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The food enzyme containing endo‐polygalacturonase and β‐glucosidase (EC 3.2.1.15 and EC 3.2.1.21) is produced with the non‐genetically modified Aspergillus tubingensis strain ARO by DSM Food Specialties B.V. The food enzyme was free from viable cells of the production organism. It is intended to be used in five food manufacturing processes. Dietary exposure was estimated to be up to 0.609 mg total organic solids (TOS)/kg body weight per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2217 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure of at least 3640. A search for the homology of the amino acid sequence of the food enzymes to known allergens was made and four matches with food allergens and 22 matches with respiratory allergens were found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Polygalacturonases (PG) are the enzymes that cause depolymerization of pectin. In current study, bacterial strain was isolated from rotten sample and then purified. It was screened for endo-polygalacturonase activity using PSAM media. It had shown positive response for endo-PG activity. Bacterial DNA was isolated and 16 S rRNA gene amplification was done using universal primer pair of 8 F and 1492 R. Bacterial strain was also amplified by 16 S rRNA gene for sequencing. BLAST of gene sequence on NCBI database had shown that bacterial isolate was identified as Bacillus pumilus. Endo-polygalacturonase enzyme was produced by submerged fermentation to optimize culture conditions by RSM in JMP-12 software. The optimized parameters had shown maximum endo-polygalacturonase activity of 153.2 U/mL/min after using 3 g of orange peels as substrate, 3 mL of inoculum size, 6.5 pH buffer, 40°C temperature and 3 days of incubation. After optimizing fermentation conditions, endo-polygalacturonase was precipitated using 70 % concentration of ammonium sulfate. This partially precipitated sample was dialyzed with dialysis tube and used to find activity of endo-polygalacturonase (1620.6 U/mL/min). This sample was applied to gel filtration chromatography for further purification. Endo-polygalacturonase activity increased many times 2231.44 U/mL/min after gel filtration. The Molecular weight of endo-polygalacturonase was 46 kDa after SDS PAGE characterization. High Vmax value and alkaliphilic nature of endo-polygalacturonase will make it good candidate for food and feed industry near future. [Display omitted] • A bacterial strain was isolated from rotten sample. • Bacillus pumilus was identified through 16 S rRNA gene sequencing. • Maximum enzyme activity of 153.2 U/mL/min was achieved under optimized conditions. • The characterized endo-polygalacturonase has great potential in industrial applications. • Polygalacturonase had shown maximum activity after column chromatography. [ABSTRACT FROM AUTHOR]

A novel endo -polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH 2 -terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na+, Ca2+, Mg2+, Mn2+, and Zn2+) positively affect PGC-AN64 activity, while Ba2+, Cd2+, and Cr3+ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification. [Display omitted] • Orange peel waste is used as substrate for pectinase production from A. niger HO32. • A novel thermostable endo -polygalacturonase PGC AN64 was purified and characterized. • PGC AN64 is a monomer of 64 kDa and its optimal activity was at pH 6.5 and 70 °C. • It exhibited a good substrate specificity and k cat /K m than PECLYVE V and PECLYVE CP. • This pectinase is considered as possible candidate for clarification of orange juice. [ABSTRACT FROM AUTHOR]

The food enzyme has four declared activities: endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase (endo‐cleaving); EC 3.2.1.15), pectinesterase (pectin pectylhydrolase; EC 3.1.1.11), pectin lyase ((1–4)‐6‐O‐methyl‐α‐d‐galacturonan lyase; EC 4.2.2.10) and non‐reducing end α‐l‐arabinofuranosidase (α‐l‐arabinofuranoside non‐reducing end α‐l‐arabinofuranosidase; EC 3.2.1.55). It is produced with the non‐genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant has requested to extend its use to include four additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme–TOS was estimated only for the remaining six processes. The dietary exposure was calculated to be up to 0.612 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (204 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 333. Based on the previous evaluation, the assessment of the new data and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme endo‐polygalacturonase ((1 → 4)‐α‐d‐galacturonan glycanohydrolase EC 3.2.1.15) is produced with the genetically modified Aspergillus oryzae strain AR‐183 by AB ENZYMES GmbH. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. As the food enzyme‐total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme‐TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.087 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 11,494. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme endo‐polygalacturonase ((1→4)‐α‐D‐galacturonan glycanohydrolase (endo‐cleaving); EC 3.2.1.15)) is produced with the non‐genetically modified Aspergillus tubingensis strain MUCL 55013 by Soufflet Biotechnologies. The food enzyme is free from viable cells of the production organism. It is intended to be used in 10 food manufacturing processes: processing of fruits and vegetables for the production of juices, other fruit and vegetable products, wine, distilled spirits from wine, alcoholic beverages other than grape wine; processing of plant‐derived products for the production of refined and unrefined sugar, edible oils from plants, green coffee beans by demucilation, coffee extracts and tea and other herbal and fruit infusions. Since residual amounts of total organic solids (TOS) are removed in three processes, dietary exposure was calculated only for the remaining seven food manufacturing processes. Exposure was estimated to be up to 7.834 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2,097 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure of at least 268. A search for the similarity of the amino acid sequence of the food enzyme to known allergens found 14 matches, one of which was to a food allergen. The Panel considered that the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya, but that the risk will not exceed that of consumption of papaya. In addition, oral allergy reactions cannot be excluded in pollen‐sensitised individuals. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Abstract Background Hedera nepalensis is a traditional medicinal plants, and the dried leaves of it are generally used for the cure and treatment of many diseases, also widely known as Chang-Chun-Teng in Chinese. Until now, structural characterization of water-soluble polysaccharides isolated from leaves of Hedera nepalensis have been scarcely studied, even though the chemical compounds derived from it and their biological activities have been widely studied. Methods Water-soluble polysaccharides (WHNP) were isolated from the dried leaves of Hedera nepalensis, and their structural features were investigated. One neutral polysaccharide fraction (WHNP-N) and three major pectin fractions (WHNP-A2b, WHNP-A2c and WHNP-A3b) were obtained from WHNP, respectively. There was no analysis of the neutral fraction (WHNP-N), while the structural characterization of three major pectin fractions (WHNP-A2b, WHNP-A2c and WHNP-A3b) were further studied by monosaccharide composition, HPGPC, NMR and methylation analyses. Results The results indicated that two fractions WHNP-A2b (Mw = 45.8 kDa) and WHNP-A3b (Mw = 58.6 kDa) were mainly composed of rhamnogalacturonan I (RG-I). In WHNP-A2b, RG-I domains primarily substituted with α-L-1,5/1,3,5-arabinan, type II arabinogalactan (AG-II), β-D-1,4-galactan and/or type I arabinogalactan (AG-I) as side chains, while RG-I-type pectin of WHNP-A3b mainly branched with α-L-1,5/1,3,5-arabinan, β-D-1,4-galactan and AG-II side chains. WHNP-A2c (Mw = 12.4 kDa) was primarily comprised of galacturonic acid (GalA, 60.8%), and enzymatic analysis indicated that this fraction mainly consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HG) domains with mass ratios of 1.8:1.0:0.6. On the other hand, WHNP-A2c was found to be rich in RG-I domains, which contained α-L-1,5/1,3,5-arabinan, AG-II, β-D-1,4-galactan and/or AG-I as side chains. The HG domains of WHNP-A2c was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with a 1 to 7 degree of polymerization after endo-polygalacturonase degradation. Conclusion Our results reveal the structural characteristics of these polysaccharide fractions, which will contribute to elucidating their structure–activity relationships. Graphical Abstract

The food enzyme with the declared activities endo‐polygalacturonase ((1–4)‐α‐D‐galacturonan glycanohydrolase; EC 3.2.1.15) and pectin lyase ((1–4)‐6‐O‐methyl‐α‐D‐galacturonan lyase; EC 4.2.2.10) is produced with the non‐genetically modified Aspergillus tubingensis strain NZYM‐PE by Novozymes A/S. It is intended to be used in four food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, refined olive oil production and wine and wine vinegar production. Since residual amounts of total organic solids (TOS) are removed during production, dietary exposure was not calculated for refined olive oil production. For the remaining three food processes, it was estimated to be up to 0.132 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,430 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure above 10,833. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 13 matches were found, including one food allergen (papaya). The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya, but that the risk will not exceed that of consumption of papaya. In addition, oral allergy reactions cannot be excluded in pollen‐sensitised individuals. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Background: Hedera nepalensis is a traditional medicinal plants, and the dried leaves of it are generally used for the cure and treatment of many diseases, also widely known as Chang-Chun-Teng in Chinese. Until now, structural characterization of water-soluble polysaccharides isolated from leaves of Hedera nepalensis have been scarcely studied, even though the chemical compounds derived from it and their biological activities have been widely studied. Methods: Water-soluble polysaccharides (WHNP) were isolated from the dried leaves of Hedera nepalensis, and their structural features were investigated. One neutral polysaccharide fraction (WHNP-N) and three major pectin fractions (WHNP-A2b, WHNP-A2c and WHNP-A3b) were obtained from WHNP, respectively. There was no analysis of the neutral fraction (WHNP-N), while the structural characterization of three major pectin fractions (WHNP-A2b, WHNP-A2c and WHNP-A3b) were further studied by monosaccharide composition, HPGPC, NMR and methylation analyses. Results: The results indicated that two fractions WHNP-A2b (Mw = 45.8 kDa) and WHNP-A3b (Mw = 58.6 kDa) were mainly composed of rhamnogalacturonan I (RG-I). In WHNP-A2b, RG-I domains primarily substituted with α-L-1,5/1,3,5-arabinan, type II arabinogalactan (AG-II), β-D-1,4-galactan and/or type I arabinogalactan (AG-I) as side chains, while RG-I-type pectin of WHNP-A3b mainly branched with α-L-1,5/1,3,5-arabinan, β-D-1,4-galactan and AG-II side chains. WHNP-A2c (Mw = 12.4 kDa) was primarily comprised of galacturonic acid (GalA, 60.8%), and enzymatic analysis indicated that this fraction mainly consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HG) domains with mass ratios of 1.8:1.0:0.6. On the other hand, WHNP-A2c was found to be rich in RG-I domains, which contained α-L-1,5/1,3,5-arabinan, AG-II, β-D-1,4-galactan and/or AG-I as side chains. The HG domains of WHNP-A2c was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with a 1 to 7 degree of polymerization after endo-polygalacturonase degradation. Conclusion: Our results reveal the structural characteristics of these polysaccharide fractions, which will contribute to elucidating their structure–activity relationships. [ABSTRACT FROM AUTHOR]

The food enzyme endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain RF6197 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.156 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6,410. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and matches were found with a number of pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

A large-scale screening of pectinolytic activity in the yeast Saccharomyces paradoxus isolated from various natural sources in Europe, Asia, North America, and the Hawaiian Islands was carried out. Of the 98 studied strains, pectinolytic activity was absent only in five Hawaiian and two European strains. Most strains were able to secrete active endo-polygalacturonase. North American strains UCDFST 52-225, UCDFST 61-359, UCDFST 61-220, 95-3, and UCDFST 62-186 had very high pectinolytic activity, comparable to or even higher than that of the experimentally obtained tetraploid strain S. cerevisiae VKPM Y-718. Comparative analysis of the nucleotide and amino acid sequences of pectinase genes showed that the North American and Far Eastern populations of S. paradoxus were more genetically diverse than the European and Hawaiian ones. Phylogenetic analysis confirmed the species specificity of the PGU genes of Saccharomyces yeasts. Of the eight Saccharomyces species, high pectinolytic activity is characteristic of S. bayanus and S. paradoxus. Five North American strains with the highest pectinolytic activity are of interest for further molecular genetic studies and breeding work with wine yeasts. The ecological role of endo-polygalacturonase is discussed. [ABSTRACT FROM AUTHOR]

The endo -polygalacturonase D (PgaD) from Aspergillus niger JL15 was recombinantly expressed in Escherichia coli BL21, exhibiting an optimal activity at 55 °C and pH 4.0. Hydrolysis products of citrus pectin by recombinant PgaD included galacturonic acid (GalA), digalacturonic acid (GalA2), trigalacturonic acid (GalA3), and tetragalacturonic acid (GalA4). The hydrolysates exhibited significant antioxidant capacity and dose-dependent competitive inhibition of α-glucosidase. GalA2 and GalA3 acted as competitive inhibitors of α-glucosidase, with inhibition constant of 0.0589 mmol.L−1 and 0.6732 mmol.L−1, respectively. Molecular dynamics (MD) simulations revealed that both GalA2 and GalA3 penetrated the catalytic pocket of α-glucosidase and formed stable hydrogen bonds with key catalytic residues D352 and D215. The binding free energies of GalA2-α-glucosidase and GalA3-α-glucosidase complexes were − 10.3 ± 0.6 kcal·mol−1 and -10.8 ± 0.7 kcal·mol−1, respectively. These findings might offer new ideas for the development of α-glucosidase inhibitors sourced from citrus pectin, as well as enhance utilization of the renewable plant polysaccharide resources. [Display omitted] • Citrus pectin hydrolysates produced by rePgaD exhibited significant antioxidant activity. • The hydrolysates, along with GalA2 and GalA3, acted as competitive inhibitors of α-glucosidase. • GalA2 and GalA3 effectively bind to the catalytic pocket of α-glucosidase, forming stable NCI with key sites. • This study offers new insights into probing α-glucosidase inhibitors sourced from citrus pectin. [ABSTRACT FROM AUTHOR]

The food enzyme endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Aspergillus niger strain EPG by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in fruit and vegetable processing for juice production. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.122 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,014 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure at least 8,311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 38 matches were found, two of which are food allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya or maize, but that the risk will not exceed that of consumption of papaya or maize. In addition, oral allergy reactions cannot be excluded in pollen‐sensitised individuals. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme containing endo‐polygalacturonase ((1–4)‐α‐d‐galacturonan glycanohydrolase; EC 3.2.1.15) and cellulase (4‐(1,3;1,4)‐β‐d‐glucan 4‐glucanohydrolase; EC 3.2.1.4) activities is produced with the non‐genetically modified Talaromyces cellulolyticus strain NITE BP‐03478 by Meiji Seika Pharma Co., Ltd. It is intended to be used in eight food manufacturing processes: baking processes, brewing processes, fruit and vegetable processing for juice production, wine and wine vinegar production, fruit and vegetable processing for products other than juices, fruit and vegetable processing for refined olive oil production, coffee bean demucilation and grain treatment for starch production. Since residual amounts of total organic solids (TOS) are removed during three food processes (refined olive oil production, coffee bean demucilation and grain treatment for starch production), dietary exposure was not calculated for these food processes. For the remaining five food processes, dietary exposure was estimated to be up to 3.193 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 806 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 252. A search for the similarity of the amino acid sequences of the food enzyme to known allergens was made and six matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, especially in individuals sensitised to pollen. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

The food enzyme endo‐polygalacturonase (1→4)‐α‐d‐galacturonan glycanohydrolase EC 3.2.1.15 is produced with the genetically modified Aspergillus oryzae strain AR‐183 by AB ENZYMES GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids (TOS) are removed by repeated washing or distillation, dietary exposure to the food enzyme TOS from coffee demucilation and from the production of flavouring extracts was considered not necessary. For the remaining three food processes, dietary exposure was estimated to be up to 0.087 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 11,494. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to pollen allergens, cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of pectin followed by ultrafiltration of hydrolysate was applied with the aim to produce fractions with potent antioxidant capacity. Pectin was isolated from waste sugar beet pulp by acidic extraction, washed by diafiltration and concentrated by ultrafiltration. Enzymatic hydrolysis was performed with endo-polygalacturonase, and hydrolysate was processed by ultrafiltration into four fractions using membranes in series of decreasing cut-offs from 10 to 1 kDa. Hydrolysis with endo-polygalacturonase increased total antioxidant capacity by twofold in comparison to un-hydrolyzed pectin. Antioxidant capacity of all fractions was considerably higher than that of pectin—from 14.7 to 25-fold, for fraction containing fragments 10 kDa > Mw > 5 kDa and Mw < 1 kDa, respectively. Considerable increase of total antioxidant capacity of pectin through the integration of enzymatic modification and ultrafiltration fractionation indicated great potential of applied green protocol for the production of high-value hydrolysates of pectin from waste sugar beet pulp. [ABSTRACT FROM AUTHOR]

For the first time, a large-scale screening has been carried out of pectinolytic activity in Saccharomyces bayanus strains isolated in various regions of the world from fermentation processes and natural sources. The ability to secrete active endo-polygalacturonase was shown to be a feature of this species. Regardless of the source and place of isolation, S. bayanus var. uvarum, S. bayanus var. bayanus and S. eubayanus have the PGU1bPGU2bPGU3b genotype. According to phylogenetic analysis, the pectinase genes (PGU) of the hybrid brewer's yeast S. pastorianus are originated from the cryophilic S. bayanus yeast and not from S. cerevisiae. Five selected S. bayanus strains with the highest pectinolytic activity are promising for further molecular genetic studies and breeding of wine yeasts. [ABSTRACT FROM AUTHOR]

The food enzyme has two declared activities, endo‐polygalacuronase ((1→4)‐α‐D‐galacturonan glycanohydrolase; EC 3.2.1.15) and endo‐1,3(4)‐β‐glucanase (3‐(1→3;1→4)‐β‐D‐glucan 3(4)‐glucanohydrolase; EC 3.2.1.6) and is produced with the non‐genetically modified Aspergillus fijiensis strain NZYM‐RE by Novozymes A/S. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in eight food manufacturing processes, i.e. distilled alcohol production, brewing processes, baking processes, cereal‐based processes, wine and wine vinegar production, fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices and refined olive oil production. Since residual amounts of total organic solids (TOS) are removed during distilled alcohol production and refined olive oil production, dietary exposure was not calculated for these two processes. For the remaining six food manufacturing processes, dietary exposure was estimated to be up to 0.553 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 3,677 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure of at least 6,649. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and nine matches were found. The Panel considered that, under the intended conditions of use (other than distilled alcohol production), the risk of allergic reactions by dietary exposure to this food enzyme, particularly in individuals suffering from the oral allergy syndrome or sensitised to papaya, cannot be excluded. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

In this study, a potential producer of prebiotics, a novel endo-polygalacturonase pePGA from Penicillium rolfsii BM-6, was successfully expressed in Komagataella phaffii, characterized and applied to produce pectic oligosaccharides. The optimum temperature and pH of pePGA were 60 °C and 6.0. The purified recombinant enzyme showed a good pH stability and was stable from pH 3.5 to 8.0. The Km, Vmax and kcat values of pePGA were 0.1569 g/L, 12,273 μmol/min/mg and 7478.4 s−1, respectively. More importantly, pePGA-POS, the pePGA hydrolysis products from commercial pectin, had good prebiotic and antibacterial activities in vitro. The pePGA-POS was able to significantly promote the growth of probiotics; meanwhile, the growth of Escherichia coli JM109, Staphylococcus aureus and Bacillus subtilis 168 was effectively inhibited by pePGA-POS. In addition, pePGA-POS also had the DPPH radical scavenging capacity. These properties of pePGA-POS make pePGA attractive for the production of prebiotics. [ABSTRACT FROM AUTHOR]

The food enzyme has four declared activities (endo-polygalacturonase ((1→4)-α-D-galacturonan glycanohydrolase (endo-cleaving); 3.2.1.15), pectinesterase (pectin pectylhydrolase; 3.1.1.11), pectin lyase ((1→4)-6-O-methyl-α-D-galacturonan lyase; 4.2.2.10) and non-reducing end α-L-arabinofuranosidase (α-L-arabinofuranoside non-reducing end α-L-arabinofuranosidase; 3.2.1.55) and is produced with the non-genetically modified Aspergillus niger strain PEC by DSM Food Specialties B.V. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in the manufacture of alcoholic beverages from fruits other than grapes, fruit and vegetable processing for juice production, and wine and wine vinegar production. Dietary exposure was estimated to be up to 0.25 mg TOS/kg bodyweight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 204 mg TOS/kg bw per day, the highest dose tested which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 800. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and several matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, particularly for individuals sensitised to several pollen allergens or papaya allergens. Based on the data provided, the Panel concluded that this food enzyme did not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Pectins are nutrient components of plants and are widely used in the food industry. In this study, one major pectin fraction (WLJP-A0.2b) with Mw of 40.6 kDa was purified from Lonicera japonica Thunb. The structural feature and antioxidant activity of it was investigated. Monosaccharide composition, Fourier transform infrared (FT-IR) spectra, enzymatic hydrolysis, and nuclear magnetic resonance (NMR) spectra analysis indicated that WLJP-A0.2b consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HG) domains, with mass ratio of 0.4:1.0:2.1. The RG-I domain contained highly branched α-L-1,5-arabinan, β-D-1,4-galactan and type II arabinogalactan (AG-II) side chains. The HG domain was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with degree of polymerization 1–8 after degradation by endo-polygalacturonase. Radical scavenging assays indicated that WLJP-A0.2b exhibited antioxidant activity through the synergistic effects of different pectin domains. Oligogalacturonides, especially de-esterified oligogalacturonides, showed better antioxidant activities than RG-II and RG-I domains. Moreover, de-esterified oligogalacturonides remarkably reduced H2O2-induced reactive oxygen species production in HEK-293T cells. These results provide useful information for screening of natural antioxidants from Lonicera japonica Thunb. and application of pectin in functional food field.

Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1–10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.

Simple Summary: Grape skins, usually discarded during wine making, are a valuable source of cellulose (20–50%), hemicelluloses (15–20%), lignin (17–30%) and other compounds, e.g., polyphenols, which can be used as biomaterials in the manufacturing of a variety of new products, such as bioethanol or pharmaceutical products. However, to obtain these biomaterials, the complex polysaccharides of the grape cell walls must be broken down into smaller molecules to allow the extraction of compounds. The degradation process is often performed enzymatically or hydrothermally. Microorganisms that produce the required enzymes while using this waste product as a growth medium can have interesting economic advantages. Here, we created two genetically engineered wine yeast strains that produce grape cell wall degrading enzymes. These yeasts, when grown on grape pomace, induced enzymatic structural changes to the grape cell walls. A collection of antibodies binding to the different cell wall molecules were used to monitor the impact on the cell wall structure of the enzymes, confirming increased extractability of key cell wall polymers when relatively low levels of enzymes are present, illustrating the potential to develop and optimise yeast for grape waste valorisation applications. Industrial wine yeast strains expressing hydrolytic enzymes were fermented on Chardonnay pomace and were shown to unravel the cell walls of the berry tissues according to the enzyme activities. The yeasts produced a native endo-polygalacturonase (Saccharomyces cerevisiae × Saccharomyces paradoxus hybrid, named PR7) and/or a recombinant endo-glucanase (S. cerevisiae strains named VIN13 END1 and PR7 END1). The impact of the enzymes during the fermentations was evaluated by directly studying the cell wall changes in the berry tissues using a Comprehensive Microarray Polymer Profiling technique. By the end of the fermentation, the endo-glucanase did not substantially modify the berry tissue cell walls, whereas the endo-polygalacturonase removed some homogalacturonan. The recombinant yeast strain producing both enzymes (PR7 END1) unravelled the cell walls more fully, enabling polymers, such as rhamnogalacturonan-I, β-1,4-D-galactan and α-1,5-L-arabinan, as well as cell wall proteins to be extracted in a pectin solvent. This enzyme synergism led to the enrichment of rhamnogalacturonan-type polymers in the subsequent NaOH fractions. This study illustrated the potential utilisation of a recombinant yeast in pomace valorisation processes and simulated consolidated bioprocessing. Furthermore, the cell wall profiling techniques were confirmed as valuable tools to evaluate and optimise enzyme producing yeasts for grape and plant cell wall degradation. [ABSTRACT FROM AUTHOR]

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo -PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo -PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo -PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo -polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo -polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions. [ABSTRACT FROM AUTHOR]

In this study, a potential producer of prebiotics, a novel endo-polygalacturonase pePGA from Penicillium rolfsii BM-6, was successfully expressed in Komagataella phaffii, characterized and applied to produce pectic oligosaccharides. The optimum temperature and pH of pePGA were 60 °C and 6.0. The purified recombinant enzyme showed a good pH stability and was stable from pH 3.5 to 8.0. The Km, Vmax and kcat values of pePGA were 0.1569 g/L, 12,273 μmol/min/mg and 7478.4 s−1, respectively. More importantly, pePGA-POS, the pePGA hydrolysis products from commercial pectin, had good prebiotic and antibacterial activities in vitro. The pePGA-POS was able to significantly promote the growth of probiotics; meanwhile, the growth of Escherichia coli JM109, Staphylococcus aureus and Bacillus subtilis 168 was effectively inhibited by pePGA-POS. In addition, pePGA-POS also had the DPPH radical scavenging capacity. These properties of pePGA-POS make pePGA attractive for the production of prebiotics.

The distribution and properties of the pectinase-encoding PGU genes in different species of Saccharomyces yeasts was studied. Application of molecular karyotyping and Southern hybridization revealed that S. arboricola, S. cariocanus, S. cerevisiae, S. kudriavzevii, and S. paradoxus species had a single PGU gene located on chromosome X. The other three species had polymeric PGU genes of different chromosomal localization: S. mikatae and S. jurei (chromosomes X and VIII), S. bayanus (X, I, and XIV). This is the first report on comparative analysis of the nucleotide and amino acid sequences of the PGU genes was carried out in all eight species of the genus Saccharomyces. Species-specificity of the PGU genes was revealed, as well as their intraspecific polymorphism in S. kudriavzevii and S. paradoxus, associated with the geographical origin of the strains. The most divergent proteins were Pgu1a (S. arboricola) and Pgu1b, Pgu2b, Pgu3b (S. bayanus), for which the level of similarity to the Pgu proteins of other Saccharomyces species did not exceed 89%. The highest similarity (>95%) was noted for the Pgu proteins of S. cerevisiae, S. paradoxus, and S. cariocanus, as well as S. mikatae and S. jurei. Significant intraspecific polymorphism of endo-polygalacturonase secretion was observed in the studied Saccharomyces species, except for the species S. bayanus, all studied strains of which had a relatively high activity. The ability to secrete active endo-polygalacturonase is probably a specific feature of this species. [ABSTRACT FROM AUTHOR]

A molecular genetic study of pectinase PGU genes from 74 strains of the yeast Saccharomyces bayanus var. uvarum isolated from various fermentation processes and natural sources in different regions of Europe and the United States has been performed. Unlike the S. cerevisiae yeasts, each having a single PGU gene, the S. bayanus var. uvarum strains have three divergent genes, PGU1b, PGU2b, and PGU3b, which are located respectively on chromosomes X, I, and XIV. The high pectinolytic activity of these yeasts appears to be related to the presence of several PGU polymeric genes in their genomes. [ABSTRACT FROM AUTHOR]

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZαA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The enzyme had optimum activity at pH 5.5 and 40 °C with stability between pH 5.0 and 8.0 and at temperatures up to 50 °C. The enzyme activity was enhanced by 41% with the addition of 1 mM Co++, and inhibited by Fe++ with a 63% reduction. The mode of the enzyme action showed internal cleavage of α-1,4 glycoside bonds of polygalacturonic acid and citrus peel pectin. Trigalacturonate and hexagalacturonate were the main hydrolysis products, with a yield of 0.44±0.01 and 0.21±0.01 mg released per mg polygalacturonic acid substrate, respectively. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products.

Protein engineering has been a research hotspot to improve the catalytic efficiency of industrially important enzymes. In the present study, a novel computational strategy was developed to in silico screen mutants with enhanced binding interaction between enzyme and substrate as well as catalytic efficiency. Through homology modeling and molecular dynamics (MD) simulation, four key residues related to substrate binding were identified in the endo-polygalacturonase BiPG28A from Bispora sp. MEY-1. Further analyses of the conformation, hydrogen bond interactions, and binding free energy revealed that lysine at position 129 (subsite − 2) has the strongest affinity to substrate. Biochemical and calorimetry experiments confirmed the functional role of Lys129 in substrate binding through non-covalent interactions. The common role of Lys129 was also verified in another GH28 endo-polygalacturonase. Distinguished from other protein engineering strategies involving structure resolution and construction of certain enzymes, this computational strategy represents an insightful and efficient approach to develop a “designed” enzyme with significantly enhanced binding affinity and catalytic efficiency. [ABSTRACT FROM AUTHOR]

Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum . Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7 U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants K m and V max of PoxaEnPG28A were calculated as 1.57 g/L and 14,641.29 U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries. [ABSTRACT FROM AUTHOR]

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50 °C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endopolygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+. [ABSTRACT FROM AUTHOR]

Endo-polygalacturonase (endo-PG) production by Aspergillus niger T0005/007-2 in solid medium with 170 mm of height was evaluated in a cylindrical double surface bioreactor in 96-h experiments. Cell concentration close to 92 mg.g -¹ dm (mg per g of dry medium) in the standard condition (static) was achieved, whereas in tests under forced aeration of 1.4 and 2.8 L.min-1. Kg-1 mm (L of air per minute per Kg of moist medium) and with the central shaft fungal biomass attained approximately 100 mg.g-1 dm. Superior endo-PG activity was obtained with the central-shaft system, 78 U.g-1 dm (units per g of dry medium). Forced aeration and pressure pulse showed no positive effect on the production of endo-PG, 45 U.g-1 dm and 28 U.g-1 dm, respectively. None of the conditions evaluated was efficient for medium temperature control. Endo-PG was stable up to 40ºC. The activity decreased in 50% after 120 minutes at 50ºC, which is a temperature normally found during this process.

Endo-polygalacturonases (PGs) are hydrolytic enzymes involved in the degradation of pectin, one of the major components of plant cell wall. While PGs from fungi, bacteria and plants have been extensively studied, PGs from insects are much less known, although they are likely to play an important role in insect-plant interactions. Presence of PGs has been reported for both piercing-sucking and chewing insect species, and possibly more commonly in mirid bugs (Heteroptera: Miridae). A screening of some common mirid species and other insects, belonging to different orders and families, was conducted using agarose diffusion assays run at different pHs. All the mirid species tested [Lygus rugulipennis Popp., L. pratensis (L.), Orthops kalmi (L.), Adelphocoris lineolatus (Goeze) and Closterotomus norwegicus (Gmelin)] showed PG activity, mainly at pH 7-8, whereas no activity was recorded for the other insect species, except Sitophilus sp. (Coleoptera: Curculionidae). PG activity in females of L. pratensis was significantly higher than in males, whereas there were no differences between the sexes in the other species. In all these species, PGs were present both in the salivary glands and the gut, with a higher activity in the salivary glands, confirming the role of these enzymes in the feeding behaviour of mirid bugs. Inhibition of mirid PGs by polygalacturonase-inhibiting proteins (PGIPs) from different plant sources was analysed at pH 7. PGIPs are extracellular plant proteins known for their ability to inhibit fungal PGs and restrict fungal colonization. Two PGIPs from Phaseolus vulgaris (PvPGIP3 and PvPGIP4) inhibited PGs of all the mirid bugs tested. This information may be helpful for the development of innovative insect-resistant plant varieties, for use in low-impact IPM.

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8-100% for S. cerevisiae, 86.1-95.7% for S. bayanus (var. uvarum), 94-98.3% for S. kudriavzevii, and 96.8-100% for S. paradoxus/ S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking. [ABSTRACT FROM AUTHOR]

Pectinase (endo-polygalacturonase) is the key enzyme splitting plant pectin. The corresponding single gene PGU1 is documented for the yeast S. cerevisiae. On the basis of phylogenetic analysis of the PGU nucleotide sequence available in the GenBank, a family of divergent PGU genes is found in the species complex S. bayanus: S. bayanus var. uvarum, S. eubayanus, and hybrid taxon S. pastorianus. The PGU genes have different chromosome localization. [ABSTRACT FROM AUTHOR]

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZαA vector and constitutively expressed in Pichia pastoris. The recombinant endo- PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The enzyme had optimum activity at pH 5.5 and 40 °C with stability between pH 5.0 and 8.0 and at temperatures up to 50 °C. The enzyme activity was enhanced by 41% with the addition of 1 mM Co++, and inhibited by Fe++ with a 63% reduction. The mode of the enzyme action showed internal cleavage of α-1,4 glycoside bonds of polygalacturonic acid and citrus peel pectin. Trigalacturonate and hexagalacturonate were the main hydrolysis products, with a yield of 0.44±0.01 and 0.21±0.01 mg released per mg polygalacturonic acid substrate, respectively. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products. [ABSTRACT FROM AUTHOR]

The kinetic characteristics of Rhizopus oryzae endo-polygalacturonase, RPG1, hydrolyzing galacturonic acid oligomers (Gal p A) n were determined. RPG1 generates (Gal p A) 3 as a dominant product of polygalacturonic acid and (Gal p A) 4–6 hydrolysis. The enzyme can hydrolyze (Gal p A) 3 , but hydrolysis occurs at a significantly lower rate relative to oligomers with a higher degree of polymerization. Hydrolysis of the α-1,4 glycosidic bond by RPG1 is an endothermic process with a Δ H app , of 1.03±0.04 kcal/mol. Determination of kinetic constants by isothermal titration calorimetry showed that for oligomers (Gal p A) 3–6 , the K m decreased and the k cat increased as the length of the (Gal p A) oligomer increased. Fixed time point assays followed by chromatographic analysis provided apparent k cat values similar to those found using isothermal titration calorimetry. Assays to determine to what extent the enzyme is subject to product inhibition demonstrated that the enzyme is competitively inhibited by (Gal p A) 2 when using (Gal p A) 4 as substrate. The apparent K i of 767 μM is significantly higher than the K m values obtained for the series of galacturonic acid oligomers. [ABSTRACT FROM AUTHOR]

The cell wall in plants offers protection against invading organisms and is mainly composed of the polysaccharides pectin, cellulose, and hemicellulose, which can be degraded by plant cell wall degrading enzymes (PCWDEs). Such enzymes are often synthe-sized by free living microorganisms or endosymbionts that live in the gut of some animals, including certain phytophagous insects. Thus, the ability of an insect to degrade the cell wall was once thought to be related to endosymbiont enzyme activity. However, recent studies have revealed that some phytophagous insects are able to synthesize their own PCWDEs by endogenous genes, although questions regarding the origin of these genes remain unclear. This study describes two pectinases from the sugarcane weevil, Sphenophorus levis Vaurie, 1978 (Sl-pectinases), which is considered one of the most serious agricultural pests in Brazil. Two cDNA sequences identified in a cDNA library of the insect larvae coding for a pectin methylesterase (PME) and an endo-polygalacturonase (endo-PG)- denominated Sl-PME and Sl-endoPG, respectively-were isolated and characterized. The quantitative real-time reverse transcriptase polymerase chain reaction expression profile for both Sl-pectinases showed mRNA production mainly in the insect feeding stages and exclusively in midgut tissue of the larvae. This analysis, together Western blotting data, suggests that Sl-pectinases have a digestive role. Phylogenetic analyses indicate that Sl-PME and Sl-endoPG sequences are closely related to bacteria and fungi, respectively. Moreover, the partial genomic sequences of the pectinases were amplified from insect fat body DNA, which was certified to be free of endosymbiotic DNA. The analysis of genomic sequences revealed the existence of two small introns with 53 and 166 bp in Sl-endoPG, which is similar to the common pattern in fungal introns. In contrast, no intron was identified in the Sl-PME genomic sequence, as generally observed in bacteria. These data support the theory of horizontal gene transfer proposed for the origin of insect pectinases, reinforcing the acquisition of PME genes from bacteria and endo-PG genes from fungi. [ABSTRACT FROM AUTHOR]

The role of putrescine (PUT) in regulating fruit softening, antioxidative enzymes and biochemical changes in fruit quality was investigated during ripening and cold storage of mango (Mangifera indica cv. Samar Bahisht Chaunsa). Fruit were treated with various PUT concentrations (0.0, 0.1, 1.0 and 2.0mM) and were allowed to ripen at 32±2°C for 7 days, or stored at 11±1°C for up to 28 days. Respiration rate and ethylene production were measured daily during ripening and cold storage. Cell wall degrading enzymes such as exo-polygalacturonase (exo-PG), endo-polygalacturonase (endo-PG), pectin esterase (PE), endo-1,4-β-d-glucanase (EGase), antioxidative enzymes including superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT), fruit firmness as well as biochemical fruit quality characteristics were estimated during ripening and cold storage at 2 and 7 day intervals, respectively. PUT treatments reduced respiration rate, ethylene production and maintained higher fruit firmness during ripening as well as cold storage. PUT-treated fruit exhibited significantly suppressed activities of cell wall enzymes (exo-, endo-PG and EGase), but retained higher PE activity during ripening and cold storage. Total phenolic and antioxidant contents were significantly higher in PUT-treated fruit during ripening as well in the cold storage period than in the controls. Activities of antioxidative enzymes (CAT, POX and SOD) were also significantly higher in PUT-treated fruit during ripening as well as cold storage. SSC and SSC:TA were lower in PUT-treated fruit, while TA and ascorbic acid content showed the reverse trend. In conclusion, pre-storage 2.0mM PUT treatment inhibited ethylene production and suppressed the activities of cell wall enzymes, while resulting in higher activities of antioxidative enzymes and maintaining better fruit quality during ripening and cold storage. [ABSTRACT FROM AUTHOR]

Tomato and carrot were subjected to a split-stream process designed to produce a tomato-carrot suspension with reduced consistency. Raw tomatoes, containing pectinmethylesterase and endo-polygalacturonase, were mixed with thermally pretreated (blanched versus cooked) carrots containing different levels of solubilized pectin. After mixing the vegetables, tomato pectinases were shown to act on both tomato and carrot pectin in case an incubation step at medium temperature level (30 min, 40 °C), to allow enzyme action, was performed. Carrot pectin, when present in a mix of tomato and blanched (5 min, 95 °C) carrot, was solubilized as well as depolymerized, whereas depolymerization of the thermo-solubilized carrot pectin by the tomato pectinases was observed in the tomato-carrot purée containing cooked (30 min, 95 °C) carrots. The final serum pectin properties were however similar for both purée types. Carrot contributed more to the consistency of the purée mix compared with tomato but by stimulating the action of the tomato pectinases at mild temperature (30 min, 40 °C), this contribution was lost which resulted in a consistency reduction of the purée mix. This purée liquefaction was larger for the tomato-carrot purée containing blanched instead of cooked carrots. Based on the results, it is suggested that the liquefying effect is related to solubilization and degradation of pectin that is counteracted by a reduction in particle size. The purée mix containing cooked carrot showed in this respect smaller particle sizes than the mix containing blanched carrot. [ABSTRACT FROM AUTHOR]

Highlights: [•] A first acetylesterase reported in Bacillus licheniformis DSM13 active on pectin. [•] The enzyme deacetylates the homogalacturonan of pectin, hence, a pectin acetylesterase. [•] The bacterial PAE (BliPAE) deacetylates a wide range of acetyl-rich pectins. [•] 1H NMR and HILIC-MSn analyses show that BliPAE specifically deacetylates the O-3 galacturonic moieties. [ABSTRACT FROM AUTHOR]

Abstract: Potato cell wall was used as low-value source for the enzymatic extraction of galactan-rich rhamnogalacturonan I (RG I). The effects of selected reaction parameters of endo-polygalacturonase from Aspergillus niger-catalyzed isolation of RG I and their interactions were investigated by response surface methodology. Models were developed to relate independent parameters (cell wall concentration, enzyme amount, reaction time) to responses (yield, neutral sugar content, saccharide molar composition, weak acidic fraction proportion). The most significant parameters that affected extracted polysaccharide yield and its galactose (Gal) and arabinose (Ara) contents were the cell wall concentration and enzyme amount. The interaction between the cell wall concentration and the reaction time was the most determinant for the yield. However, the cell wall concentration and the enzyme amount exhibited significant interaction effect on Gal and Ara contents. Comparison of predicted and experimental values validated the established predicted models, which can be used to identify the conditions for the isolation of RG I-type pectic polysaccharides with selected structural and saccharide composition properties. The monosaccharide composition and the linkage patterns confirmed the isolation of galactan-rich RG I type pectic polysaccharides. The present study is expected to increase the capability to generate RG I targeting specific composition and functional properties. [Copyright &y& Elsevier]

Highlights: [•] PG/PL digestion with HILIC–MS/ELSD is a powerful approach for pectin analysis. [•] Novel GalA oligomeric structures released by PG/PL digestion are identified using LC–MS. [•] Descriptive parameters enable us to distinguish between substitution patterns in SBPs. [•] The degree of hydrolysis by PG/PL provides information on SBP distribution patterns. [ABSTRACT FROM AUTHOR]