Immobilization of enological pectinase in calcium alginate hydrogels: A potential biocatalyst for winemaking

A biocatalyst was obtained by immobilizing an enological commercial pectinase within calcium alginate hydrogels using an entrapment technique, and its catalytic activity was evaluated during different storage conditions. Hydrogel beads were stored at 4 °C in three different ways: (i) wet, in citrate buffer solution (pH 3.8); (ii) dehydrated by using a vacuum stove; and (iii) freeze-dried. Biocatalyst surface and their internal morphology were characterized by Scanning Electron Microscopy and a good enzyme distribution throughout alginate matrix was observed. Fourier Transform Infrared Spectroscopy results confirmed the presence of absorption bands associated with amino groups present in enzymes. Immobilization procedure did not modify the optimal pH and temperature (pH = 4.0 and 20 °C) for pectinase activity, comparing to free enzyme. Entrapped pectinase showed activity until six reaction cycles with 40% residual activity. Storage stability studies demonstrated that wet entrapped pectinase retained its initial enzymatic activity up to 11 weeks, whereas that lyophilized hydrogels retained its original activity after 8 months of storage. These results suggest that immobilized pectinase may be successfully exploited in various industrial applications, with special concern in grape juice clarification process. Thus, the turbidity of grape must decreased significantly using the immobilized pectinase during 150 min at 20 °C. This biocatalyst could be easily removed after clarification process and it can be reused, minimizing production economic costs in wine industry.Fil: Martín, María Carolina. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; ArgentinaFil: Lopez, Olivia Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Ciolino, Andrés Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Morata, Vilma Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; ArgentinaFil: Villar, Marcelo Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Ninago, Mario Daniel. Universidad Nacional de Cuyo. Facultad de Ciencias Aplicadas a la Industria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentin

Ultrafiltration Fibers like Bioreactors

A polysulfone ultrafiltration membrane with pectinase physically immobilized on it by a dynamic formation method was used to examine the potential of these reactive membranes in applications involving solutions containing pectin. The effect of various operational parameters such as: pH of enzyme and pectin solutions, NaCl, retentate flow rate, and enzyme (Ce) and substrate (Cp) concentrations on the production of reducing compounds expressed as galacturonic acid (Ca), was investigated. It was found that the maximum Ca values were obtained when: (i) enzyme solution to immobilize, without NaCl, had pH values between 4.2 and 4.6 and enzyme concentration from 5.0 to 7.5 mg/mL; (ii) pectin solution had initial pH values between 4.2 and 5.0 and a concentration of 3 mg/mL; and (iii) retentate flow rate was 0.025 L/minFil: Carrin, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Ceci, Liliana Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; ArgentinaFil: Lozano, Jorge Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentin

PECTOPLATE: the simultaneous phenotyping of pectin methylesterases, pectinases and oligogalacturonides in plants during biotic stresses

Degradation of pectin, a major component of plant cell wall, is important for fungal necrotrophs to achieve a successful infection. The activities of pectin methylesterases (PMEs) from both plants and pathogens and the degree and pattern of pectin methylesterification are critical for the outcome of plant–pathogen interaction. Partial degradation of pectin by pectin degrading enzymes releases oligogalacturonides (OGs), elicitors of plant defense responses. Few analytical techniques are available to monitor pectin methylesterification-modulating machineries and OGs produced during plant pathogen interaction. In the present study, ruthenium red is presented as useful dye to monitor both Botrytis cinerea mycelium growth and the induction of PME activity in plant tissue during fungal infection. Moreover a simple, inexpensive and sensitive method, named PECTOPLATE, is proposed that allows a simultaneous phenotyping of PME and pectinase activities expressed during pathogen infection and of pectinase potential in generating OGs. The results in the manuscript also indicate that PME inhibitors can be used in PECTOPLATE as a tool to discriminate the activities of plant PMEs from those of pathogen PMEs expressed during pathogenesis

Purifikasi Dan Karakterisasi Enzim Pektinase Dari Aspergillus Ustus Bl5 [Purification and Characterization of Pectinase From Aspergillus Ustus Bl5]

Pectinase is an enzyme that could hydrolyze pectin into galacturonic acid. Natural pectinase was produced by microbes such as bacteria, yeast, fungi and Actinomycetes. Application of pectinase in industry were mainly in juice industry, textile, pulp, tea, cocoa and coffee fermentation. In this research, we conducted purification and characterization of pectinase produced by Aspergillus ustus BL5 in submerged fermentation using commercial pectin. The result showed that the optimum of pectinase production was reached at 120 hours fermentation process with specific activity 0.59 U/mg. The crude extract of pectinase was then concentrated using PEG 6000 and purified by Sephadex G-75 gel filtration chromatography. There were 2 fractions contained pectinase which the activity was 4.15 U/mg (pectinase A) and 3.3 U/mg (pectinase B), respectively. Compare to crude extract, the yield product of pectinase A and B increased 6.94 and 5.53 times, respectively. The purified pectinase A have optimum temperature at 50 oC and optimun pH at 5

AMOBILISASI PEKTINASE DARI Bacillus firmus MENGGUNAKAN MATRIKS OPP (OXIDIZED POLYPROPYLENE)-KITOSAN

A Pectinase is an enzyme that can hydrolyze pectin compounds into galacturonic acid. In order to enhance the pectinase efficiency, the enzyme can be immobilized in certain matrix. The pectinase was isolated from Bacillus firmus, and then purified with ammonium sulphate with 20-60% saturated level, followed by dialysis. The aims of the research were to determine the optimum conditions of immobilized pectinase by physical adsorption on oxidized polypropylene (OPP) coated chitosan, which included the shaking time and enzyme concentration. Initial protein used for immobilizing free pectinase was 1.367 mg/mL and the activity was 241.1 units. The results showed that the optimum condition of pectinase immobilization was achieved on shaking time of 3 hours and pectinase concentration of 1.094 mg/mL with amount of pectinase adsorbed at 53,98 mg/g within activity of 220.2 units

The Draft Genome of the Invasive Walking Stick, Medauroidea extradendata, Reveals Extensive Lineage-Specific Gene Family Expansions of Cell Wall Degrading Enzymes in Phasmatodea.

Plant cell wall components are the most abundant macromolecules on Earth. The study of the breakdown of these molecules is thus a central question in biology. Surprisingly, plant cell wall breakdown by herbivores is relatively poorly understood, as nearly all early work focused on the mechanisms used by symbiotic microbes to breakdown plant cell walls in insects such as termites. Recently, however, it has been shown that many organisms make endogenous cellulases. Insects, and other arthropods, in particular have been shown to express a variety of plant cell wall degrading enzymes in many gene families with the ability to break down all the major components of the plant cell wall. Here we report the genome of a walking stick, Medauroidea extradentata, an obligate herbivore that makes uses of endogenously produced plant cell wall degrading enzymes. We present a draft of the 3.3Gbp genome along with an official gene set that contains a diversity of plant cell wall degrading enzymes. We show that at least one of the major families of plant cell wall degrading enzymes, the pectinases, have undergone a striking lineage-specific gene family expansion in the Phasmatodea. This genome will be a useful resource for comparative evolutionary studies with herbivores in many other clades and will help elucidate the mechanisms by which metazoans breakdown plant cell wall components

Phosphoenolpyruvate Carboxykinase Is Involved in the Decarboxylation of Aspartate in the Bundle Sheath of Maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C4 photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-14C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn2+, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-14C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn2+ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK

Amobilisasi Pektinase Dari Bacillus Firmus Menggunakan Matriks Opp (Oxidized Polypropylene)-kitosan

A Pectinase is an enzyme that can hydrolyze pectin compounds into galacturonic acid. In order to enhance the pectinase efficiency, the enzyme can be immobilized in certain matrix. The pectinase was isolated from Bacillus firmus, and then purified with ammonium sulphate with 20-60% saturated level, followed by dialysis. The aims of the research were to determine the optimum conditions of immobilized pectinase by physical adsorption on oxidized polypropylene (OPP) coated chitosan, which included the shaking time and enzyme concentration. Initial protein used for immobilizing free pectinase was 1.367 mg/mL and the activity was 241.1 units. The results showed that the optimum condition of pectinase immobilization was achieved on shaking time of 3 hours and pectinase concentration of 1.094 mg/mL with amount of pectinase adsorbed at 53,98 mg/g within activity of 220.2 units

Impact of carbon and nitrogen sources on pectinase production of post-harvest fungi

In the present investigation, emphasis has been given on to study the pectinase enzyme production of post-harvest fungi isolated from mango and papaya fruits, under the influence of carbon and nitrogen sources. It was found that, among carbon sources fructose and sucrose induced pectinase activity, while lactose, CMC and starch inhibited the pectinase activity of test fungi. Nitrate source like sodium nitrate, sodium nitrite and calcium nitrate were found to be stimulated the pectinase activity while, ammonium sources in the form of nitrate, phosphate and sulphate were proved inhibitory for pectinase production of all post-harvest fungi