Evaluation of pectolytic activities of enological interest in industrial enzyme preparations

 The purpose of this research was to determine the activity of several pectolytic enzymes, such as pectin methyl esterase, endopolymethylgalacturonase, endopolygalacturonase, exopolymethylgalacturonase, endopolygalacturonase, pectin transeliminase and pectic acid transeliminase, from within preparations that are available on the Portuguese market. The selected preparations were as follows: 15 pectolytic enzyme preparations, one hemicellulolytic preparation and one glucanase preparation. The measurements of enzyme activity were carried out using model solutions with 50 mg·l^–1 SO₂, pH 3.2, 25°C, with and without 12% ethanol (v/v), and a 24-h incubation period. Quantitatively, the results proved to be heterogeneous, significantly varying from preparation to preparation: all pectolytic enzyme activities in the 17 preparations analysed in this study were significantly different. The presence of ethanol reduced the enzymatic activity, apart from that of endopolymethylgalacturonase, and, in general, an increase of enzyme concentration raised the pectolytic enzyme activity. Received: 17 March 1997 / Revised version: 11 June 1997     

Analysis of cell-wall pectin from hot and cold break tomato preparations

The initial processing of tomatoes includes a break step, which involves rapid heating of the freshly chopped tomatoes to >90 °C for hot break, or 60–77 °C for cold break. It is believed that pectolytic enzyme deactivation is the key element in the hot break step; therefore the pectin content of the different products should be qualitatively distinct. There are two general sources of pectin in a tomato fruit: the middle lamella which is rich in homogalacturonan, and the cell-wall. In this study, a 0-min or 24-min hold time (room temperature) for the chopped tomatoes preceded the break step to replicate hot and cold break, respectively, with minimum variation in the processing. The cell-wall pectin was then isolated and analyzed for the carbohydrate composition, degree of polymerization, and degree of esterification. The results showed no observable differences in pectin isolated from the two preparations, indicating that there is no significant pectolytic activity in the cell wall during the 24 min hold time; thus, the pectolytic enzymes probably act on the lower ester, middle lamella pectin in cold break products.     

Regulation of the expression of endopolygalacturonase gene PGU1 in Saccharomyces

Previous work in our laboratory has shown that Saccharomyces bayanus strain SCPP is the only reported yeast expressing the three types of pectolytic enzymes: pectin esterases, pectin lyases and polygalacturonases. One of these enzymes, the endopolygalacturonase (endoPG), hydrolyses plant-specific polysaccharide pectin. The endoPG encoding gene (PGU1) is also present in Saccharomyces cerevisiae. It has been shown that this endoPG is required for the development of pseudohyphae. Using genomic DNA, the PGU1-1 and PGU1-2 promoters of these strains have been amplified and used to construct gene fusions with the beta-galactosidase gene. On the basis of beta-galactosidase measurements, we compared the expression of both promoters in different environmental conditions in order to identify their modulation. We have shown that the PGU1 gene is upregulated by the presence of the pectin and the product resulting from endopolygalacturonase activity. Moreover, expression of the PGU1 is also enhanced under respiratory and filament formation conditions.     

ASPERGILLUS NIGER ENDOPOLYGALACTURONASE

ABSTRACT A purified endopolygalacuronase (endo PG) from Aspergillus niger has been characterized. The optimum temperature was 45°C and the optimum pH was 4.1–4.2 with a highest stability at pH 6. The purified enzyme was a typical endo PG: the enzyme hydrolyzes about 56.7% and 7% of the glycosidic linkages of polygalacturonic acid and pectin (DE = 72.8%), respectively, and a loss of 50% in viscosity of polygalacturnoic acid was reached when only 2.3% of the glycosidic linkages of the substrate were split. The products at the maximum hydrolysis were mono-, di- and trigalacturonic acids. Endo PG has a V_max of 239 μ mole reducing groups per min per mg of protein and a K_m of 0.44 mg per ml of polygalacutronic acid (DP_n= 44). Sodium, potassium and ammonium ions stimulated endo PG activity, while divalent ions, or ethylene — diamine – tetracetic acid had no effect. Pectinolytic enzymes (saponifying enzymes and depolymerases) are widespread in nature; they are produced by bacteria, fungi and higher plants (Rombouts and Pilnik 1972; MacMillan and Sheiman 1974). They are involved in natural processes, as fruit physiological changes (Pilnik and Voragen 1970) and they are responsible for the phytopathogenicity of many microorganisms (Rexova-Benkova and Markovic 1976). These enzymes are very useful for their applications in food processing. Commercial preparations of fungal pectinolytic enzymes are employed in the production of fruit and vegetable juices (Rombouts and Pilnik 1978). These preparations contain mainly pectinesterases (pectin-pectyl hydrolase, E.C. 3. 1. 1. 11 and endopolygalacturonase (poly (1, 4 α D galacturonide) glycanohydrolase, E.C. 3. 2. 1. 15); for this reason, special attention was paid to endopolygalacturonase (endo PG (ENDO, 1964a, 1964b, 1964c; Yamasaki et al. 1966). Purified endo PG was also used as a tool for specific degradations of pectic substances (Talmadge et al. 1973) or material containing galacturonic acids (Kikuchi and Yokotsuka, 1973; Kikuchi and Sugimoto, 1976). In a previous paper (Thibault and Mercier 1978) we have described a rapid method for the purification of endo PG from a commercial preparation of pectolytic enzymes. This method is based on a remark of English et al. (1972) who observed that endo PG was delayed during chromatography on agarose gel (Bio-Gel A 0.15 m). We demonstrated that dialyzed endo PG from a crude preparation can be optimally bound by ion-exchange (Thibault 1978) to the agarose gel (Sepharose 6B) when the column is eluted at pH 4.3–4.4 (20 mM acetate buffer). The endo PG released by a 0–0.15 M NaCl gradient is therefore purified 62 times if the activity is determined by a viscosimetric method and 36 times if the activity is expressed in n. katals (cf. enzyme assay methods for the definitions). The present paper reports characterization and some physico-chemical properties of the purified enzyme.     

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1)

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.     

Studies on rats fed a dietary fibre preparation from sugar beet

 Groups of 15 female rats were fed ad libitum for 4 weeks with a standard diet containing 0, 2.5, 5 or 10% of a dietary fibre (DF) preparation made on a laboratory scale from sugar beet pulp. This preparation had a total DF content of 72.2%. Its major components were 36.7% cellulose, 16.9% pectin, 16.8% hemicelluloses (HC) and 11.0% protein. The DF preparation from sugar beet exhibited a water-binding capacity (WBC) of 8.9 g H₂O/g. As the proportion of DF preparation in the diet increased, up to 15.8% total DF, 4.6% cellulose and 1.9% pectin were found in the feeds. The WBC of the diets was estimated to be 1.4–2.9 g H₂O/g. At the end of the experiment, 20.3–64.1% total DF, 10.3–38.2% cellulose, 0.2–7.8% pectin, 4.3–9.2% HC pentoses and 4.3–10.3% HC hexoses were determined in caecum contents (ca. 0.6 g dry weight/rat). The following proportions were found in faeces (3rd week; 1.4–1.9 g dry weight/rat): 34.5–56.9% total DF, 19.5–36.1% cellulose, 6.4–8.4% HC pentoses, 7.4–8.3% HC hexoses. The WBC of faeces ranged from 3.7 g H₂O/g to 4.9 g H₂O/g. About 30–50% of the daily intake of DF appeared in the faeces. Higher amounts of total DF, pectin and HC pentoses were fermented by gastrointestinal microflora as the concentration of DF preparation from sugar beet in the diet increased. In addition, the fermentation of different DF components could be shown by the monosaccharide composition of caecum contents and faeces. Received: 21 October 1997     

Determination of kinetic parameters of pectolytic enzymes at low pectin concentrations by a simple method

A simple and sensitive method for determination of the pectolytic enzyme activity was improved for soluble and immobilized forms of the enzyme. During enzymatic hydrolyzation of pectin, samples were collected from substrate solution at certain time intervals. Pectin in the samples was precipitated by alcohol and then pectin concentration was determined by measuring the refractive index of a prepared aqueous solution of these precipitates. The validity of this technique was evaluated using the kinetic behavior of soluble and immobilized enzymes. The kinetics of free and Duolite A568-immobilized pectinase was investigated. Michaelis-Menten constants and maximal reaction rates were found as K _m=20.71 g l ^-1 and V _max=81.30 g l ^-1s ^-1 for free enzyme and K _m=1.02 g l ^-1 and V _max=0.035 g s ^-1 g ^-1 particles for immobilized pectinase at 20 °C.     

Production of Cellulase-Free Polygalacturonase Preparation by Sclerotium rolfsii for Bioscouring of Cotton

SUMMARY

Cultivation media for the production of both a cellulase containing and a cellulase-free polygalacturonase (PG) preparation by a highly productive strain of Sclerotium rolfsii (CBS 350.80) were developed. These enzyme preparations both contained a PG activity higher than 500 IUmL^?1 and were applied in a bioscouring sequence of knitted cotton at various activity levels. The progress of bioscouring was evaluated by weight loss, water absorption time and determination of residual pectin. Released sugars have been determined and compared to bioscouring effects. No significant differences among the tested preparations were obtained for water absorption and pectin removal. However, considerably higher weight losses were observed for the preparations containing cellulase. A minimal enzyme dosage of 100 IUg^?1 cotton was necessary to achieve suitable scouring results under the applied conditions.     

Characterisation and purification of a cinnamate esterase from Aspergillus niger industrial pectinase preparation

Pectolytic enzymatic preparations used in enology come from Aspergillus niger. They have a desirable principal activity and several secondary activities. Among these secondary activities, three classes of esterases were detected: an esterase specific for ester linkage between cinnamic acid and alcohol (cinnamate esterase); an esterase acting on ester linkage between two phenolic rings (depsidase); and an esterase hydrolysing ester linkage between phenolic acids and straight-chain alcohols (phenyl esterase). Only the first class of enzyme is able to hydrolyse the cinnamic acid tartrate ester of the white must. The authors tried to purify the cinnamate esterase from the other esterase of an A niger industrial pectolytic preparation. This purification was done in two steps. A gel filtration on S200 HR Sephacryl was first used. This step eluted two fractions: esterases and pectin esterase. The second step was affinity chromatography on a 4B Sepharose EAH grafted with chlorogenic acid. This step eluted two fractions: an unretained fraction containing the phenyl esterase and the depsidase; and a retained fraction with the cinnamate esterase. This enzyme has a molecular weight of 240 kDa and contains two submits of 120 kDa. A new classification for these three esterases is suggested. © 1998 Society of Chemical Industry.     

Untersuchungen an Kartoffelpulpe als Ballaststoffquelle. Zum Einfluß Von pektolytischen und cellulolytischen Enzymen

Investigations on potato pulp as a dietary fiber source. The influence of pectolytic and cellulolytic enzymes . The influence of treatment with pectolytic and cellulolytic enzyme preparations was investigated with reference to the separation of water and the composition of potato pulp. In contrast to pectinesterase, pectate lyase or cellulase it was found an intensive action on the pulp after incubation with Pectinex Ultra SP-L or pectinase/cellulase combinations. The content of pectin, starch and protein as well as the water binding capacity are varied in dependence of the used enzyme preparations. The occurring changes in the supermolecular structure of the potato pulp tissue are investigated by scanning electron microscopy. The grown biological structure is partly or extensive destroyed especially after combined action of pectinases and cellulases. The content of starch in the potato pulp preparations remains relatively high even after intensive treatment with cell wall degrading enzymes.     

Enhanced inactivation of pectin methyl esterase in orange juice using modified supercritical carbon dioxide treatment

The aim of the study is to quantify the effect of ethanol addition and exposure surface on the inactivation of pectin methyl esterase (PME), a juice clarifying enzyme, in orange juice using supercritical carbon dioxide (SC‐CO₂). Addition of ethanol to the SC‐CO₂ at 2% (v/v) caused greater inactivation than SC‐CO₂ alone, with a maximum reduction of PME activity of 97% at 30 MPa and 40 °C for 60 min. As the surface area to volume ratio was increased, the rate of inactivation of PME increased. Analysis of first‐order reaction kinetic data revealed that D values were greatly influenced by ethanol addition and agitation. With the addition of 2% ethanol, the D value reduced by half, that is, 56 min from 109 min. With impeller agitation of the sample at 1100 ± 100 rpm, the D value for PME was further reduced to 43 and 30 min without and with ethanol, respectively. The activity of PME treated with SC‐CO₂ remained unchanged after 14 days of storage at 4 °C. Treatment did not significantly change pH or colour, but did significantly increase the cloud values of the juice, resulting in a cloud stabilised juice with similar qualities to fresh juice.     

Enzymatic degradation studies of pectin and cellulose from red beets.

The influence of structural features of the cell wall polysaccharides pectin and cellulose on the enzymatic degradation of red beet was evaluated. Alcohol-insoluble substances and acetone-insoluble residues were prepared from red beets and characterized with respect to the content of dietary fibre, pectin fractions, neutral saccharide composition and water absorption. The high-methylated and high-acetylated pectin component was partly soluble in water and in EDTA. Pectin was hardly extractable from alcohol-insoluble substances as well as from red beets. Isolated pectin could not be completely degraded by pectolytic enzymes. After de-acetylation, the pectic acid from red beets was degradable in a similar rate like citrus pectic acid. From alcohol-insoluble substances, several cellulose and lignin fractions were prepared and analysed. A cellulose preparation from red beets was intensely degraded by cellulases with high activities as shown by the release of reducing end-groups, viscosity and scanning electron microscopy. Cell wall preparations from red beets were able to bind high amounts of water. A decrease in water absorption during enzymatic action or changes in viscosity and flow behaviour are sensitive markers for decomposition or depolymerization processes. Furthermore, an inhibitor of microbial enzymes was isolated from red beets and acetone-insoluble residues. The main reason for the poor enzymatic liquefaction or maceration of red beets by pectolytic and cellulolytic enzymes is the high degree of acetylation of the pectin component.     

Enzymatic degradation of plant cell wall polysaccharides: The kinetic effect of competitive adsorption

Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP^® from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomponent enzymes from Pectinex Ultra SP^® (arabinofuranosidase, endoglucanase II, pectin lyase, polygalacturonase I, rhamnogalacturonan acetyl esterase, rhamnogalacturonase a, rhamnogalacturonase b and xylanase I) were added in order to increase the yield. Surprisingly, however, the yield is not increased when any of the monocomponent enzymes are added. To describe the results a new model designated ‘the competitive activity adsorption model’ is proposed. The model is based on the fact that the enzymes are adsorbed to the substrate before action. A combination of the Langmuir adsorption isotherm and basic enzyme kinetics shows that different enzymes that adsorb competitively will have an inhibitory effect on each other and consequently decrease the hydrolysis rate and thereby the yield. The model has been confirmed by an experiment in which the fibre has been pre-treated with rhamnogalacturonan acetyl esterase.     

Experimental design and response surface modeling applied for the optimisation of pectin hydrolysis by enzymes from A. niger CECT 2088

Optimisation of pectin hydrolysis using pectolytic enzymes produced by Aspergillus niger CECT 2088 was carried out. The effect of enzyme concentration (0.018–0.072%, w/v), substrate concentration (0.004–0.896%, w/v), pH (4.49–5.91), temperature (32.2–67.8 °C) and reaction time (18.2–71.8 min) on the enzymatic process was studied. The experiments were arranged according to a central composite statistical design. Response surface methodology (RSM) was used to assess factor interactions and empirical models regarding four product response variables (i.e., reactor conversion, reducing sugar concentration, endopectolytic productivity and enzymatic depolymerization productivity). Multi-response optimisation was also performed on the reactor and endopectolytic productivity data set of the factorial design. The highest reactor conversion (61%) and endopectolytic productivity (7.2 cSt mg⁻¹ ml⁻¹) were achieved with 1 h reaction time at 46 °C and pH 4.8 at a substrate/enzyme ratio of 11.6.     

Characterisation of gold kiwifruit pectin isolated by enzymatic treatment

Pectin was extracted from gold kiwifruit by four commercial enzyme preparations (Celluclast 1.5 L, Cytolase CL, Cellulyve TR 400 and NS33048). The chosen enzymes were used either in single or in combination, with and without protease addition. The recovered pectin was characterised and compared for the yield, total nonstarch polysaccharide and neutral sugar composition, protein and ash, pectin, molecular weight distribution and the viscosity. Results indicated that enzyme‐extracted gold kiwifruit pectin was rich in galacturonic acid. Purified pectin yield, and their physicochemical composition and rheological property (viscosity), was significantly affected by the type of enzyme used. The pectin extracted by Celluclast 1.5 L demonstrated to be the most viscous and recorded the highest in molecular weight (M_w) (1.65 × 10⁶ g mol^–1) compared with the other extracts. The extract M_w and their distribution were discussed and related to their viscosity behaviour.     

Effect of enzymatic hydrolysis on the yield of cloudy carrot juice and the effects of hydrocolloids on color and cloud stability during ambient storage

The color and cloud stability of cloudy carrot juice were improved by enzymatic hydrolysis and addition of hydrocolloids. Cellulytic and pectolytic enzyme preparations were used to prepare the carrot juice and their optimum dosages were 1.6 g kg^?1 and 0.035 g kg^?1, respectively. Hydrocolloids including guar gum, pectin and flaxseed gum were each added to the carrot juice and assessed for their ability to stabilize the carrot juice. Pectin had no effect on the cloud stability of carrot juice. Guar gum and flaxseed gum stabilized the cloudy carrot juice owing to macromolecular steric repulsion, while flaxseed gum reduced the creaming of cloudy carrot juice owing to its emulsification. The color and β‐carotene content of carrot juice did not change during 6 months of storage. Copyright © 2004 Society of Chemical Industry     

The extraction of yellow gentian root (Gentiana lutea L .)

 Several solvents have been investigated for the preparation of bitter compounds of gentian roots (Gentiana lutea L.) for food applications. The highest concentrations of the bitter compounds, amarogentin and gentiopicroside, were obtained with ethanol : water 55 : 45 (v/v), propylene glycol: water 30 : 70 (v/v) and ethanol: propylene glycol: water 20 : 20 : 60 (v/v/v). Enzyme treatment prior to solvent extraction gave a greater extract yield (3.5%) but the amarogentin and gentiopicroside concentrations remained the same. The volatile fraction was affected by the solvent used through the formation of esters of organic acids from the plant. Received: 22 January 1997 / Revised version: 18 March 1997     

Enzymatic degradation studies of pectin and cellulose from red beets

The influence of structural features of the cell wall polysaccharides pectin and cellulose on the enzymatic degradation of red beet was evaluated. Alcohol‐insoluble substances and acetone‐insoluble residues were prepared from red beets and characterized with respect to the content of dietary fibre, pectin fractions, neutral saccharide composition and water absorption. The high‐methylated and high‐acetylated pectin component was partly soluble in water and in EDTA. Pectin was hardly extractable from alcohol‐insoluble substances as well as from red beets. Isolated pectin could not be completely degraded by pectolytic enzymes. After de‐acetylation, the pectic acid from red beets was degradable in a similar rate like citrus pectic acid. From alcohol‐insoluble substances, several cellulose and lignin fractions were prepared and analysed. A cellulose preparation from red beets was intensely degraded by cellulases with high activities as shown by the release of reducing end‐groups, viscosity and scanning electron microscopy. Cell wall preparations from red beets were able to bind high amounts of water. A decrease in water absorption during enzymatic action or changes in viscosity and flow behaviour are sensitive markers for decomposition or depolymerization processes. Furthermore, an inhibitor of microbial enzymes was isolated from red beets and acetone‐insoluble residues. The main reason for the poor enzymatic liquefaction or maceration of red beets by pectolytic and cellulolytic enzymes is the high degree of acetylation of the pectin component.     

Effect of pH on the in vitro interactions between bile acids and pectin

 The interactions between pectin and bile acids (BA) were investigated after in vitro incubation at 37°C in the pH range of 5.0 – 8.0, separation of the non-absorbed BA using membrane filtration and their estimation by HPLC and HPTLC. In general, the interactions increased with the degree of esterification and the molecular weight (viscosity) of the pectin. They were reduced if the carboxyl groups in the polysaccharide molecules are arranged in a blockwise manner and in the presence of Ca²⁺. The highest interactions were found for unconjugated BA, followed by the corresponding glycine-conjugates. Taurine-conjugates showed the lowest interaction with pectin. Chenodeoxycholic acid and deoxycholic acid reacted more effectively than the trihydroxy bile acid cholic acid in the in vitro experiments. The results show that structural parameters of both BA and pectin play an important role in the extent of their interaction. Received: 28 November 1996 / Revised version: 20 January 1997     

Degradation of apple cell wall material by commercial enzyme preparations

The action of commercial enzyme preparations on the release of cell wall constituents from alcohol-insoluble substance prepared from apples without skins and cores as well as their influence on the water binding of remaining residues is described as a model for the enzymatic cell wall destruction during production of liquid fruit products. Besides 'normal' enzyme concentrations adapted from the usual industrial dosage, 'tenfold' enzyme concentrations were applied. Dependent on enzyme spectrum and activities, concentrations of dietary fibre, e.g., pectin, increased in the soluble fractions using conditions of enzymatic 'mash treatment'. A further release of these cell wall constituents occurred when cellulase containing enzyme preparations were used under conditions of 'pomace treatment', especially with the 'tenfold' enzyme dosage. The partial enzymatic degradation of the cell wall material is connected with a decrease in water binding of the remaining residues during both simulated mash treatment of pomace treatment. Alcohol-insoluble substance from apples is a suitable model for the determination of complex enzymatic actions of enzyme preparations containing pectolytic, hemicellulolytic, and/or cellulolytic activities under standardised conditions.