Purification and Thermodynamic Characteristics of an Exo-Polygalacturonase from Trichoderma Pseudokoningii

2020 ◽  
Vol 42 (5) ◽  
pp. 767-767
Author(s):  
Wesam H Abdulaal and Yaaser Q Almulaiky Wesam H Abdulaal and Yaaser Q Almulaiky
Keyword(s):  
Food Industry ◽  
Potential Role ◽  
Fruit Juice ◽  
Gel Filtration ◽  
Thermodynamic Characteristics ◽  
Orange Peel ◽  
Sds Page ◽  
Trichoderma Pseudokoningii ◽  
Km Value

Polygalacturonases (PGs) are necessary to degrade the insoluble viscous pectin components during the clarification process of fruit juice and are produced by some plants and various microbes, such as bacteria, yeasts and fungi. In this study, an exo-polygalacturonase (exo-PGP4a) was purified from T. Pseudokoningii using DEAE-Sepharose and Sephacryl S-200 columns. We show that the enzyme produced in this study by solid-state fermentation of citrus Orange peel was purified 20-fold with 12.8% recovery. The apparent molecular mass of the enzyme was determined to be 25 kDa using gel filtration and SDS-PAGE. The optimum temperature and pH of the exo-PGP4a were 45and#176;C and 6, respectively. The exo-PGP4a showed half-lives of 50.95 and 21.32 min at 55 and 75and#176;C, respectively. The activation energy for denaturation (Ea*) was 42.596 kJ/mol. The Km value of the enzyme for PGA hydrolysis was 2 mg/ml, and the Vmax was 3.27 and#181;mol min-1 mg-1. Several metal cations, such as Cu2+and Zn2+, were found to enhance the enzymatic activity of the exo-PGP4a, while Pb2+, Ca2+, Ni2+, Cd2+, Co2+ and Hg2+ ions were found to be inhibitory. In this study, we suggest the exo-polygalacturonase has potential role of the clarification of Orange, Apple, Grape, and Peach juices in the food industry.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Streptococcus salivarius Promotes Mucin Putrefaction and Malodor Production by Porphyromonas gingivalis

2006 ◽  
Vol 85 (10) ◽  
pp. 910-914 ◽  
Author(s):  
N. Sterer ◽  
M. Rosenberg
Keyword(s):  
Porphyromonas Gingivalis ◽  
Potential Role ◽  
Oral Microbiota ◽  
Streptococcus Salivarius ◽  
Oral Malodor ◽  
Gram Positive ◽  
Gram Negative ◽  
Sds Page ◽  
Micro Organisms

Although the contribution of the oral microbiota to oral malodor is well-documented, the potential role of Gram-positive micro-organisms is unclear. In the current study, we tested the hypothesis that Gram-positive micro-organisms contribute to malodor production by deglycosylating oral glycoproteins, rendering them susceptible to subsequent proteolysis. To this end, we examined the effect of Streptococcus salivarius on Porphyromonas gingivalis-mediated putrefaction of a model glycoprotein (pig gastric mucin). Malodor was scored by two odor judges, and volatile sulfides were determined with the use of a sulfide monitor. Mucin degradation was followed by electrophoresis on SDS-PAGE. Results showed that the addition of S. salivarius or β-galactosidase promoted mucin degradation and concomitant malodor production. Addition of glycosidic inhibitors (p-APTG and glucose) inhibited this process. These results suggest that Gram-positive micro-organisms such as S. salivarius contribute to oral malodor production by deglycosylating salivary glycoproteins, thus exposing their protein core to further degradation by Gram-negative micro-organisms.

scholarly journals Partial purification and characterization of a peroxidase activity from human placenta

1990 ◽  
Vol 268 (3) ◽  
pp. 739-743 ◽  
Author(s):  
J L Nelson ◽  
A P Kulkarni
Keyword(s):  
Peroxidase Activity ◽  
Potential Role ◽  
Gel Filtration ◽  
Partial Purification ◽  
Human Haemoglobin ◽  
Prostaglandin Synthase ◽  
Membrane Bound ◽  
Cytochrome P 450

Peroxidases can metabolize a variety of xenobiotics to reactive intermediates capable of binding to protein or DNA. The potential role of these enzymes in fetotoxicity has not been explored. In this study, the presence of peroxidase activity was observed in human term and pre-term placenta. Human term placental peroxidase activity (HTPP) was partially purified by concanavalin A affinity chromatography from CaCl2 extracts of the particulate fraction. HTPP appears to be a membrane-bound glycoprotein. Arachidonic acid-dependent oxidation of guaiacol was not observed, suggesting that the peroxidase activity was not due to prostaglandin synthase. Moreover, HTPP preparations were devoid of catalase and spectrally dissimilar from human haemoglobin, cytochrome P-450, eosinophil peroxidase and myloperoxidase, suggesting an endogenous origin. An Mr of approx. 119,000 was determined for HTPP by gel filtration. Cathodic slab-PAGE of cetyltrialkylammonium bromide-solubilized HTPP yielded two peroxidase-staining bands.

scholarly journals Purification and Characterization of a Prominent Polygalacturonase Isozyme Produced by Phomopsis cucurbitae in Decayed Muskmelon Fruit

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 488F-489
Author(s):  
J.X. Zhang ◽  
B.D. Bruton ◽  
C.L. Biles
Keyword(s):  
Latent Infection ◽  
Gel Filtration ◽  
Central American ◽  
Purification And Characterization ◽  
Fruit Storage ◽  
Sds Page ◽  
Fruit Decay ◽  
Isozyme Activity

Phomopsis cucurbitae is a latent infecting pathogen that infects unripe muskmelon fruit, but causes decay after harvest. This fungus causes severe losses during muskmelon fruit storage and marketing in the U.S., Japan, and some Central American countries. Previous studies showed that the fungus produced the cell wall-degrading enzyme polygalacturonase (PG) in both culture and muskmelon fruit tissue. The role of P. cucurbitae PG in the fruit decay process and its relation to latent infection is not well-understood. A prominent PG isozyme produced by the fungus in decayed fruit was purified to homogeneity by a sequence of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. This isozyme exhibited endo-activity, a molecular weight of 54 kDa according to SDS-PAGE, and a pI of 4.2 based on IEF-PAGE. Isozyme activity was optimal at 40–45°C and pH 5.0. It had a Km of 44.7 g/ml and a Vmax of 0.313. The purified isozyme also effectively macerated mature muskmelon fruit tissues. This isozyme was the most prominent of the PG isozymes produced by P. cucurbitae in decaying fruit, and may play an important role in postharvest decay.

Purification, application and immunolocalization of thermostable xylanases

2014 ◽  
Author(s):  
◽  
Stephanie Govender
Keyword(s):  
Reducing Sugars ◽  
Animal Feed ◽  
Paper Industry ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Thermomyces Lanuginosus ◽  
Sds Page ◽  
Km Value ◽  
Enzyme Dosage ◽  
Paper And Pulp

Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry.

scholarly journals Purification and Characterization of α-amylase from a Novel Thermoalkalophilic Strain of Bacillus sonorensis GV2 Isolated from Mushroom Compost

2019 ◽  
pp. 1-14
Author(s):  
Gitanjali Vyas ◽  
Nivedita Sharma ◽  
Nisha Sharma
Keyword(s):  
Corn Starch ◽  
Gel Filtration ◽  
Mushroom Compost ◽  
Raw Starch ◽  
Peptide Mass ◽  
Sds Page ◽  
Wide Range ◽  
Bacillus Sonorensis

A novel thermoalkalophilic α-amylase producing bacterial strain Bacillus sonorensis GV2 |KJ775811.1| was isolated from mushroom compost. The purification of α-amylase was performed through different chromatography techniques. After purification with SDS-PAGE and Sephadex G-75 gel filtration, the molecular weight of monomeric α-amylase was found to be 45 kDa. The enzyme showed an optimal activity over a wide range of temperature and pH of 35-60oC and 7-11, respectively. The effect of divalent ions i.e. Mg2+ and Ca2+ showed a positive increase in enzyme activity. This enzyme is unique in a sense that it also exhibited a considerable raw corn starch hydrolyzing activity at 55oC. The end products when subjected to TLC which were identified as main maltooligosaccharides, proving the endo action of an enzyme. The Vmax and Km values of Bacillus sonorensis GV2 α-amylase were found to be 1347 μmol/mg/min and 3.46  mMol/ml. The MALDI peptide mass fingerprint analysis of the reduced and carboxymethylated amylase digested with chymotrypsin indicated that this partial amino acid sequence was homologous by a score of 6 with UDP transferalyase. All these findings suggests about the potential role of this α-amylase for raw starch degrading applications in the relevant industry.

scholarly journals Changes in Pectins in Apple Fruit during Development and Ripening

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 816A-816
Author(s):  
Jong-Pil Chun ◽  
Jae-Chang Lee ◽  
Yong-Soo Hwang
Keyword(s):  
Ion Exchange ◽  
Potential Role ◽  
Gel Filtration ◽  
Apple Fruit ◽  
Fruit Softening ◽  
Pectin Degradation ◽  
Degree Of Esterification ◽  
Fruit Development And Ripening ◽  
Chromatographic Evidence

Pectins isolated from three cultivars with different maturity were compared to find a potential role of pectin modification on the fruit softening during fruit development and ripening. There was an increase of total pectins in developing fruit and no significant decrease of pectins was confirmed even after storage in `Tusgaru' (30 days) and `Fuji' (120 days), whereas soluble pectins, except NaOH-soluble ones, gradually increased in all cultivars. Gel-filtration profile and ion exchange chromatographic evidence of soluble pectins revealed that pectin degradation in apple fruit may not be associated with softening. However, a degree of esterification probably has an important role on softening of fruits. Further results will be discussed in the presentation.

scholarly journals Quality and Authenticity Control of Fruit Juices-A Review

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1014 ◽  
Author(s):  
Marilena Dasenaki ◽  
Nikolaos Thomaidis
Keyword(s):  
Food Industry ◽  
Fruit Juice ◽  
Cost Effective ◽  
Control Area ◽  
Fruit Juices ◽  
Juice Quality ◽  
Hyphenated Techniques ◽  
Food Fraud ◽  
The Past

Food fraud, being the act of intentional adulteration of food for financial advantage, has vexed the consumers and the food industry throughout history. According to the European Committee on the Environment, Public Health and Food Safety, fruit juices are included in the top 10 food products that are most at risk of food fraud. Therefore, reliable, efficient, sensitive and cost-effective analytical methodologies need to be developed continuously to guarantee fruit juice quality and safety. This review covers the latest advances in the past ten years concerning the targeted and non-targeted methodologies that have been developed to assure fruit juice authenticity and to preclude adulteration. Emphasis is placed on the use of hyphenated techniques and on the constantly-growing role of MS-based metabolomics in fruit juice quality control area.

scholarly journals Purification and Characterization of Peroxidase From Anthracnose Disease Infected Papaya (Carica papaya L.)

2014 ◽  
Vol 6 (2) ◽  
pp. 49-57 ◽  
Author(s):  
L Bari ◽  
P Hassan ◽  
N Absar ◽  
S Khatun ◽  
MI Hossain
Keyword(s):  
Ammonium Sulphate ◽  
Gel Filtration ◽  
Potassium Cyanide ◽  
Ammonium Sulphate Precipitation ◽  
Reducing Conditions ◽  
Sds Page ◽  
Peroxidase Enzyme ◽  
Km Value ◽  
Temperature Optima

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAEcellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM. DOI: http://dx.doi.org/10.3329/bjmb.v6i2.17643 Bangladesh J Med Biochem 2013; 6(2): 49-57

Production of pectin lyase from Geobacillus pallidus p26, purification, characterization and fruit juice application

2014 ◽  
Vol 7 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Safinur Yildirim Çelik ◽  
Nazan Demir ◽  
Yaşar Demir ◽  
Ahmet Adiguzel ◽  
Medine Gulluce
Keyword(s):  
Molecular Weight ◽  
Fruit Juice ◽  
Hot Spring ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Lyase ◽  
Anion Exchange Column ◽  
Sds Page ◽  
Temperature Optima ◽  
Geobacillus Pallidus

Abstract A bacterial strain was isolated from Pasinler hot spring, Erzurum, Turkey. The purified thermophilic isolate was identified as Geobacillus pallidus P26 and used to produce extracellular pectin lyase (EC 4.2.2.10). Pectin lyase enzyme was purified 34 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 56 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was verified by SDS-PAGE. The pH- and temperature optima of enzyme were determined (pH 9.0 and 60°C, respectively). Pectin lyase was mostly stable at 50 oC for 24 hours. Its’ activity decreased to 50 % for 24 h at 60°C. KM and Vmax were calculated as 24.8 mg/mL and 2.28 μmol/L min, respectively. Purified pectin lyase was inhibited by Fe3+, Zn2+, Cu2+, Ca2+, Co2+ and Hg2+ but not by Mg2+. The purified pectin lyase enzyme was used for getting fruits juices. It was found that yields of fruits juices increased when they were compared with control.

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