Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

2018 ◽  
Vol 14 (9-10) ◽  
Author(s):  
Neslihan Saki ◽  
Mustafa Akin ◽  
Esma H. Alici ◽  
Gulnur Arabaci
Keyword(s):  
Ammonium Sulfate ◽  
Polyphenol Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Edible Mushroom ◽  
Bottom Phase ◽  
Phase Partitioning ◽  
Three Phase ◽  
Ammonium Sulfate Saturation ◽  
Wild Edible Mushroom

AbstractPolyphenol oxidase (PPO) from the wild edible mushroom Lepiota procera is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km – 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 °C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications.

scholarly journals Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

2019 ◽  
Vol 13 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Valentina Dobreva ◽  
Boriana Zhekova ◽  
Georgi Dobrev
Keyword(s):  
Solid State ◽  
Ammonium Sulfate ◽  
Rhizopus Arrhizus ◽  
Two Phase ◽  
Sodium Tartrate ◽  
Phase Partitioning ◽  
Potassium Sodium ◽  
Three Phase ◽  
Ammonium Sulfate Saturation ◽  
Potassium Sodium Tartrate

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Isolation of cathepsin D using three-phase partitioning in t-butanol/water/ammonium sulfate

1989 ◽  
Vol 180 (1) ◽  
pp. 169-171 ◽  
Author(s):  
Gavin R. Jacobs ◽  
Robert N. Pike ◽  
Clive Dennison
Keyword(s):  
Ammonium Sulfate ◽  
Cathepsin D ◽  
Phase Partitioning ◽  
Three Phase

scholarly journals Antifungal Activity Toward Fusarium culmorum in Soluble Wheat Extracts

2000 ◽  
Vol 90 (6) ◽  
pp. 666-671 ◽  
Author(s):  
F. M. Doohan ◽  
A. Mentewab ◽  
P. Nicholson
Keyword(s):  
Antifungal Activity ◽  
Ammonium Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fusarium Culmorum ◽  
Gel Filtration ◽  
Head Blight ◽  
Inhibitory Compounds ◽  
Molecular Masses

This study investigated antifungal activity in soluble extracts from seed of a range of wheat cultivars differing in susceptibility to Fusarium head blight. Antifungal activity was assessed in terms of β-D-glucuronidase (GUS) activity of a Fusarium culmorum GUS transformant using a sensitive laboratory assay. Significant antifungal activity was detected in seed extracts from WEK0609, CM 820036, and Arina. Initial characterization of the Arina seed extract indicated that it contained antifungal proteinaceous compounds. The Arina extract yielded two (60 and 80%) ammonium sulfate fractions containing inhibitory compounds. Gel filtration chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of antifungal fractions showed that the antifungal activities detected in the Arina 60 and 80% ammonium sulfate fractions were associated with putative proteinaceous compounds with apparent molecular masses of approximately 60 and 28 kDa, respectively.

Application of three-phase partitioning to the purification and characterization of polyphenol oxidase from antioxidant rosemary (Rosmarinus officinalis L.)

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Yonca Yuzugullu Karakus ◽  
Busra Kahveci ◽  
Arda Acemi ◽  
Gulden Kocak
Keyword(s):  
Enzyme Activity ◽  
Polyphenol Oxidase ◽  
Cetyltrimethylammonium Bromide ◽  
Rosmarinus Officinalis ◽  
Phase Partitioning ◽  
Three Phase ◽  
Diammonium Salt ◽  
The Common ◽  
Rosmarinus Officinalis L

AbstractPolyphenol oxidase (PPO) has been purified from the rosemary plant (Rosmarinus officinalis L.) through three-phase partitioning (TPP) and has been biochemically characterized. The optimized TPP consisted of 50% (w/v) ammonium sulfate and equal volumes of crude extract and tert-butanol prepared at pH 6.5 and room temperature. Using this system, PPO was purified 14-fold, with 230% recovery of activity from the middle phase. The partitioned enzyme had a molecular mass of 53 kDa. The highest enzyme activity was detected at 30 °C and pH 7.0 against catechol. In substrate specificity tests, the enzyme displayed activity towards catechol, 4-methylcatechol, caffeic acid, hydroquinone, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), pyrogallol, syringaldezine, and 3,4-dihydroxy-L-phenylalanine but no activity towards L-tyrosine. The enzyme was inhibited by the common PPO inhibitors; salicylhydroxamic acid (SHAM), cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and the organic solvent dimethyl sulfoxide (DMSO). Enzyme activity increased in the presence of the organic solvents acetone, ethanol, and methanol.

scholarly journals Monoclonal antibodies to human vitamin K-dependent protein S

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1583-1590
Author(s):  
RD Litwiller ◽  
RJ Jenny ◽  
JA Katzmann ◽  
RS Miller ◽  
KG Mann
Keyword(s):  
Monoclonal Antibodies ◽  
Ammonium Sulfate ◽  
High Performance ◽  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Sds Page ◽  
Dependent Protein ◽  
Hybridoma Technology

Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.

scholarly journals Monoclonal antibodies to human vitamin K-dependent protein S

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1583-1590 ◽  
Author(s):  
RD Litwiller ◽  
RJ Jenny ◽  
JA Katzmann ◽  
RS Miller ◽  
KG Mann
Keyword(s):  
Monoclonal Antibodies ◽  
Ammonium Sulfate ◽  
High Performance ◽  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Sds Page ◽  
Dependent Protein ◽  
Hybridoma Technology

Abstract Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S- complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high- performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.

scholarly journals The effect of different substrates on chitinase activity from Bacillus sp. WS4F

2021 ◽  
Vol 924 (1) ◽  
pp. 012034
Author(s):  
P M Astuti ◽  
S Setyahadi ◽  
A K Wardani ◽  
A Sutrisno
Keyword(s):  
Ammonium Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pathogenic Fungi ◽  
Chitinase Activity ◽  
Plant Diseases ◽  
Partial Purification ◽  
Bacillus Sp ◽  
Ammonium Sulfate Precipitation ◽  
Shrimp Shells

Abstract One of the roles of chitinase is as an antifungal which is widely used as a biocontrol agent for plant diseases caused by pathogenic fungi. Bacillus sp. WS4F has chitinase activity which can inhibit the growth of Ganoderma boninense, a fungus that attacks oil palm and causes basal stem rot (BSR). This study aims to investigate the effect of different substrates on the activity of the chitinase from Bacillus sp. WS4F. Two kinds of substrates i.e. chitin flakes and shrimp shells were used in this study. Enzyme activity of chitinase was analyzed after partial purification of enzyme was performed using ammonium sulfate precipitation followed by dialysis. The highest activity of chitinase was achieved by the substrate using shrimp shells. The ammonium sulfate precipitation (60-80% saturation) 0.0949 U/mL for activity enzyme and 0.2639 mg/mL for protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 64.389 kDa.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Sign in / Sign up

Export Citation Format

Share Document