Enzyme pectic

Pectic enzymes are inactivated by pasteurization. Citms juices require higher temperatures for enzyme deactivation than for pasteurization. Heat treatment at 85—94°C for 30 s inactivates pectic enzymes (9) and is more than adequate for pasteurization. 

Pectic enzymes are classified according to their mode of attack on the galacturonan part of... 

The availability of these novel enzymes, next to the known pectic enzymes, offer new opportunities to use them as analytical tools in revealing the structure of oligo- and polysaccharides [31,32]. In contrast with frequently used chemical degradation methods, which usually have a poor selectivity, these enzymes act in a deflned way. To be able to recognize different structural units within the polymer, endo-acting types of enzyme are preferred, although accessory enzymes might be essential as well [30]. 

Rombouts, F, M. and Pilnik, W. (1980). Pectic enzymes. In Economic Microbiology. Vol. 5 Microbial enzymes and bioconversions, pp. 228-282. Edited by A. H. Rose. New York Academic Press. 

Ried, J. L. and Collmer, A. (1986). Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica. AppI Environ Microbiol 52, 305-310. 

Rexova-Benkova, L. and Markovic, 0. (1976). Pectic enzymes, in Advances in carbohydrate chemistry and biochemistry 33, pp. 323-385. Edited by R. S. Tipson and D. Horton. New York Academic Press. 

Collmer A, Keen NT (1986) The role of pectic enzymes in plant pathogenesis. Ann Rev Phytopathol 24 883-409... 

Yang Z, Cramer CL, Lacy GH (1992) Erwinia carotovora subsp. carotovora pectic enzymes In planta gene activation and roles in soft rot pathogenesis. Mol Plant-Microbe Interact 5 104-112... 

Figure 1 indicates that pectin methyltransferase (PMT) activity from freeze-thawed microsomes measured without exogenous substrate was maximal at neutral pH (6.5 to 7.5). When exogenous pectic substrates of various DE had been added, similar optimal neutral pH was observed, and the activity was slightly stimulated (1.2 to 1.8 times). A second optimal pH occured at pH 5.5, but in the presence of low methylated pectin (DE 0.1). As suggested by Lineweaver and Ballou [8] to explain the behaviour of another pectic enzyme -i.e. pectin methylesterase (PME), the mobility and the activity of PMT might be influenced by the presence of polyanionic substrates. On the other hand, the existence of several forms of pectin methyltransferase in flax microsomes might be responsible for such variations of the activity. 

The r2 isolate of Fusarium oxysporum f. sp. radicis-lycopersici (FORL) produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We have detected three forms that have lyase activity, an absolute requirement for calcium, and pis of 9.20, 9.00 and 8.65. The two most alkaline forms had a weak preference for pectin whereas the other was more active on pectate. The three lyases were produced when the fungus grew on pectin and on restricted galacturonic acid (data presented in the "XV Congreso Nacional de Microbiologia" [21] and sent for publication). 

Two characteristics of the lyase that we have purified may be significant. First, the small molecular size of the protein may confer it a high mobility that could be helpful to its movement through host cell walls. In second place, it is an endo-type enzyme, fact that has been considered essential for maceration of plant tissues [35]. In this sense it is noteworthy that between the battery of pectic enzymes produced by FORL, this pectin lyase is the only protein that behaves as an endo-type. 

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers - tyroglobulin, 2- apoferritin, 3- p-amylase, 4-alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers 1- myosin, 2- p-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

Evidence of pectic enzymes secreted by FORL species has been obtained (3). The total proteins of FORL subjected to isoelectric focusing were resolved into several bands widely distributed between pi 5.9 and 7.45. FORL (rj) presented one mayor band of activity with a pi of 7.0 however, FORL (r ) presented one characteristic and principal band at 7.45, slightly more basic than these reported by others authors (4). 

Candida boidinii - a new found producer of pectic enzymes complex. 

Candida boidinii is a further yeast producer of pectic enzymes complex. The production is induced by the presence of pectin as a C-source in the medium the primary methabolic path is the utilization of methanol and the secondary the utilization of pectate chains. The pectic enzymes were bound on the cell walls or released on the cultivation medium. The main enzyme of pectic complex, polygalacturonase, was briefly characterized and the possibility to influence the production of its multiple forms discussed. 

This study reports on the production of pectic enzymes and partial characterization of polygalacturonases produced by Candida boidinii. Candida boidinii belongs to the so called methylotrophic yeasts with famous utilization of methanol. Pectin, the natural substrate of pectic enzymes complex, can serve for microorganisms as a C - source by two different ways after deesterification with pectinesterase as methanol and after hydrolytic cleavage with... 

The activities of pectic enzymes present in cultivation medium (98 mg of protein extracted from 2.5 1 of pectin medium) were poor, not leading to the clarification of cultivation medium indicating the cleavage of pectate chains, with values 0.024 pmol/min.mg for polygalacturonase, 0.004 pmol/min.mg for exopolygalacturonase, 0.034 pmol/min.mg for pectinesterase and 0.005 pmol/min.mg for pectin lyase. The production of individual pectic enzymes was dependent on the C-source used in the cultivation medium (Tab. 1). 

The production of individual pectic enzymes by Candida boidinii - dependence on the C - source... 

The activity of the main enzyme of pectic enzymes complex, polygalacturonase, was dependent on the pH of the cultivation media the highest activity was reached at pH 3.51 (natural pH of pectin medium), the activity decreased to 70% and 20% by the cultivation at pH 5.49 and pH 7.01, respectively. The isoelectric focusing patterns showed the production of polygalacturonase multiple forms with isoelectric points varying from 3.5 to 7.5 (Fig. 3 A,B) with the possibility to influence their production with the change of the C-source and pH of the cultivation media. 

The poor activities of pectic enzymes in the cultivation medium led us to prove the cell cytosole and the cell walls for these activities. The cytosole contains only traces of polygalacturonase activity, but the suspension of cell walls established the activity which seems to be widely sufficient for yeast growth and development. The characterization of this cell wall bound enzymes will be the object of our next studies. 

Whitaker JR (1984) Pectic substances, pectic enzymes and haze formation in fruit juices. Enzyme Microb Technol 6 341-349... 

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