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This study investigated the optimisation of microwave pretreatment of orange and plantain peels for polygalacturonase (PG) production, by Aspergillus awamori CICC 2040, using response surface methodology. The microwave pretreatment factors interacted were particle size (PS) (

This study investigated the optimisation of alkaline pretreatment of orange and plantain peels for polygalacturonase (PG) production by Aspergillus awamori CICC 2040 using response surface methodology. The factors evaluated were particle size, PS (< 0.4250, 0.4250 < PS < 0.8025 and 0.8025 < PS < 1.1800 mm), NaOH molarity (0.010, 0.055, and 0.100 M), and time (1.0, 6.5, and 12.0 h). These factors were interacted to determine the most suitable combinations for maximum polygalacturonase activity (MPA). The pretreated orange and plantain peel powders were inoculated with 106 spores/mL Aspergillus awamori CICC 2040 was incubated at 28 oC for 5 days, and crude PG was extracted and its activity determined. The alkaline pretreatment combinations that gave MPA were

Studies were conducted to obtain a complex enzyme preparation that allows efficient hydrolysis of the main plant nonstarch polysaccharides (cellulose, xylans, and pectin). The goal of the studies was to optimize the composition of a fermentation medium for the submerged cultivation of the new mutant strain T. reesei Co-44, a highly active producer of endo-carbohydrases. A concentrate of low molecular soy components was chosen as the inducer of the key carbohydrase biosynthesis by the strain. This concentrate provided the maximum (more than eightfold) increase in polygalacturonase activity and an increased level of endoglucanase and xylanase biosynthesis. After the cultivation of T. reesei Co-44 under optimized conditions, a complex enzyme preparation, Xylorizin K4, was obtained, and its physicochemical properties were studied. The presence of endopolygalacturonase (GH28) T. reesei in Xylorizin K4 was confirmed via electrophoresis and MALDI⎯TOF mass spectrometry. The studies show the potential of Xylorizin K4 application for the production of soy-protein concentrates to eliminate the main soybean nonstarch polysaccharides.

The Lasiodiplodia theobromae genome encodes numerous glycoside hydrolases involved in organic matter degradation and conducive to pathogen infection, whereas their molecular mechanisms are still largely unknown. Here, we identified the glycoside hydrolase family 28 endopolygalacturonase LtEPG1 in L. theobromae and characterized its function in detail. LtEPG1 acts as a virulence factor during L. theobromae infection. Overexpression and silencing of LtEPG1 in L. theobromae led to significantly increased and decreased lesion areas, respectively. Further, the high transcript level of LtEPG1 during the infection process supported its virulence function. Polygalacturonase activity of LtEPG1 was substantiated by detecting its ability to degrade pectin. Furthermore, LtEPG1 functioned as microbe-associated molecular patterns during the infection process. Both transient expression of LtEPG1 in planta and infiltration of purified LtEPG1 triggered cell death in Nicotiana benthamiana. Site-directed mutation of LtEPG1 indicated that the enzymatic activity of LtEPG1 is independent from its elicitor activity. A protein kinase, KINβ1, was shown to interact in the yeast two‐hybrid system with LtEPG1. This interaction was further confirmed in vitro using a pull-down assay. Our data indicate that LtEPG1 functions as a polygalacturonase and also serves as an elicitor with two independent mechanisms. Moreover, LtEPG1 may be able to manipulate host immune responses by regulating the KINβ1-mediated signal pathway and consequently promote its own successful infection and symptom development.

Polygalacturonase (PG) activity in plants can serve as an important index for plant disease. However, the conventional method to detect PG activity is a complex process and requires a skilled technician and expensive analytical equipment. In this study, a paper-based colorimetric sensor was developed based on the principle of the ruthenium red (RR) dye method for easy and simple measurement of PG activity. The proposed paper-based sensor has a three-layer structure for detection of PG activity in samples. The sensor sensitivity was enhanced by optimizing the pH of the sodium acetate buffer used in polygalacturonic acid (PGA)-RR complex formation and the reaction temperature for PG and the PGA-RR complex. Further, for quantitative analysis of PG activity, Delta RGB analysis was conducted to detect color changes in the sensing window of the sensor. Results presented that the linear measurement range of the paper sensor was 0.02-0.1 unit with the limit of detection of 0.02 unit, which showed a similar detection range, but a lower detection limit, compared to the spectrophotometry. Furthermore, PG activity based on culture condition was measured using samples from Sclerotium cepivorum to verify the potential application of the developed paper-based sensor in the field. The measured activity showed no statistically significant difference from the values obtained from the spectrophotometry at 95% confidence level. Therefore, the paper-based colorimetric sensor can be used to predict plant diseases in Allium crops during the stage of pathogen invasion, potentially contributing to the improvement of crop production.

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

Polygalacturonases are extensively used in food industries for pectic substances degradation. They are essential for fruit juice clarification due the degradation of insoluble viscous pectinacious compounds. Polygalacturonase production by the fungus Calonectria pteridis was performed on submerged fermentation and, after purification, the enzyme showed a molecular weight of 68 kDa. The highest polygalacturonase activity occurred at pH 4.0, and the enzyme preserved more than 70 % of its activity at pH values between 3.6 and 6.0. The optimum temperature was 60 °C. The half-life values at 50 and 60 °C were 260 and 66 min, respectively. The KM value was 0.52 mg/mL for the substrate polygalacturonic acid and the presence of Hg+2 and SDS completely inhibited the enzyme activity. The purified polygalacturonase was applied for apple juice clarification and it resulted in a volume and an amount of reducing sugars released increase of 10.65 and 40.93 %, respectively.

AIM: The present investigation was aimed at isolating and identifying bacterial strains from cured vanilla beans. Additionally, the study focused on evaluating bacterial processes pertaining to the aromatic compounds production (ACP). METHODS AND RESULTS: Three bacteria were isolated from Vanilla planifolia beans, previously subjected to the curing process. According to morphological, biochemical and 16S rRNA analysis, the strains were identified as Citrobacter sp., Enterobacter sp. and Pseudomonas sp. The polygalacturonase activity (PGA) was determined using the drop, cup‐plate and DNS methods. Aromatic compounds production was analysed by cup‐plate method using FA as substrate and quantified by high performance liquid chromatography (ppm), the functional groups of vanillic acid (VA) were identified by FT‐IR and the aromatic compounds (AC) resistance was determined and reported as minimum inhibitory concentration. Citrobacter sp., Enterobacter sp. and Pseudomonas showed PGA (70·31 ± 364, 76·07 ± 12·47 and 51 ± 10·92 U ml⁻¹ respectively), were producers of VA (3·23 ± 0·49, 324 ± 41 and 265·99 ± 11·61 ppm respectively) and were resistant to AC. CONCLUSIONS: The Gram‐negative bacteria isolated from V. planifolia beans were responsible for ACP. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evidence for the role of Gram‐negative bacterial isolates from cured Mexican V. planifolia beans in the process related to ACP.

Biofuels production from organic waste requires an efficient hydrolysis to produce fermentable sugars. Physicochemical and biological pretreatment corresponds to good alternative to improve biofuel yield. Nonetheless, physical treatments can result in a negative energy balance and chemical treatments can generate inhibitors for a fermentative process, as well as difficulties in the recycling of organic matter. Thus, enzymatic pretreatments lead to fast and ecofriendly processes for conversion of waste biomass into monomeric units. In this work, fruit and vegetable wastes were hydrolyzed applying three types of enzymatic complexes: Viscozyme® L and a mixture of Multifect® B and Naturalzyme 40 XLTM. Production of reducing sugars as a temperature function and enzyme concentration was optimized by response surface analysis. Enzymatic complexes revealed high hydrolysis yield. Viscozyme® L application is highlighted; obtaining a hydrolysis higher than with the mixture of Multifect® B and Naturalzyme 40 XLTM (80% and 60% respectively) and being a promising treatment for the development of an efficient saccharification process of fruit and vegetable wastes. The optimum application conditions for Viscozyme® L were of 0.24 ppm and 40 °C and were validated with an error rate of 5%. For the mixture of Multifect® B and Naturalzyme 40 XLTM with Pectinase/Polygalacturonase activity, an optimum 0.93 ppm and 41 °C was found; these were validated with an error of 6%. Therefore, cellulases, xylanases and hemicellulases (Viscozyme® L) assured the hydrolysis of food wastes obtaining better available sugars for successive fermentative processes.

The present study was aimed at polygalacturonase production from Aspergillus nomius MR103 under solid state fermentation. A total of 57 fungal strains were obtained from mangrove soils collected from Gilakaladindi and Malakayalanka of Krishna District Andhra Pradesh. For the isolation of fungi these Soil samples were serially diluted and plated on pectin agar media plates. Among them, the isolate which showed maximum polygalacturonase activity was selected for this study. This strain was identified as A. nomius MR 103 by 18S rRNA sequences analysis. Pectin rich agro-industrial wastes like apple peel, citrus peel, orange peel, wheat bran, rice bran and sugarcane bagasse were used as substrates for polygalacturonase production by A. nomius MR 103. This strain was inoculated into the nutrient broth containing agro industrial wastes under solid state fermentation and amount of Polygalacturonase production was estimated. Maximum enzyme production of 4.83 IU/mg was recorded at pH 7.0 and temperature 35?C after 7 days of incubation, when orange peels were used as substrate. Addition of carbon and nitrogen sources to orange peel media improved the Polygalcturonase production. Sucrose as carbon and peptone as nitrogen sources were proved to be the best for maximum production of Polygalcturonase by A. nomius MR 103 on orange peel substrate. Utilization of agro-industrial by-products provided the establishment of a cost-efficient and sustainable process for enzyme production.

Effect of Gum Arabic (GA) as a non-toxic outer layer to prolong the shelf-life of tomato was carried out in the present study. Tomatoes were coated with 0% (Control) (A), 5% w/v (B), 10% w/v (C), 15% w/v (D) and 20% w/v (E) GA and stored at ambient (33°C; 70%). The polygalacturonase and β-galacturonase activities were carried out. Weight loss, titratable acidity, pH, lycopene, β-Carotene and antioxidant property of the fruits were determined at day 0, 5, 10, 15 and 20 during the experimental duration. Results indicated that polygalacturonase activity ranged from 0.753-1.138 unit/mg, β-galacturonase activity ranged from 1.55-2.78 x 103 miller units, β-carotene ranged from 1.78-23.89 mg/g, antioxidant property ranged from 66.26-192.12μg/g, titratable acidity was from 0.595-0.95% while the lycopene ranged from 0.77-17.62 mg/g. These results showed that control had significant rapid weight loss with faster rate of softening while the tomatoes coated with GA demonstrated a significant (p

Background and Aims Reactive oxygen species are involved in fruit ripening. Here, we investigated the role of hydrogen peroxide (H2O2) in promoting grape (Vitis vinifera L.) berry ripening. Methods and Results Solutions of 100, 300 and 500 mmol/L H2O2 were sprayed on Kyoho bunches 25 days post anthesis (dpa). All treatments promoted early ripening of berries, although the 300 mmol/L treatment was the most effective; at this concentration, Kyoho berries ripened 20 days earlier than the Control. Ripening‐related physiological indexes, such as berry diameter and firmness, anthocyanin, sucrose and malonaldehyde concentration, and catalase, cellulase and polygalacturonase activity, were similar for the H2O2‐treated and Control berries. The TSS concentration of berries treated with 300 mmol/L H2O2 was significantly higher than that of Control berries throughout the experiment. Superoxide dismutase activity in H2O2‐treated berries was significantly higher than that in Control berries at 35 and 70 dpa. All H2O2 treatments enhanced the ascorbic acid concentration of berries. Conclusions Exogenously applied H2O2 promotes the early ripening of Kyoho berries. Significance of the Study This study provides a simple, efficient, safe, economical and environmentally friendly medium to promote early ripening of grape berries.

White rot or stem rot caused by Sclerotinia sclerotiorum is one of the most destructive fungal diseases that have become a serious threat to the successful cultivation of oilseed Brassicas. The study was designed with an aim to investigate the association between the pathogenic aggressiveness and pathogenicity determinants of this pathogen specifically in Brassica for the first time. For this, a total of 58 isolates of S. sclerotiorum from different geographical regions were collected and purified. These isolates were inoculated on a Brassica juncea cv. RL-1359 and they exhibited high level of variation in their disease progression. The isolates were grouped and then 24 isolates were selected for the biochemical analysis of pathogenicity determinants. The isolates varied significantly with respect to their total organic acids, oxalic acid production and pectin methyl esterase and polygalacturonase activity. The oxalic acid production corresponded to the disease progression of the isolates; the isolates with higher oxalic acid production were the more aggressive ones and vice-versa. This is, in our knowledge, the first study to establish a correlation between oxalic acid production and pathogenic aggressiveness of S. sclerotiorum on B. juncea. However, the pectinases’ enzyme activity did not follow the trend as of disease progression. These suggest an indispensable role of oxalic acid in pathogenicity of the fungus and the potential to be used as biochemical marker for preliminary assessment of pathogenic aggressiveness of various isolates before incorporating them in a breeding program.

The effect of gum arabic (GA) coating was studied on apricot fruit at 20 ± 1°C for 8 days. GA coating substantially reduced weight loss, disease incidence, malondialdehyde concentration, and hydrogen peroxide compared to the control. GA coating resulted in significantly higher total phenols, ascorbic acid, and antioxidant activity in contrast to the control. GA coating also suppressed cellulase, pectin methylesterase, and polygalacturonase activity in contrast to the non-coated fruit. In addition, GA-coated apricot fruit had substantially higher catalase, peroxidase, ascorbate peroxidase, and superoxide dismutase enzymes activities in contrast with the control. Similarly, GA coating inhibited soluble solids content increase and inhibited the reduction in titratable acidity in coated fruit. The fruit coated with GA coating also had significantly better sensory and overall eating quality compared to the control apricots. In conclusion, GA could be considered an appropriate edible coating for quality conservation of apricots. PRACTICAL APPLICATIONS: Apricot is a well renowned and nutrients rich fruit. However, apricot has comparatively short shelf life potential due to its susceptibility to rapid senescence and deterioration under ambient conditions. GA is an eco-friendly and biodegradable coating which is considered relatively cheap as compared with other coating types. In our present work, application of gum arabic [GA (10%)] edible coating maintained significantly higher quality of harvested apricot fruit as compared with the control. The outcomes of the present work are of global significance with respect to apricot industry. So, GA coating could be considered an appropriate edible coating for quality conservation of the harvested apricot fruit.

Effect of edible coatings of gum Arabic, carrageenan and xanthan gum containing lemon grass essential oil 1% w/v on postharvest quality of strawberry was studied under refrigeration for a period of 12 days. Results showed all the three coatings maintained fruit quality parameters during storage compared to control. Among all the coatings, carrageenan coated fruits showed delayed weight loss (10.1 to 8%), decay percentage (78.42 to 14.29%), retained ascorbic acid (0.15 to 0.27 g kg-1), antioxidant activity (18.17 to 25.85%), firmness (9.07 to 12.43 N), L* (32.38 to 40.42), a* (16.08 to 17.22) and b* (27.36 to 33.54). Carrageenan gum also showed lowest cellulase activity (0.03 units h−1 mg protein−1), pectin methylesterase activity (1.13 A620 min−1 mg protein−1) and β-galactosidase activity (0.51 μmol min−1 mg protein−1), while showed maximum reduction in polygalacturonase activity (0.07 units h−1 mg protein−1) at the end of storage. Carrageenan gum was found effective in retention of anthocyanins and phenolic compounds during storage. Coatings loaded with antimicrobial agent inhibited psychrophilic bacteria, yeast and mold growth. It is concluded that carrageenan gum could better retain strawberry quality up to 12 days under refrigeration.

Fragments of wood drifting in the vicinity of Spitzbergen were used for the isolation of microorganisms, carried out using atypical carbon sources: colloidal chitin, cellulose and carboxymethylcellulose, xylan, casein, tributrin and olive oil. Purified cultures were subjected to a three-step identification: with classical methods, using MALDI-TOF MS Biotyper whole-cell protein fingerprinting, and molecular analysis of 16S rDNA. Subsequently, a preliminary assessment of the enzymatic potential of isolates was carried out. As a result, cellulolytic activity was observed in more than 50% of the bacterial strains, exhibiting activity of 0.30&ndash, 0.40 U/mL. Over 53% of the isolates demonstrated xylanolytic activity, of which the highest reached from 0.40 to 0.90 U. Polygalacturonase activity of 0.003&ndash, 1.6 was also demonstrated in half of the bacterial strains studied. Proteolytic activity of isolates did not exceed 0.3 U. An important highlight was the ability of fluorescent dye production by certain strains, grown on skim milk-agar, but also on pure meat extract.

To reduce environmental pollution and waste of biomass from Ginkgo biloba leaf residues (GBLRs), we developed a cost-effective enzyme system to hydrolyze GBLRs into available reducing sugars (RS). Biomass characteristics of GBLRs were investigated, which indicated that the acid hydrolyzed fraction was 49.43% of the dry weight of GBLRs. The fraction could be effectively converted into RS by an enzyme cocktail with high polygalacturonase activity without traditionally intricate pretreatment. The strain A32 isolated from the ancient ginkgo soil was used for the production of the enzyme cocktail, and a response surface methodology was used to optimize the enzymatic production. The enzyme cocktail released 87.2% of RS from GBLRs at 35 ℃ for 72 h with 1% (m/v) of loading, and the RS concentration arrived 8.95 ± 0.39 mg/ml with 9% of GBLRs loading. The cost-effective system of self-prepared enzyme cocktail is promising for facilitating GBLRs' bio-based industry.

This work aimed to evaluate the physical, chemical and biochemical characteristics of black mulberry (Morus nigra L.) cultivated in Lavras, Minas Gerais, Brazil, at different maturation stages. The fruits were harvested and separated into five different stages according to the color of the surface. Afterwards, the fruits were submitted to the physical and chemical analyses and analysis of pectin methylesterase and polygalacturonase activity. With the advancement in maturation stages of black mulberry, it was possible to observe chlorophyll degradation with concomitant anthocyanin synthesis. There was an increase in soluble solids contents, sugars and pH, decrease of total acidity and respiratory rate as well as decrease of total pectin and increase of the soluble pectin. Furthermore an increase of the activity of the enzymes pectin methylesterase and polygalacturonase and the consequent softening of the fruits was also noted. Este trabajo tuvo como objetivo evaluar las características físicas, químicas y bioquímicas de la morera negra (Morus nigra L.) cultivada en Lavras, Minas Gerais, Brasil, en diferentes etapas de maduración. Los frutos fueron cosechados y separados en cinco etapas diferentes, de acuerdo con el color de la superficie. Posteriormente, los frutos fueron sometidos a análisis físico-químicos y análisis de la actividad de la pectina metilesterasa y la poligalacturonasa. Con el avance de las etapas de maduración de la morera negra, fue posible observar la degradación de la clorofila con la síntesis concomitante de antocianina. Hubo un aumento en el contenido de sólidos solubles, azúcares y pH, una disminución en la acidez total y la frecuencia respiratoria, así como una disminución en la pectina total y un aumento en la pectina soluble. Además, se observó un aumento en la actividad de las enzimas pectina metilesterasa y poligalacturonasa y el consiguiente ablandamiento de los frutos. Este trabalho teve como objetivo avaliar as características físicas, químicas e bioquímicas da amoreira preta (Morus nigra L.) cultivada em Lavras, Minas Gerais, Brasil, em diferentes estádios de maturação. Os frutos foram colhidos e separados em cinco estádios diferentes, de acordo com a cor da superfície. Posteriormente, os frutos foram submetidos às análises físico-químicas e análise da atividade da pectina metilesterase e poligalacturonase. Com o avanço dos estádios de maturação da amoreira preta, foi possível observar a degradação da clorofila com a síntese concomitante de antocianina. Houve aumento do teor de sólidos solúveis, açúcares e pH, diminuição da acidez total e da taxa respiratória, bem como diminuição da pectina total e aumento da pectina solúvel. Além disso, foi observado um aumento da atividade das enzimas pectina metilesterase e poligalacturonase e consequente amolecimento dos frutos.

Iron is virtually an essential nutrient for all organisms, to understand how iron contributes to virulence of plant pathogenic fungi, we identified ClFTR1 and ClNPS6 in maize pathogen Curvularia lunata (Cochliobolus lunatus) in this study. Disruption of ClNPS6 significantly impaired siderophore biosynthesis. ClFTR1 and ClNPS6 did mediate oxidative stress but had no significant impact on vegetative growth, conidiation, cell wall integrity and sexual reproduction. Conidial germination delayed and appressoria formation reduced in ΔClftr1 comparing with wild type (WT) CX-3. Genes responsible for conidial germination, appressoria formation, non-host selective toxin biosynthesis and cell wall degrading enzymes were also downregulated in the transcriptome of ΔClftr1 and ΔClnps6 compared with WT. The conidial development, toxin biosynthesis and polygalacturonase activity were impaired in the mutant strains with ClFTR1 and ClNPS6 deletion during their infection to maize. ClFTR1 and ClNPS6 were upregulated expression at 12-24 and 48-120 hpi in WT respectively. ClFTR1 positively regulated conidial germination, appressoria formation in the biotrophy-specific phase. ClNPS6 positively regulates non-host selective toxin biosynthesis and cell wall degrading enzyme activity in the necrotrophy-specific phase. Our results indicated that ClFTR1 and ClNPS6 were key genes of pathogen known to conidia development and virulence factors.

This study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with sodium bicarbonate (NaHCO3 ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h.; Results: Treatment with NaHCO3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, compared with the control or the same treatment at pH 5. In contrast, pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5-treated plugs up to 72 h.; Conclusion: The rapid increase in pectin methyl esterase activity under alkaline conditions is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact, at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

The objectives of this study were to achieve the best level of each ethylene scavenger and evaluate the effect of selected levels of treatments on some quality traits of tomato during storage. Tomato fruits were subjected to four levels of treatments: palladium-promoted nano zeolite, KMnO4-promoted nano zeolite, 1-MCP, CaCl2, salicylic acid (SA) and UV-C. The sampling was done at 0, 7th, 14th, 21st, 28th and 35th days of cold storage. The results showed that palladium-promoted nano zeolite 5%, KMnO4-promoted nano zeolite 20%, 1-MCP 30 ppm, CaCl2 2%, SA 1% and UV-C 15 min levels had the most ethylene scavenging function. Effectiveness of the treatments in ethylene scavenging was in the order: palladium > KMnO4 > 1-MCP > SA = CaCl2 > UV-C. The palladium-promoted nano zeolite 5% had more positive effects on phenol content, polygalacturonase activity, lycopene content, fruit firmness and weight loss, and UV-C 15 min had effect on decay severity as compared to the other treatments. Overall, palladium-promoted nano zeolite 5% could be considered not only as favorable tool in tomato shelf life extension but also in preservation of quality characteristics of tomato fruits during storage. Moreover, the UV-C 15 min treatment could be an effective method for reducing decay severity and maintaining postharvest quality of tomato fruits.

A study into the effect of a new enzyme preparation Pectinol F-RKM 0719, used in the production of red table wines dur-ing maceration, was carried out. The investigated enzyme prepa-ration Pectinol F-RKM 0719 was previously isolated from the cul-ture liquid of the Aspergillus awamori F-RKM 0719 strain, ob-tained through multistage selection and mutagenesis at the De-partment of Biotechnology of the South Kazakhstan State Univer-sity named after M. Auezov. As a result of the development of a combined method of purification and isolation of a pectolytic en-zyme preparation, a preparation with a higher polygalacturonase activity was obtained having a sufficiently high yield of the desired product. The method is based on the sequential removal of individual groups of inactive impurities from the enzyme solutions using activated carbon and anion exchange resin. The use of hy-droxyapatite allows the activation of pectinases to be achieved by removing low molecular proteins. The absence of pectin substanc-es and carbohydrates makes it possible to use it in winemaking to clarify the best varieties of wine and remove the wine stone, which consists of the pectic substances of grape juice. The use of a new enzyme preparation in the process of maceration contributes to increasing the yield of mash from grape pulp, facilitating the pulp pressing and clarification of wine materials by reducing the vis-cosities and suspensions of the mash, as well as increasing the ex-traction of phenolic and colouring substances. The efficiency of enzymatic catalysis in the production of red table wines is demon-strated.

Background and Objective: Polygalacturonase [Poly (1, 4-"-D-galacturonide) glucanohydrolase, E.C 3.2.1.15] is a subclass of pectinase that hydrolyzes the glycosidic linkages between galacturonic acid residues in polygalacturonans. Solanum macrocarpum ripening prompts pectinase production. The objective of this study was to examine the kinetic properties of polygalacturonase from Solanum macrocarpum L. fruit. Methodology: The enzyme was partially purified by ammonium sulphate precipitation and gel filtration. Protein content, polygalacturonase activity and kinetic parameters were determined. Results: The protein content and polygalacturonase activity of the fruit juice extracts were 0.63±0.02 mg mLG1 and 45.96±6.31 U mgG1 protein, respectively. A 1.7 and 108.3 fold increase in enzyme activity was achieved by ammonium sulphate precipitation and gel filtration, respectively. The enzyme had a Vmax of 76.92 unit mgG1 protein and Km of 0.92 mg mLG1. The pH profile of the enzyme showed three activity peaks at 3.0, 5.5 and 7.0. The enzyme was most active at pH 3.0 and showed optimal activity at 30EC. Rapid release of product was observed within the first 20 min of enzyme incubation. The Zn2+, Ca2+, Mn2+, Pb2+, Fe2+, Cu2+ and EDTA exhibited inhibitory effect on polygalacturonase activity whereas Mg2+ had stimulatory effect on the enzyme. Conclusion: It was concluded that the fruit of Solanum macrocarpum is a rich source of polygalacturonase. The enzyme is favourably comparable with that of a fungi source and could be further exploited for commercial production of the enzyme

This work aimed the assessment of the use of artificial neural networks (ANNs) as alternative tool for modelling and predicting the suitability of sugar beet pulp (SBP) to produce oligosaccharides in comparison with the response surface methodology (RSM). The variables polygalacturonase to solid ratio (PGaseSR), cellulase activity to polygalacturonase activity ratio (CPGaseR), and reaction time (t) were selected as independent variables and their effects on the recovered liquors mass, the conversion of different polysaccharide into monosaccharides, and the conversion of each polysaccharide into oligomers were investigated. ANN models improved the RSM models between a 5.58% and a 61.78% for the solid yield (%) and Galactan conversion into galactooligosaccharides (%), respectively. However, RSM models presented better accuracy to predict the polysaccharides conversion into monosaccharides. The ANNs implemented in this study showed that are suitable to optimize and predict the oligosaccharides production using direct enzymatic hydrolysis from SBP.

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are openly available, this is not the case for the production of pectinases. We therefore systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study therefore presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies the most suitable base strain for further genetic optimizations.

This paper adopts the Design-Expert software to design an orthogonal experiment with a pulse voltage amplitude of 30 kV, processing time of three minutes, and a pulse width of 45 &mu, s as the center points, in order to study the effects of the pulsed electric field on the cell wall and infection activity of Rhizoctonia solani. High-voltage pulse power was used to treat the bacteria solution with the pulsed electric field. Untreated Rhizoctonia solani were used as the control group. Transmission electron microscope images were used to analyze the cell wall damage. ANOVA was performed on the experimental results and the fitting degree of the model was good (F&gt, &gt, 1). Response surface analysis was used to optimize the parameters based on chitin content and polygalacturonase activity. The optimal treatment conditions were obtained as a pulse voltage amplitude of 25 kV, processing time of 2.54 min, and a pulse width of 34.35 &mu, s. On this basis, experiments were designed to verify the optimized conditions. The results demonstrated that, under the optimal processing conditions, the damage index of the cell wall of Rhizoctonia solani was 9.59% lower in chitin content and 83.05% lower in polygalacturonase activity compared with those of the control group. All indexes were significantly different (P &lt, 0.001), which is consistent with the parameter optimization results. The results provide a theoretical basis for the pulsed electric field assisted sterilization and reference for the design of plant protection machinery in the latter stage.

The endo-polygalacturonase enzyme (endoPG: EC 3.2.1.15) plays an important role in the fruit juice and wine industries, so the development of new tools for the quantitative and qualitative analysis of its enzymatic action is necessary. In this work, we report the development of a simple, fast and practical method that did not use any chemical reagent to identify and evaluate the action of the endoPG enzyme, produced by the yeast Kluyveromyces marxianus CCT3172, using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA). This method evaluated the action of the endoPG enzyme on the polygalacturonic acid (PGA) substrate at 5 different times (0, 10, 15, 20 and 30 minutes), and at each time interval the samples were analyzed by ATR-FTIR. It was demonstrated that there was clear segregation between the samples that were and that were not subjected to the action of the endoPG enzyme, and it was also possible to distinguish the samples that were subjected to different incubation times with the enzyme. Through PCA-LDA it was possible to obtain wavelengths that are biomarkers for this enzymatic reaction and the observed changes as a function of hydrolysis duration were found to be in agreement with the breakdown of the glycosidic chain (1011 cm-1-CH-O- CH stretching) of PGA and release of oligosaccharides (1078 cm-1 C-OH elongation). The activity of the endoPG enzyme and the release of galacturonic acid were verified by the dinitrosalicylic acid (DNS) method in all samples. The efficacy of an automatic classifier using a principal component analysis-linear discriminant classifier (PCA-LDC) was evaluated to diagnose the action of the endoPG enzyme. The results showed an accuracy of 100% for the identification of the endoPG enzyme action and from 91.67% to 100% for classification according to the hydrolysis duration in which PGA was exposed to endoPG. The present study indicates that this methodology may be a new approach for the qualitative evaluation of the endoPG enzyme with the potential to be used in laboratories and industries.

Background Elymus nutans and E. sibiricus are two important forage grasses of the genus Elymus. But they are difficult to grow for commercial seed production due to serious seed shattering. We conducted a comparative transcriptome analysis of abscission zone to find possible transcription changes associated with seed shattering, explore candidate genes involved in seed shattering and identify candidate gene-based EST-SSR markers for germplasm evaluation. Results cDNA libraries from abscission zone (AZ) and non-abscission zone (NAZ) tissues of E. nutans were constructed and sequenced. A total of 111,667 unigenes were annotated and 7644 differentially expressed transcripts (DETs) were predicted, corresponding to 6936 up-regulated in AZ and 708 down-regulated in NAZ. We identified 489 candidate genes related to transcription factor, cell wall hydrolysis or modification, hydrolase activity, phytohormone signaling and response, lignin biosynthesis, and signal transduction or protein turnover. Eleven similar candidate genes involved in polygalacturonase activity, hydrolase activity, and mitogen-activated protein kinase were up-regulated in the abscission zone of the two Elymus species, suggesting these genes may have specific function for abscission zone development and seed shattering. A total of 67 polymorphic EST-SSR markers were developed and characterized based on the sequences of these candidate genes. Fourteen polymorphic EST-SSR primers were finally used to study genetic diversity in 48 E. nutans genotypes with contrasting seed shattering habit. The dendrogram based on molecular data showed that most accessions with similar seed shattering degree tended to group together. Conclusions The expression data generated from this study provides an important resource for future molecular biological research. Many DETs were associated with abscission zone development, and EST-SSR loci related to candidate genes may have potential application in identifying trait-associated markers in E. nutans in the future. Electronic supplementary material The online version of this article (10.1186/s12870-019-1691-4) contains supplementary material, which is available to authorized users.

The acquisition of susceptibility to necrotrophy over the course of ripening is one of the critical factors limiting shelf life. In this study, phytopathology and molecular biology were employed to explore the roles of pectinase in fruit susceptibility and ripening. Solanum lycopersicum fruit softened dramatically from entirely green to 50% red, which was accompanied by a continuously high expressed SlPG2 gene. The necrotrophic fungus Botrytis cinerea further activated the expression of SlPGs and SlPMEs to accelerate cell wall disassembly, while most of the polygalacturonase inhibitor proteins encoding genes expression were postponed in ripe fruit following the pathogen attack. Pectin induced the antagonistic yeast to secrete pectinolytic enzymes to increase fruit resistance against gray mold. The activities of pathogenic pectinase of B. cinerea were correspondingly depressed in the pectin-inducible yeast enzyme elicited ripe fruit. These data suggest that pectinase is a molecular target for regulation of disease resistance during fruit ripening.

Background. Polygalacturonase (EC 3.2.1.15) enzyme aids in microbial spoilage of fruits and vegetables. It is very important to find economical ways to producing the enzyme so as to achieve maximum yield in industries due to its use at different areas of production process. Methods. Isolation of polygalacturonase-producing bacterial strain from tomatoes (Lycopersicon esculentum Mill.) was studied. Polygalacturonase-producing bacterial strains were isolated and screened from tomatoes stored at normal laboratory temperature (25 ± 2°C). They were identified based on their morphological, biochemical, and molecular characteristics. The enzyme produced was partially purified by the ammonium sulphate precipitation method. Molecular weights and optimum conditions for best enzyme activity were obtained by SDS PAGE technique. Results. Five bacterial isolates resulted after screening. Bacterial strain code B5 showed highest polygalacturonase activity. Optimum conditions for polygalacturonase PEC B5 were maintained at pH 4.5; temperature 35°C; substrate concentration 0.3 mg/ml, and best activity at less than 5 min of heating. The enzyme PEC B5 was found to weigh 65 kDa and 50 kDa for crude and partially purified aliquots, respectively. The result of 16S rRNA gene sequencing revealed bacterial strain code B5 as Enterobacter tabaci NR146667 having 79% similarity with the NCBI GenBank. Conclusion. Microorganisms should be developed for large-scale production of enzymes in developing countries.

Bruchids (Callosobruchus spp.) are destructive storage pests of mung beans (Vigna radiata). Bruchids infest mature seeds during storage and in the field causing heavy losses. Bruchid resistance in mung bean has been characterized as a dominant trait controlled by a single gene. Several independent mapping studies showed that the Br locus on chromosome 5 was a key quantitative trait loci (QTL) involved in bruchid resistance. Two polygalacturonase-inhibitor protein (PGIP) family genes, VrPGIP1 and VrPGIP2, located in the Br locus may be the primary genes responsible for bruchid resistance in mung bean but no experimental proof is available. We isolated the VrPGIP1 and VrPGIP2 genes from bruchid resistant mung bean cultivar V2802 and purified the proteins by prokaryotic expression. Both VrPGIP1 and VrPGIP2 had polygalacturonase inhibitor activity and both of the PGIP proteins conferred resistance to bruchids in an artificial seed test system. VrPGIPs can inhibit the enzyme activity of polygalacturonase present in males, females and fourth instar larvae of C. maculatus. These results demonstrated that VrPGIP1 and VrPGIP2 play a critical role in bruchid resistance probably through inhibiting polygalacturonase activity.

Blueberry fruit are highly prone to deterioration, especially during storage. Calcium (Ca) can influence fruit quality of many fruit crops since it is an important mediating agent in the control of cell metabolism. Our aim was to determine if the application of foliar Ca sprays at different Ca rates (0, 400 or 800 g Ca ha-1) and timings (early: fruit set, 8 and 16d after fruit set-dafs; or late: 16, 24 and 32 dafs) affect long-term postharvest quality and condition of highbush blueberry ‘Liberty’ (V. corymbosum) fruits. During the 2014/15 and 2015/16 seasons nine-year-old plants, located in Perquenco, Araucania Region, Chile (38o43’88” LS), were used in the trials. Fruits were harvested at > 90 percent full color and stored for 15, 30 and 45d at 0 °C + 1d at 18-20 °C. Fruit firmness and mass loss were the only quality attributes that registered significant interactions between Ca rates and spray timing, along with strong positive and negative correlations, respectively with fruit Ca concentrations. Firmer fruit (up to 10% higher than control) and lesser moisture loss (1% better than control) was obtained with early Ca foliar application treatments. Ca rates and spray timing had significant interactions and increased fruit Ca levels, total phenolic content, the proportion of sound fruits and reduced the percentage of dehydrated and decayed berries after storage. Fruit Ca concentration was lower in control and with late preharvest Ca sprays, which led to higher polygalacturonase activity during storage. Results evidence that early preharvest Ca sprays increase fruit quality and reduce deterioration in cold storage.

Sunflower White Mold caused by Sclerotinia sclerotiorum and Sclerotinia minor is a devastating disease worldwide. To investigate the effect of low temperature (4 °C) on biological characteristics and aggressiveness of isolates of the two species, which were collected from the same field in Baiyinchagan, Inner Mongolia, their mycelial growth rate, oxalic acid secretion level and polygalacturonase activity were compared under normal culture temperature (23 °C) and low temperature (4 °C). Aggressiveness was also evaluated on detached leaves by inoculating the isolates produced in both temperatures. The results suggested that culture of isolates at 4 °C not only promoted mycelial growth, but also enhanced secretion of oxalic acid and polygalacturonase activity of both S. sclerotiorum and S. minor isolates compared to that cultured at 23 °C. Additionally, the corresponding aggressiveness of tested isolates of the two species also increased after culture at 4 °C. However, S. sclerotiorum always showed faster mycelial growth, higher oxalic acid levels and greater polygalacturonase activity than S. minor at both 23 °C and 4 °C, indicating that S. sclerotiorum is generally the more aggressive species than S. minor.

The effects of aqueous ozone (AO) treatment on the firmness, cellwall polysaccharide content, enzymatic activity, and gene expression of cell wall-degrading enzymes were investigated in fresh-cut apple during cold storage. AO treatment (1.4 mg L−1 for 5 min) delayed degradation of cellwall polysaccharides to reduce softening of fresh-cut apple. Water-soluble pectin content increased more slowly, while protopectin content, 4% KOH-soluble fraction, and cellulose content decreased at a lower rate in AO-treated fresh-cut apple compared with the control. However, no significant influence was shown on the 24% KOH-soluble fraction. AO treatment promoted increased pectin methylesterase activity, and distinctly inhibited β-galactosidase and α-arabinofuranosidase activities during storage. Polygalacturonase activity was not affected by AO treatment. Meanwhile, AO maintained the intactness of cell walls through repression of Mdβ-Gal and Mdα-Af transcript levels, thereby preserving the textural quality of fresh-cut apple during cold storage.

Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5 h of reaction using 0.05 g/mL of immobilized biocatalyst, which released 3 mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90 h) and application in juice clarification. Plum (4.7 °Bx) and grape (10.6 °Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity. Fil: Ramírez Tapias, Yuly Andrea. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina Fil: Rivero, Cintia Wanda. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina Fil: Gallego, Fernando López. Heterogeneous Biocatalysis Group; España. Ikerbasque; España Fil: Guisán, José M.. Institute of Catalysis and Petrochemistry; España Fil: Trelles, Jorge Abel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Biotecnología Sustentable; Argentina

The removal of fruit cluster leaves was shown to be a valuable method for controlling fruit quality and producing high-grade apples. A chemical defoliant that functions as an activation switch was shown to turn on the genes necessary for fruit cluster leaf defoliation. Elucidating the mechanism involved in leaf defoliation is crucial to our understanding of the use of chemical defoliants in fruit trees. To gain insight into chemical defoliant-mediated leaf defoliation, we first confirmed the occurrence of ethylene production by applying a chemical defoliant on fruit cluster leaves. Then, we used RNA-seq analysis to obtain a series of transcriptome profiles for genes and proteins involved in leaf senescence induction. Within 2 days of applying the chemical defoliant to apple trees, 1-aminocyclopropane carboxylic acid (ACC) oxidase, ACC synthase, a chlorophyll binding protein, and polygalacturonase-related genes were up-regulated at least sixfold. An in vitro enzyme assay showed that lanolin oil activates ACC synthase activity, a key regulatory enzyme in the ethylene pathway. We also showed that chemical defoliant decreased the light saturation point and total chlorophyll content. Then, we used a polygalacturonase activity assay to confirm the effects of chemical defoliant on leaf senescence in vivo. Furthermore, treatment with chemical defoliant resulted in a significant increase in the chromaticity value of a*, whereas L* and b* decreased in the apple fruit. Taken together, we conclude that chemical defoliant could selectively affect fruit cluster leaves, which suggested that it can be used as a selective defoliant.

BACKGROUND The koji mold Aspergillus sojae, an industrially important microorganism, can produce high levels of pectinases utilizing agro-industrial wastes. This study introduces apricot and peach pomace, two agro-industrial wastes barely considered as raw material for the generation of value-added products, and focuses on its utilization together with orange peel for polygalacturonase production in submerged cultures using A. sojae. RESULTS A Doehlert response surface methodology design conducted in shake flasks and applied individually with these three by-products led to 60–80 U mL−1 polygalacturonase activity. In bioreactor studies performed with a mixture of apricot pomace and orange peel, by fixing stirrer speed to 600 rpm and cascading airflow to the dissolved oxygen tension up to 1.7 vvm, oxygen limitation problems were overcome and polygalacturonase activity values of 380 U mL−1 were achieved. CONCLUSION A simple and efficient strategy to minimize oxygen limitation with the lowest possible shear stress is provided for stirred-tank bioreactors working with highly viscous broths, so as to ultimately enhance microbial enzyme production. The polygalacturonase activity yields obtained in our study are among the highest reported in the literature. © 2016 Society of Chemical Industry