38 The tomato, citrus, alfalfa, and papaya pectinesterases do not hydrolyze the glycol and glycerol esters of pectin.39... 

By using the same experimental procedure, the action pattern of pectinesterase produced by Clostridium multifermentans48 was examined. As none of the separation procedures used were suitable for removing the pectinesterase from exopectate lyase,51 a specific lyase was obtained by inactivating the pectinesterase by heating for 30 minutes at 38° and pH 7.0 under these conditions, the activity of the lyase was retained. All the pectinesterase preparations used were contaminated with the lyase, which could not be differentially inac-... 

Fig. 2.— Activity47 of Tomato Pectinesterase (PE) in the Presence (1) and Absence (2) of Exopectate Lyase. [Reaction mixtures contained 0.125% of poly(D-galacturonic acid methyl ester) methyl glycoside, 0.5 mM CaCl2, 2.8 units of pectinesterase, and, in the reaction mixture represented by curve 1, 1.5 unit of lyase.] Reprinted, with permission, from M. Lee and J. D. Macmillan, Biochemistry, 9, 1930-1934 (1970). Copyright by the American Chemical Society.

In the action of pectinesterase, evidently, end-product inhibition takes place.57 For highly purified tomato pectinesterase, Lee and Macmillan50 found an inhibition constant Kt = 7 mM D-galac-topyranosiduronate units, the inhibition being competitive (see Fig. 3). 

The activity of pectinesterase is affected by pH, temperature, presence of salts and various inhibitors. 

The presence of salts of univalent and bivalent cations increases, by several-fold, the activity of pectinesterases from higher plants, which is minimal in the absence of salts.38,50,57,6, 63,64 66,69,7° The activating effect of salts on pectinesterases of microbial origin is not so great, an increase by 1.5- to 2-fold being reported.51 56,63 76-78,80 Table III shows the concentrations of sodium chloride and calcium chloride that caused the maximal activation of pectinesterases of plant and microbial origin. The mechanism of activation has not yet been satisfactorily explained. 

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... 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

Four forms of pectinesterase were found in different tomato varieties (Marion, Homestead, and Pixie)83 by separation on columns of DEAE-Sephadex A-50, using elution with 150 mM sodium chloride at pH 6.0. By gel filtration on columns of Sephadex G-100, the molecular weights of the individual forms were found to be 22,000-27,000, and 35,000. 

By use of starch-gel electrophoresis, the total extract of bananas, and the fractions obtained after separation on DEAE-Sephadex A-50, were found to contain six multiple forms of pectinesterase having electrophoretic patterns different from those of tomato pectinesterase.103... 

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. 

Among the microbial pectinesterases, the one from Coniothyrium diplodiella56 was purified by repeated chromatography on columns of Duolite A-2 and DEAE-cellulose. Two electrophoretically homogeneous forms of pectinesterase were obtained, having an identical pH optimum and the same behavior towards pectins of different d.e. 

Macmillan and coworkers51 105 106 purified pectinesterase produced by Clostridium multifermentans by using practically all of the available methods and materials (calcium phosphate gel, DEAE-cellulose, DEAE-, QEAE-, CM-, and SE-Sephadex, Sephadexes G-75, G-100, G-150, and G-200, Sepharose 4B, and zonal centrifugation). However, they could not separate pectinesterase from exo-pectate lyase, and, hence, they postulated that either (a) a complex of the two enzymes having an apparent molecular weight of 400,000 exists, or (b) the two enzymes are identical in their molecular species. On the basis of the mode of action of this pectinesterase in comparison with that of those from tomatoes and from Fusarium ox-ysporum, the existence of a complex of pectinesterase and exopectate lyase in Clostridium multifermentans appears to be the more probable. 

The original test for the presence of pectinesterase was the formation of gel after an enzymic de-esterification of pectin in the presence of Ca2+ ions. This behavior was observed by Fremy107 more than 100 years ago, after he found the presence of pectase in carrots. 

---