Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains cndo-PG I, endo-PG II and pectin esterase (PE) (with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.     

Studies on Pectolytic Enzymes of Molds

The process of apple juice clarification by pectolytic enzymes has been successfully observed turbidimetrically and macroscopically by heating of reaction mixtures. It has been shown that the process of apple juice clarification varies with the varieties and conditions of apple juices as well as with the sources of enzyme preparations. From a study of the turbidimetry of apple juice clarification, α method for determination of clarification values been described.     

Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation

The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.     

Pectinolytic enzyme production by Bacillus species and their potential application on juice extraction

Five Bacillus strains isolated from decaying vegetable material were cultivated on wheat bran and endo-polygalacturonases, exo-polygalacturonase and pectin lyase activities in the crude enzymatic solution obtained were determined. Highest activity was observed for all enzymes when fermentation was carried out at 28 °C, the highest activity values were obtained after 120 h of cultivation for exo-PG and after 48 h for endo-PG and PL. The use of the enzymatic solution for treatment of fruits and vegetable mash afforded a high juice extraction and a pulp with good pressing characteristics.     

Immobilization of polygalacturonase from Aspergillus niger onto activated polyethylene and its application in apple juice clarification

The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.     

Pectic Enzymes in the Clarification of Apple Juice

To know the role of pectic enzymes in the clarification reaction of apple juice, a simplified model for apple juice, that is, aqueous re-suspension of ultracentrifugal precipitates of apple juice, was employed. It was found that the precipitates (i.e., suspended materials) contained 36% of protein and that the surface of the suspended materials was negatively charged at pH 3.5. Positively charged colloids at pH 3.5 such as gelatin enhanced the clarification reaction or mutually coagulated with the suspended materials. While negatively charged colloids at pH 3.5, such as sodium alginate completely inhibited the clarification reaction. The direct participation of pectic enzymes in the clarification of apple juice was shown, and a supposed mechanism of the enzymic clarification was presented.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Immobilized pectolytic enzymes on acrylic supports

The paper deals with the pectolytic enzymes immobilization on different acrylic supports, and the application of immobilized preparations in the apple juice pectinization process. The correlation between the protein content (C_p) and specific catalytic activities of immobilized enyzme preparations suggest a specific immobilization process only in the case of PONILEX ASH type acrylic supports. The active immobilization degree on PONILEX ASH type supports of Ultrazym 100 G and technical pectinase extract ranged from 99.80 to 296.40% for the Pectinesterase (PE) activity, and from 101.85 to 252.94% for the chain splitting (CS) activity, proving that the ionic immobilization process is a selective one. The simulated operational stability of the immobilized pectolytic enzymes tested by the PE and CS activity values proves the preservation of enzyme catalytic activity.     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation

Seven different strains were selected for their ability to degrade citrus pectin. Alkaline pectinases were produced by five bacterial soil isolates, whereas two fungal strains produced pectinase in an acidic environment. The bacteria were isolated from soil of a plum orchard in Northern Ireland. These isolates produced significant amounts of pectin lyase (PL) and polygalacturonase (PG) with maximum activities of 30.1 and 29.1 U/ml respectively. Fungal strains Aspergillus sp. and PN-1 produced four different pectinolytic activities; endo-PG, exo-PG, pectin esterase (PE) and PL. The Aspergillus sp. produced higher amounts of pectinase than PN-1. The Aspergillus sp. excreted highly stable pectinases, which may be of importance for industrial applications.     

Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.     

Stability/activity features of the main enzyme components of rohapect 10L

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions.     

Expression and Characterization of Fifteen <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> 99-880 Polygalacturonase Enzymes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40°C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 μmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later.     

Effect of oxygen transfer rate on the composition of the pectolytic enzyme complex of Aspergillus niger

Optimal agitation and aeration conditions [assuring O₂ transfer rates (OTR) from 12 to 179 mmol L^?1 h^?1] were determined for pectin lyase (PL) synthesis of an Aspergillus niger strain. Components of the pectolytic enzyme complex were also investigated in order to determine whether their O₂ demand is identical with or different from that of pectin lyase. Should the latter be the case, a possibility would be given to produce enzyme complexes of different agitation and aeration conditions. According to our results, mycelium yield of Aspergillus niger attained a maximum at an OTR of 100 mmol L^?1 h^?1. The yields of the various pectolytic enzymes reached maxima at different OTRs. Pectin lyase production was the highest (0.555 µmol min^?1 mL^?1) at an OTR of 60 mmol L^?1 h^?1. Endopolygalacturonase (PG) production showed a maximum at the OTR of 49 mmol L^?1 h^?1 with a second peak at 100?135 mmol O₂ L^?1 h^?1. Pectin esterase (PE) synthesis showed a maximum at on OTR of 12?14 mmol L^?1 h^?1, while both apple juice clarifying and macerating activities gave two maxima at 14 and 60 mmol L^?1 h^?1 due to the optima of PE and endo-PG. Macerating activity showed a high value at OTR optimal for PL production as well.     

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.     

Dextransucrase production using cashew apple juice as substrate: effect of phosphate and yeast extract addition

Cashew apples are considered agriculture excess in the Brazilian Northeast because cashew trees are cultivated primarily with the aim of cashew nut production. In this work, the use of cashew apple juice as a substrate for Leuconostoc mesenteroides cultivation was investigated. The effect of yeast extract and phosphate addition was evaluated using factorial planning tools. Both phosphate and yeast extract addition were significant factors for biomass growth, but had no significant effect on maximum enzyme activity. The enzyme activities found in cashew apple juice assays were at least 3.5 times higher than the activity found in the synthetic medium. Assays with pH control (pH = 6.5) were also carried out. The pH-controlled fermentation enhanced biomass growth, but decreased the enzyme activity. Crude enzyme free of cells produced using cashew apple juice was stable for 16 h at 30°C at a pH of 5.0.     

Studies on Pectic Enzymes of Microorganisms

Endo-polygalacturonase (endo-PG) of Aspergillus saitoi was purified through ammonium sulfate fractionation, Amberlite IRC-50 column chromatography, and several combinations of Sephadex column chromatography.

The purified endo-PG, which was almost homogeneous ultracentrifugally and electrophoretically, had the sedimentation constant of 2.2 S and the absorption maximum at 277 mμ. Its optimum pH and temperature were 4.8~5.0 and 45°C, respectively, and it was most stable between pH 4.0 and 6.0, but over 90% of the activity was lost at 50°G for 10 min.

The purified enzyme was a typical endo-PG, and hydrolyzed about 60% and 17% of glycosidic linkage of polygalacturonic acid and pectin, respectively. This enzyme preparation had no pectinesterase, trans-eliminase, and apple juice-clarifying activities, but macerated potato tuber slices singly.     

Pectic Enzymes in the Clarification of Apple Juice

Pectinesterase of Sclerotinia arachnidis was purified by 44 times. The clarification of apple juice with the purified pectinesterase and the formerly purified endo-polygalacturonase of Aspergillus saitoi was examined. The coexistence of pectinesterase and endo polygalacturonase was necessary for the complete clarification of apple juice. During the clarification reaction rapid decrease of viscosity and resultant coagulation of the suspended materials in apple juice were observed. The coagulated materials slowly precipitated leaving clear juice.

It was suggested from the result of electrophoresis that the positive charge existed inside of the suspended materials. The decrease in viscosity was supposed to correlate to pectin’s depolymerization which would result in revealing of positive charge inside. The probable mechanism of coagulation was attributed to the electrostatic neutralization between positive charge thus appeared and negative charge of still undegraded pectin.     

Purification and Characterization of Extracellular Pectinesterases from Phytophthora infestans

Constitutively produced extracellular pectinesterases from culture filtrates of the potato late blight fungus Phytophthora infestans were purified and characterized. One enzyme (PE II) was purified to homogeneity. Sodium dodecyl sulfate electrophoresis of the second enzyme (PE I) revealed two protein bands; there are indications that both proteins are pectinesterases, which were not separable by a number of different techniques. Thus, P. infestans might produce three pectinesterases in vitro. Enzyme activities were optimal in the neutral pH range and were largely dependent on the presence of NaCl or CaCl₂ in the reaction medium. The molecular weight of the PE I-complex was between 45 and 48 kilodaltons, and the one of PE II was between 35 and 40 kilodaltons. Further investigations will help us to clarify the role of these enzymes during pathogenesis.     

Use of a preparation from fungal pectin lyase in the food industry

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.