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The utilization of microbial pectinase in different industries has been increased in its world demand. The major sources of pectinase are microorganisms mainly bacteria, fungi and yeast. The utilization of low-cost agro-industrial wastes as substrates has been preferable in pectinase production. Pectinase production faced various parameters optimization constraints such as temperature, pH and production times which are the main factors in pectinase production. The pectinase enzyme is getting attention due to its several advantages; hence, it needs to be explored further to take its maximum advantage in different industries. This review discusses the pectin substance structure, substrate for pectinase production, factors influencing pectinase production, the industrial application of microbial pectinase and also discusses challenges and future opportunities of applying microbial pectinase in industry., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Setegn Haile and Abate Ayele.)

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g -1 without enzyme treatment, the GICF yield increased to 70.44 mg g -1 via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction., Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris., Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG., Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The K M and k cat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 μM and 179 s-1 , respectively. Cu 2+ was found to enhance SsExo-PG activity while Ag 2+ and Fe 2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures., Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

Aims: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M., Methods and Results: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent K m , V max and K cat values for hydrolyzing polygalacturonic acid were 2·56 mg ml -1 , 163·1 U mg -1 and 277 s -1 respectively. The polygalacturonase presented exo activity and was activated by Mg 2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour., Conclusions: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C., Significance and Impact of the Study: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application., (© 2019 The Society for Applied Microbiology.)

Pectinases are a group of enzymes, which catalyze the breakdown of pectin with numerous applications in various industries. Microbes are the predominant pectinase producers. In the present study, bacterial species were isolated from the soil of a vegetable and fruit dump yard area in a market. The species screened and isolated were identified as Bacillus tequilensis SALBT, and the media and culture conditions were optimized for enhanced production of total pectinases. Maximum pectinolytic activity was observed with 1.5% (w/v) pectin concentration with a combination of yeast extract as nitrogen source and MgSO 4 as a metal ion source. Carbon/nitrogen in 2:1 ratio (w/v) yielded the maximum pectinase production with pH and temperature of the medium of 7.5°C and 40°C, respectively. Pectinase activity was determined by the dinitrosalicylic acid method. The pectinase production was relatively stable in the presence of various surfactants like Tween (20, 40, 60, and 80) and sodium dodecyl sulfate (SDS), whereas Triton X-100 showed an inhibitory effect. Mass production of the enzyme in optimized media and partial purification was performed by ammonium sulfate precipitation followed by dialysis. The approximate molecular weight of the partially purified pectinase was found to be 35 kDa by SDS-polyacrylamide gel electrophoresis. Application studies such as demucilaging coffee beans and juice clarification were also performed. The findings revealed that B. tequilensis SALBT with pectinase activity has the ability to remove the mucilage layer of pulped coffee seeds, and the partially purified pectinases found to be effective in clarifying juice., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It was found that Bacillus sp. Y1 could secrete alkaline pectinase with suitable enzyme system for powerful and fast degumming of ramie fiber. In this study, the medium components and fermentation conditions were optimized by some statistical methods including mixture design, fractional factorial design, central composite design and response surface methodology, and single factor method for enhancing the alkaline pectinase production. The optimized conditions for pectinase production were that the culture was shaken at 34°C for 60 h in 50 mL of medium containing 10.5% (w/v) carbon source (consisting of 3.8% starch, 4.2% wheat bran, and 2.5% sucrose), 0.37% (NH 4 ) 2 SO 4 , 0.3% MgSO 4 , and 0.1% Tween-80, with initial pH 8.2 and inoculation amount of 1.3 mL (with the OD600 of the seed medium about 5.77). Using the optimizing conditions, the activities of polygalacturonate lyase (PGL) and polygalacturonase (PG) in fermentation liquor were increased to 2.00-fold and 3.44-fold, respectively, and the fermentation time shortened 12 hours (from 72 h to 60 h), which showed good application potential in degumming of ramie.

Background: Pectinase enzyme has immense industrial prospects in the food and beverage industries., Objective: In our investigation, we find out the optimum process parameters suitable for better pectinase generation by Bacillus subtilis MF447840.1 using submerged fermentation., Method: 2% (OD600 nm = 0.2) of pure Bacillus subtilis MF447840.1 bacterial culture was inoculated in sterile product production media. The production media components used for this study were 1 g/l of pectin, 2 g/l of (NH4)2SO4, 1 g/l of NaCl, 0.25 g/l of K2HPO4, 0.25 g/l of KH2PO4 and 1 g/l of MgSO4 for pectinase generation. We reviewed all recent patents on pectinase production and utilization. The various process parameters were observed by changing one variable time method., Results: The optimum fermentation condition of different parameters was noticed to be 5% inoculums, 25% volume ratio, temperature (37°C), pH (7.4) and agitation rate (120 rpm) following 4 days incubation., Conclusion: Maximum pectinase generation was noticed as 345 ± 12.35 U following 4 days incubation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Pectin degrading enzyme has been increasing interest in an industrial application as biocatalysts, such as juice, textile, and wine industry. Bacillus paralicheniformis CBS3, isolated from popular traditional Korean food (kimchi), produced a novel extracellular thermostable alkaline endopolygalacturonase (BPN3). In this study, BPN3 was purified to 22.04-fold with a recovery yield of 18.93% and specific activity of 2216.41 U/mg by gel filtration and anion exchange column chromatography. The molecular mass of BPN3 was approximately 53 kDa as analyzed by SDS-PAGE and pectic zymography. The N-terminal sequence of BPN3 was AIPVILAX. BPN3 was stable over a broad pH range (8.14-11.47), was thermally stable at 50-60 °C, and functioned optimally in pH 9.1 at 60 °C. BPN3 had K m and V max values of 0.039 mg/mL and 747.9 ± 1.2 U mg - 1 , respectively, whereas pectin from apple as substrate. BPN3 activity was remarkably affected by metal ions, modulators, and detergents. Digalacturonic acid (GA 2 ) was the major oligosaccharide produced by hydrolysis of BPN3. Immobilized BPN3 was active over a pH range (8.1-11.5), temperature (50-60)°C, and remained stable with 63.34 and 43.41% of its relative activity during second and third cycle, respectively. Desized cotton exhibited highest reducing sugar liberation through optimized conditions of bioscouring. Bioscouring effectiveness of BPN3 was characterized by the comparison of weight loss for purified BPN3 with commercial pectinase and comparison of BPN3 with grey fabric. BPN3 was simple to purify, had high thermal stability, and was stable over a broad pH range that suggests its suitability for bioscouring application as an industrial catalyst.

Aim: The aim of this study was to evaluate the physicochemical properties of the crude pectinase activity from three Bacillus isolates of ruminant dung origin and study their synergism with crude xylanases from the same Bacillus spp. and a commercial cellulase to evaluate their accessory role in improved biomass saccharification., Methods and Results: Pectinolytic crude culture filtrate obtained from three ruminant dung isolates, Bacillus safensis M35, Bacillus altitudinis R31 and Bacillus altitudinis J208, on crude pectin containing medium possessed polygalacturonate hydrolase, pectate lyase and pectin lyase activities. Studies regarding their stability under various temperature and pH conditions revealed their mild acidic to alkaline and mesophilic nature with enzyme activity falling within the pH range 6·0-9·0 and temperature range 30-60°C. The pectinase activity was categorized as endolytic as it brought about ~50% reduction in relative viscosity of pectic polymer within initial 10 min of incubation. Synergism of pectinase activity with crude xylanase activities and/or commercial cellulase was clearly demonstrated as ~1·6 to ~1·9-fold increase in agrowaste biomass saccharification was obtained confirming the role of pectinases as accessory enzymes., Conclusion: Synergism of the broad-spectrum endopectinase activity obtained from three Bacillus isolates with accessory crude xylanases from the same isolates and commercial cellulase enhanced the agrowaste saccharification and confirmed the accessory role of crude pectinase as they formed an efficient enzyme cocktail functioning in a contributive manner for improvement of agrowaste biomass saccharification., Significance and Impact of the Study: Mesophilic crude endopectinases obtained from Bacillus spp. isolated from ruminant dung possessed activity in broad pH and temperature ranges as well as broad substrate specificity. Moreover, their synergism with crude xylanase and Primfast ® 200 cellulase demonstrated the potential to form efficient enzyme cocktail for application in plant biomass saccharification process., (© 2018 The Society for Applied Microbiology.)

Medium compositions for a heat-tolerant acidic pectinase production from Bacillus sp. ZJ1407 were optimized via response surface methodology (RSM) and its enzymatic properties were investigated. A 2-level factorial design was used to estimate the main effect of factors, and to screen the significant factors. A central composite design was used to find out the optimal concentrations of screened key factors. Lactose, tryptone and (NH 4 ) 2 SO 4 were found to have a significant influence on the pectinase activity (p <0.05). The optimal medium compositions were as follows: lactose 44.8g/l, tyrptone 30.9g/l, (NH 4 ) 2 SO 4 1.35g/l, MnSO 4 ·H 2 O 0.2g/l, MgSO 4 0.4g/l and NaCl 3.5g/l. Pectinase was purified to homogeneity by ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephadex G-100 size-exclusion chromatography. The molecular weight of the purified pectinase determined by SDS-PAGE was about 23kDa, and its final specific activity was 110.47U/mg. Its optimal temperature and pH were 37°C and 5.0, respectively. Pectinase was very stable within a pH range of 3.0-5.0, and showed a high thermo-stability at 80 and 90°C. Ba 2+ could significantly promote the activity of pectinase, and Mn 2+ heavily inhibited its activity. This study provides new insight into the future development and use of pectinase from Bacillus sp. ZJ1407., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are V max =4.18μM.s -1 ,K m =3.25mgmL -1 and k cat =2.58s -1 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands., Methods: A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized., Results: Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 μmol/min, respectively., Conclusion: This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Alkaline pectinase has important applications in the pretreatment of waste water from food processing and in both the fabric and paper industries. In this study, a 2-level factorial design was used to screen significant factors that affect the activity of alkaline pectinase, and the response surface methodology (RSM) with a Box-Behnken design (BBD) was used to optimize their concentrations. Starch, peptone, KH 2 PO 4 and K 2 HPO 4 ·3H 2 O were found to significantly affect the activity of alkaline pectinase. Their optimal concentrations were as follows: 4.68% starch, 1.6% peptone, 0.26% KH 2 PO 4 and 0.68% K 2 HPO 4 ·3H 2 O. Under the above conditions, the activity of alkaline pectinase was significantly improved to 734.11 U/mL. Alkaline pectinase was purified to homogeneity with a recovery rate of 9.6% and a specific activity of 52372.52 U/mg. Its optimal temperature and pH were 50°C and 8.6, respectively. The purified enzyme showed strong thermo-stability and good alkali resistance. In addition, the activity of alkaline pectinase was improved in the presence of Mg 2+ . Cu 2+ , Mn 2+ , and Co 2+ significantly inhibited its activity. This study provides an important basis for the future development and use of a heat-tolerant alkaline pectinase from B. subtilis ZGL14.

Pectin is a plant cell wall constituent that is mainly composed of polygalacturonic acid (PGA), a linear α1,4-d-galacturonic acid (GalUA) backbone. Polygalacturonase (PG) hydrolyzes the α1,4-linkages in PGA. Nearly all plant PGs identified thus far are secreted as soluble proteins. Here we describe the microsomal PG activity in pea (Pisum sativum) epicotyls and present biochemical evidence that it was localized to the Golgi apparatus, where pectins are biosynthesized. The microsomal PG was purified, and it was enzymatically characterized. The purified enzyme showed maximum activity towards pyridylaminated oligogalacturonic acids with six degrees of polymerization (PA-GalUA6), with a Km value of 11 μM for PA-GalUA6. The substrate preference of the enzyme was complementary to that of PGA synthase. The main PG activity in microsomes was detected in the Golgi fraction by sucrose density gradient ultracentrifugation. The activity of the microsomal PG was lower in rapidly growing epicotyls, in contrast to the high expression of PGA synthase. The role of this PG in the regulation of pectin biosynthesis or plant growth is discussed., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

An extracellular exo-polygalacturonase (exo-PG) produced by Penicillium notatum was purified (3.07-folds) by ammonium sulfate fractionation, ion exchange, and gel filtration chromatography. Two distinct isoforms of the enzyme, namely exo-PGI and exo-PGII, were identified during column purification with molecular weights of 85 and 20 kDa, respectively, on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme displayed its optimum activity at pH 6.0 and 50 °C and was found to be stable in the slightly acidic pH (ranging from 4.5 to 6.0). Michaelis-Menten parameters, i.e., K m (app) and V max for pectin hydrolysis, were calculated to be 16.6 mg/mL and 20 μmol/mL/min, respectively. The enzyme followed biphasic deactivation kinetics. Phase I of the exo-PGI showed half-lives of 6.83 and 2.39 min at 55 and 80 °C, respectively, whereas phase II of the enzyme exhibited a half-life of 63.57 and 22.72 min at 55 and 80 °C, respectively. The activation energy for denaturation was 51.66 and 44.06 kJ/mol for phase I and phase II of the exo-PGI, respectively. The enzyme activity was considerably enhanced by Mn 2+ , whereas exposure to a hydrophobic environment (urea and sodium azide solution) drastically suppressed the enzyme activity. Results suggest that exo-PGI might be considered as a potential candidate for various applications, particularly in the food and textile industries.

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., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The viability of the scaling-up of pectinases production by Aspergillus flavipes at 5L-bioreactor scale has been demonstrated by keeping constant the power input, and a drastic increase in the endo- and exopectinolytic enzyme production was recorded (7- and 40-fold, respectively). The main process variables were modelled by means of logistic and Gompertz equations. In order to overcome the limitations of the conventional downstream strategies, a novel extraction strategy was proposed on the basis of the adequate salting-out potential of two biocompatible cholinium-based ionic liquids (N 1112OH Cl and N 1112OH H 2 PO 4 ) in aqueous solutions of Tergitol, reaching more than 90% of extraction., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 endo-polygalacturonase. 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+., (© 2017 S. Karger AG, Basel.)

Xanthomonas oryzae pv.oryzae (Xoo) causes the serious bacterial blight disease of rice. Xoo secretes a repertoire of plant cell wall degrading enzymes (CWDEs) like cellulases, xylanases, esterases etc., which act on various components of the rice cell wall. The major cellulases and xylanases secreted by Xoo have been identified and their role in virulence has been determined. In this study, we have identified some of the pectin degrading enzymes of Xoo and assessed their role in virulence. Bioinformatics analysis indicated the presence of four pectin homogalacturonan (HG) degrading genes in the genome of Xoo. The four HG degrading genes include one polygalacturonase (pglA), one pectin methyl esterase (pmt) and two pectate lyases (pel and pelL). There was no difference in the expression of pglA, pmt and pel genes by laboratory wild type Xoo strain (BXO43) grown in either nutrient rich PS medium or in plant mimic XOM2 medium whereas the expression of pelL gene was induced in XOM2 medium as indicated by qRT-PCR experiments. Gene disruption mutations were generated in each of these four genes. The polygalacturonase mutant pglA- was completely deficient in degrading the substrate Na-polygalacturonicacid (PGA). Strains carrying mutations in the pmt, pel and pelL genes were as efficient as wild type Xoo (BXO43) in cleaving PGA. These observations clearly indicate that PglA is the major pectin degrading enzyme produced by Xoo. The pectin methyl esterase, Pmt, is the pectin de-esterifying enzyme secreted by Xoo as evident from the enzymatic activity assay performed using pectin as the substrate. Mutations in the pglA, pmt, pel and pelL genes have minimal effects on virulence. This suggests that, as compared to cellulases and xylanases, the HG degrading enzymes may not have a major role in the pathogenicity of Xoo., Competing Interests: The authors have declared that no competing interests exist.

Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

An extracellular endo-polygalacturonase (PGase) was purified, after a single purification step, from the constitutive and hyperpectinolytic CT1 mutant of Penicillium occitanis. This enzyme named PG2 has a molecular weight of 42kDa. It was optimally active at 35°C and pH6 with more than 85% of activity at pH7 in contrast to the majority of fungal PGase, generally acting at 50°C and pH5. The specific activity obtained was among the highest ones, 31397.26U/mg. The PGase activity increased with the decrease of the degree of methylation (DM) of pectin, but it was also able to degrade the highly methyl-esterified substrates, 70% (DM) and 90% (DM), with almost 80% and 40% of residual activity respectively. Interestingly, PG2 is completely inhibited by DEPC, suggesting the implication of a Histidine residue in the active site. The sequencing of P. occitanis whole genome allowed us to identify the pga2 gene encoding PG2 and to localize the His residue, target of DEPC, while it was absent in the PG1 that resisted to DEPC. Besides that, the potentialities of PG2 have been put in use in juice clarification of pear, banana and citrus juice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme., Results: F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2)., Conclusions: Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca 2+ , and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. E a ( d ) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and Δ G ( d ) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus . Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme.

Polygalacturonase is one of the important enzymes used in various industries such as food, detergent, pharmaceutical, textile, pulp and paper. A novel liquid/liquid extraction process composed of surfactant and acetonitrile was employed for the first time to purify polygalacturonase from Durio zibethinus. The influences of different parameters such as type and concentration of surfactants, concentrations of acetonitrile and composition of surfactant/acetonitrile on partitioning behavior and recovery of polygalacturonase was investigated. Moreover, the effect of pH of system and crude load on purification fold and yield of purified polygalacturonase were studied. The results of the experiment indicated the polygalacturonase was partitioned into surfactant top rich phase with impurities being partitioned into acetonitrile bottom rich phase in the novel method of liquid/liquid process composed of 23% (w/w) Triton X-100 and 19% (w/w) acetonitrile, at 55.6% of TLL (tie line length) crude load of 25% (w/w) at pH 6.0. Recovery and recycling of components also was measured in each successive step of liquid/liquid extraction process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 97.3% while phase components were also recovered and recycled above 95%. This study demonstrated that the novel method of liquid/liquid extraction process can be used as an efficient and economical extraction method rather than the traditional methods of extraction for the purification and recovery of the valuable enzyme., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL-1 and 101.01 Units·mg·protein-1·min-1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3-7 and >50% activity in 10-50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.

The present study aimed to investigate and compare the enzymatic production of endophytic bacteria isolated from healthy and brittle leaf disease affected date palm leaves (pectinase, cellulase, lipase, and amylase). The findings revealed that the enzymatic products from the bacterial isolates of healthy date palm leaves were primarily 33% amylolytic enzyme, 33 % cellulase, 25 % pectinase, and 25 % lipase. The isolates from brittle leaf disease date palm leaves, on the other hand, were noted to produce 16 % amylolytic enzyme, 20 % cellulose, 50 % pectinase, and 50 % lipase. The effects of temperature and pH on amylase, pectinase, and cellulose activities were investigated. The Bacillus subtilis JN934392 strain isolated from healthy date palm leaves produced higher levels of amylase activity at pH 7. A Box Behnken Design (BBD) was employed to optimize amylase extraction. Maximal activity was observed at pH and temperature ranges of pH 6-6.5 and 37-39 °C, respectively. Under those conditions, amylase activity was noted to be attained 9.37 U/ml. The results showed that the enzyme was able to maintain more than 50 % of its activity over a temperature range of 50-80 °C, with an optimum at 70 °C. This bacterial amylase showed high activity compared to other bacteria, which provides support for its promising candidacy for future industrial application.

A novel aqueous two-phase system composed of a surfactant and sorbitol was employed for the first time to purify pectinase from Psidium guajava. The influences of different parameters, including the type and concentration of the surfactant and the concentration and composition of the surfactant/sorbitol ratio, on the partitioning behavior and recovery of pectinase were investigated. Moreover, the effects of system pH and the crude load on purification fold and the yield of purified pectinase were studied. The experimental results indicated that the pectinase was partitioned into surfactant-rich top phase, and the impurities were partitioned into the sorbitol-rich bottom phase with the novel method involving an ATPS composed of 26% (w/w) Triton X-100 and 23% (w/w) sorbitol at 54.2% of the TLL crude load of 20% (w/w) at pH 6.0. The enzyme was successfully recovered by this method with a high purification factor of 15.2 and a yield of 98.3%, whereas the phase components were also recovered and recycled at rates above 96%. This study demonstrated that this novel ATPS method can be used as an efficient and economical alternative to the traditional ATPS for the purification and recovery of the valuable enzyme.

Characterization, kinetic and thermodynamic parameters of purified pectinase from Bacillus subtilis, isolated from a marine sediment sample collected from Chinchani beach at Tarapore, India, were studied. Marine pectinase produced under submerged growth conditions was purified by ammonium sulfate precipitation followed by gel filtration chromatography using DEAE cellulose. Partial characterization of the marine pectinase was carried out in terms of effect of pH, temperature, substrate concentration, and metal ions. It was found that pectinase from marine B. subtilis showed maximal activity in alkaline buffer at pH 9.0 and at 40°C. It was also found that metal ions, namely, Mn(2+) and Fe(2+), stimulate pectinase activity. Marine pectinase followed Michaelis-Menten kinetics. The kinetics and thermodynamic parameters of the purified marine pectinase from B. subtilis were studied as the characterization of the enzyme is vital for its use in industrial processes.

Polygalacturonase (PG) production by Penicillium chrysogenum during solid-state fermentation was accompanied by decomposition of orange peels. A leaching procedure was developed through the selection of solvent, time and intensity of stirring. A maximum PG activity was observed after 48 h peel inoculation. Further cultivation decreased the enzyme activity significantly, up to 60% of the maximum PG activity. During fermentation, a rapid acidification of the solid medium which inhibited the pectinolytic enzyme, was observed. Buffering agents with different pH values and different ionic strengths were examined to identify the most suitable medium to avoid this problem. Buffer addition counteracted acidification and enhanced active protein production, which was observed for all of the applied pH values (6.5-8.0) of the buffering agent. The most satisfactory results were obtained when using the highest pH at 8.0. The protein content and PG activity increased from 3.5 mg/g and 1.09 U/g to 7.7 mg/g and 7.11 U/g during cultivation, with uncontrolled and pH-controlled medium, respectively. Measurements at wide pH and temperature ranges indicated an optimum for PG activity at pH 5.0 and 43°C; however, high thermal stability corresponded to lower temperatures, and a temperature of 37°C is thus recommended. Under these conditions, the operational stability was determined to be t1/2=570 h.

Xylanase and polygalacturonase were concurrently produced by a novel alkalo-thermotolerant Bacillus pumilus AJK. They were purified and characterized to evaluate their potential for various industrial applications. Xylanase was purified to 19-fold with 67 % recovery and polygalacturonase up to 23-fold with 75 % recovery. Existence of multiple forms of xylanase was indicated by its elution-profile through Sephadex G-100 as two peaks, xylanase-I and xylanase-II, with molecular weights of ~24.5 and ~13 kDa, respectively, and by the presence of two pH optima, one at pH 6.0 and other at pH 8.5. The molecular weight of polygalacturonase was ~40 kDa by gel-filtration chromatography. Zymographic studies confirmed the presence of seven isozymic forms of xylanase. Xylanase and polygalacturonase are stable over a broad range of pH and temperature.

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

Formation of dense, highly hydrated biofilm structures pose a risk for public and environmental health. Extracellular polymeric substances encompassing biofilms offer 1000-fold greater resistance as compared to the planktonic cells. Using enzymes as anti-biofouling agents, will improve penetration of antimicrobials and increase susceptibility of biofilms to components of immune system. The challenge of using enzymes derived from unrelated bacteria for the degradation of capsular matrix of Klebsiella pneumoniae has not been dealt in the past. Thus, statistical optimization was done to enhance depolymerase production by Aeromonas punctata, directed against the exopolysaccharide matrix of Klebsiella pneumoniae B5055, capable of substituting the available phage borne depolymerase enzyme. Optimization via central composite design (CCD) resulted in 16-fold enhancement in depolymerase yield (166.65 µmoles ml(-1)  min(-1) ) over unoptimized medium. Out of the 19 variables, media composition giving maximum expression levels of the enzyme consisted of 1 mg ml(-1) galactose and ammonium chloride, 1.5 mg ml(-1) each of capsular polysaccharide (CPS) and magnesium sulfate. Tryptic peptide analysis of the purified 29 kDa band by Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) showed a high homology with a protein of unknown function from Aeromonas cavaie Ae398. Further improvements in the enzyme can lead to its successful development as prophylactic and/or a therapeutic agent., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A multimodular pectinase of glycoside hydrolase family 28, S6A, was identified in Penicillium oxalicum SX6 that consists of an N-terminal catalytic domain of pectin methylesterase, a Thr/Ser-rich linker region, and a C-terminal catalytic domain of polygalacturonase. Recombinant S6A and its two derivatives, S6PE (the catalytic domain of pectin methylesterase) and S6PG (the catalytic domain of polygalacturonase), were produced in Pichia pastoris. S6A was a bifunctional protein and had both pectin methylesterase and polygalacturonase activities. Three enzymes showed similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 50 °C) and excellent stability at pH 3.5-6.0 and 40 °C. Most metal ions tested (Na(+), K(+), Ca(2+), Li(+), Co(2+), Cr(3+), Ni(2+), Cu(2+), Mn(2+),Mg(2+), Fe(3+), Zn(2+), and Pb(2+)) enhanced the pectin methylesterase activities of S6PE and S6A, but had little or inhibitory effects on the polygalacturonase activities of S6A and S6PG. In comparison with most fungal pectin methylesterases, S6A had higher specific activity (271.1 U/mg) towards 70 % DM citrus pectin. When S6PE and S6PG were combined at the activity ratio of 1:4, the most significant synergistic effect was observed in citrus pectin degradation and degumming of sisal fiber, which is comparable with the performance of S6A (95 v.s. 100 % and 16.9 v.s. 17.2 %, respectively). To the best of our knowledge, this work represents the first report of gene cloning, heterologous expression, and biochemical characterization of a bifunctional pectinase with separate catalytic domains.

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T(660 nm) = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Ustilago maydis is a pathogenic fungus that produces the corn smut. It is a biotrophic parasite that depends on living plant tissues for its proliferation and development. Polygalacturonases are secreted by pathogens to solubilize the plant cell-wall and are required for pathogen virulence. In this paper, we report the isolation of a U. maydis polygalacturonase gene (Pgu1) and the functional and structural characterization of the encoded enzyme. The U. maydis Pgu1 gene is expressed when the fungus is grown in liquid culture media containing different carbon sources. In plant tissue, the expression increased as a function of incubation time. Pgu1 gene expression was detected during plant infection around 10 days post-infection with U. maydis FB-D12 strain in combination with teliospore formation. Synthesis and secretion of active recombinant PGU1 were achieved using Pichia pastoris, the purified enzyme had a optimum temperature of 34 °C, optimum pH of 4.5, a Km of 57.84 g/L for polygalacturonic acid, and a Vmax of 28.9 µg/min mg. Structural models of PGU1 based on homologous enzymes yielded a typical right-handed β-helix fold of pectinolytic enzymes classified in the glycosyl hydrolases family 28, and the U. maydis PGU1 is related with endo rather than exo polygalacturonases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The endo-galacturonase A gene (pgaA) was cloned using the cDNAs synthesized from total RNA of Aspergillus niger JL-15 by reverse transcription as template. The open reading frame (ORF) of pgaA was 1113bp, encoding a peptide of 370 amino acids with the predicted molecular mass of 38.8kDa. The pgaA was successfully expressed in Pichia pastoris GS115 under the control of AOX1 promoter. After induction by methanol for 96h, the activity of the recombinant endo-galacturonase A (rePgaA) in culture supernatant was 2091.0U/mg. SDS-PAGE analysis showed that the molecular mass of rePgaA was about 40.0kDa. Enzymatic properties assays showed that the optimum temperature and pH for rePgaA were 50°C and pH 5.0, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of rePgaA for citrus pectin were 3.20mgml(-1) and 40.97μmolmin(-1)ml(-1), respectively. The rePgaA mediated a rapid decrease in viscosity of pectin solution with release of small amount of reducing sugar. High performance liquid chromatography (HPLC) analysis revealed that digalacturonate (G2) and trigalalcturonate (G3) were the main hydrolysis products released from pectin by rePgaA. The rePgaA showed very low activity on G2 and G3, which suggested it was a typical endo-acting enzyme., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A novel endo-polygalacturonase (endo-PG I) from Achaetomium sp. Xz8 was identified, overexpressed in Pichia pastoris, and characterized in this report. Recombinant endo-PG I is distinguished from other enzyme counterparts by its high activity towards polygalacturonic acid (49,934 U/ml) and high yield in the 15-l fermentor (2.13 g/l). It exhibits optimal activity at 45 °C and remained active over a broad temperature range of 0-80 °C. Distinct from most fungal polygalacturonases that have acidic pH optima, endo-PG I is optimally active at pH 6, similar to the pH of fresh papaya juice (5.7). Endo-PG I alone reduced the viscosity of papaya juice by 17.6%, and increased its transmittance by 59.1%. When combined with a commercial pectin methylesterase, it showed much higher efficiency with a synergy degree of more than 1.25. All these favourable enzymatic properties make endo-PG I attractive for potential applications in the juice industry., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Plant peels could be a potential source of novel pectinases for use in various industrial applications due to their broad substrate specificity with high stability under extreme conditions. Therefore, the extraction conditions of a novel pectinase enzyme from pitaya peel was optimized in this study. The effect of extraction variables, namely buffer to sample ratio (2:1 to 8:1, X₁), extraction temperature (-15 to +25 °C, X₂) and buffer pH (4.0 to 12.0, X₃) on specific activity, temperature stability, storage stability and surfactant agent stability of pectinase from pitaya peel was investigated. The study demonstrated that the optimum conditions for the extraction of pectinase from pitaya sources could improve the enzymatic characteristics of the enzyme and protect its activity and stability during the extraction procedure. The optimum extraction conditions cause the pectinase to achieve high specific activity (15.31 U/mg), temperature stability (78%), storage stability (88%) and surfactant agent stability (83%). The most desirable conditions to achieve the highest activity and stability of pectinase enzyme from pitaya peel were the use of 5:1 buffer to sample ratio at 5 °C and pH 8.0.

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0-6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn(2+). The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K m, V max, K cat, and K cat/K m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.

Unlabelled: Of the twenty-three morphotypes of yeasts isolated from soil capable of utilizing pectin as sole carbon source at 6°C, two yeast isolates, one psychrotolerant (PT1) and one psychrophilic (SPY11), were selected according to their ability to secrete pectinolytic enzymes under some oenological conditions (temperature 6 and 12°C and pH 3.5) and ability or inability to grow above 20°C, respectively. As compared to their optimal activity, the three pectinolytic enzymes viz., pectin methyl esterase (PME), endopolygalacturonase (endo-PG) and exopolygalacturonase (exo-PG) isolated and assayed at pH 3.5 from PT1 were found to retain 39, 60 and 60% activity at 12°C and 40, 79 and 74% activity at 28°C, respectively. Likewise, the enzymes PME and endo-PG at pH 3.5 from SPY11 displayed 46 and 86% activity at 12°C and 50 and 60% activity at 28°C, respectively. All these enzymes showed 20-90% of residual activity at pH 3.5 and 6°C. The yeast isolates PT1 and SPY11 were identified as Rhodotorula mucilaginosa and Cystofilobasidium capitatum, respectively, on the basis of morphological, physiological and molecular characteristics. This study presents the first report on pectinolytic activities under major oenological conditions from psychrotolerant isolate R. mucilaginosa PT1 and psychrophilic isolate C. capitatum SPY11., Significance and Impact of the Study: The cold-active pectinolytic enzymes (PME, endo-PG and exo-PG) from the newly isolated and identified psychrophilic yeast Cystofilobasidium capitatum SPY11 and psychrotolerant yeast Rhodotorula mucilaginosa PT1that exhibited 50-80% of their optimum activity under some major oenological conditions pH (3.5) and temperatures (6 and 12°C) could be applied to wine production and juice clarification at low temperature. The psychrotrophic yeasts themselves could be applied to cold process for the production of enzymes thus saving cost of energy and protecting process from contamination., (© 2013 The Society for Applied Microbiology.)

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Aspergillus niger NRC1ami pectinase was evaluated according to its hydrolysis efficiency of dry untreated orange peels (UOP), HCl-treated orange peels and NaOH-treated orange peels (HOP and NOP). Pectinase was entrapped in polyvinyl alcohol (PVA) sponge and the optimum pH and temperature of the free and immobilized enzymes were shifted from 4, 40 °C to 6, 50 °C respectively. The study of pH stability of free and immobilized pectinase showed that the immobilization process protected the enzyme strongly from severe alkaline pHs. The immobilization process improved the enzyme thermal stability to great instant. The unique feature of the immobilization process is its ability to solve the orange juice haze problem completely. Immobilized enzyme was reused 12 times in orange juice clarification with 9% activity loss from the original activity. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the partially purified form were significantly changed after immobilization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hyper tannase and pectinase-producing yeast Rhodotorula glutinis MP-10 was isolated from persimmon (Diospyros kaki L.) fruits. The main pectinase activity of yeast was exo-polygalacturonase. No pectin methyl esterase and too low pectin lyase activities were detected for this yeast. The maximum exo-activities of tannase and polygalacturonase were determined as 15.2 and 26.9 U/mL for free cells and 19.8 and 28.6 U/mL for immobilized cells, respectively. Immobilized cells could be reused in 13 successive reaction cycles without any loss in the maximum tannase and polygalacturonase activities. Besides, too little decreases in activities of these enzymes were recorded between 14 and 18 cycles. At the end of 18 successive reaction cycles, total 503.1 U/mL of polygalacturonase and 349.6 U/mL of tannase could be produced using the same immobilized cells. This is the first report on the use of free and/or immobilized cells of a microorganism for the co-production of tannase and pectinase.

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS-PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0-5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3h. The addition of Mn(2+), K(+), Zn(2+), Ca(2+) and Al(3+) inhibited the enzyme activity; it increased in the presence of Mg(2+) and Cu(2+) ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K(m) and V(max) values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T(650) from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bioaffinity immobilization exploits the affinity of the enzyme to a macro-(affinity ligand). Such a macro-(affinity ligand) could be a lectin, a water-soluble polymer, or a bioconjugate of a water-soluble polymer and the appropriate affinity ligand. Successive layering of the enzyme and the macro-(affinity ligand) on a matrix allows deposition of a large amount of enzyme activity on a small surface. Illustrative protocols show affinity layering of a pectinase and horseradish peroxidase on Concanavalin A-agarose and Concanavalin A-Sephadex matrices, respectively.