scholarly journals Proteinases from buckwheat (Fagopyrum esculentum moench) seeds: Purification and properties of the 47 kDa enzyme

2006 ◽  
Vol 58 (3) ◽  
pp. 171-177 ◽  
Author(s):  
Gordana Timotijevic ◽  
Svetlana Radovic ◽  
Vesna Maksimovic
Keyword(s):  
Affinity Chromatography ◽  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Membrane Fraction ◽  
Aspartic Proteinase ◽  
Ammonium Sulfate Precipitation ◽  
Milk Clotting ◽  
Purification And Properties ◽  
Fagopyrum Esculentum Moench ◽  
Pepstatin A

Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .

Purification and properties of a β-hexosidase from Sporobolomyces singularis

1969 ◽  
Vol 47 (11) ◽  
pp. 1021-1025 ◽  
Author(s):  
J. A. Blakely ◽  
S. L. MacKenzie
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Properties

A β-hexosidase has been isolated from Sporobolomyces singularis by conventional techniques involving ammonium sulfate precipitation and chromatography on columns of Sephadex G-200 and DEAE-Sephadex A-50. Electrophoresis on polyacrylamide gel was used as the final preparative step. The sedimentation coefficient (s°20,w) of the enzyme was 7.6 and its molecular weight was in the range 140 000–145 000. Although the β-hexosidase performed the functions of a β-D-galactoside galactohydrolase (β-galactosidase), it also catalyzed the hydrolytic function normally performed by a β-D-glucoside glucohydrolase; both these functions appear to reside in the same molecule.

Purification of Oidiodendron kalrai proteases

1976 ◽  
Vol 22 (3) ◽  
pp. 327-333 ◽  
Author(s):  
P. M. Cino ◽  
R. P. Tewari
Keyword(s):  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Column Chromatography ◽  
Crude Extract ◽  
Proteolytic Enzymes ◽  
Deae Cellulose ◽  
Ammonium Sulfate Precipitation ◽  
Proteolytic Activities ◽  
Yeast Phase ◽  
Cellulose Column

Oidiodendron kalrai yeast-phase cells demonstrate proteolytic activity. Some of the proteolytic enzymes of the crude extract were purified by a combination of ammonium sulfate precipitation, Sephadex G-200, and diethylaminoethyl (DEAE) cellulose column chromatography. At least six proteins exhibiting a range of proteolytic activities could be identified by these procedures. Purity of the enzyme fractions obtained from the DEAE-cellulose columns was tested by running polyacrylamide gels.

scholarly journals Isolation and Purification of Jacalin from Artocarpus Heterophyllus Lam

2010 ◽  
Vol 7 (1) ◽  
pp. 71-81
Author(s):  
M.R. Othman ◽  
L.M. Min ◽  
W.J.N. Fernando
Keyword(s):  
Affinity Chromatography ◽  
Ammonium Sulfate ◽  
Large Scale ◽  
Maximum Yield ◽  
Ammonium Sulfate Precipitation ◽  
Protein Determination ◽  
Artocarpus Heterophyllus ◽  
Isolation And Purification ◽  
Lowry Method ◽  
Excessive Quantity

This paper presents investigation results of saturation conditions needed for purification of jacalin lectin from the extract seeds of Artocarpus heterophyllus by ammonium precipitation and affinity chromatography on Galactose-Affi gel Hz. Three different aspects of parameters encompassing the percentage of saturation of ammonium sulfate precipitation, the presence of ammonium sulfate on Lowry method and the suitable galactose concentration for optimum elution of the protein from Galactose-Affi gel Hz were investigated. With three different sets of fractional saturation of jacalin purification using ammonium sulfate precipitation, the maximum yield of 0.463 g/g was achieved at 0-90% saturation range in the absence of dialysis. Maximum yield of 0.425 g/g was obtained at 30-60% and 0-90% saturation range in the presence of dialysis. The result from this work also indicates that excessive quantity of NH4SO4 interferes with Lowry method for protein determination substantially. The 0-90% saturation range was found to be more potentially appropriate for large scale application than 30-60% saturation, since the former involves only 1 step NH4SO4 addition. From the affinity chromatography, elution of 0.2 M galactose (in 0.15 M NaCl) from Galactose-Affi gel Hz produced the maximum peak profile and jacalin concentration. A reduction or increase in galactose concentration of more than 0.2 M did not increase concentration of purified jacalin purified using this method.

scholarly journals Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography

2018 ◽  
Vol 1089 ◽  
pp. 43-59 ◽  
Author(s):  
Lentel Pringels ◽  
Valérie Broeckx ◽  
Kurt Boonen ◽  
Bart Landuyt ◽  
Liliane Schoofs
Keyword(s):  
Affinity Chromatography ◽  
Ammonium Sulfate ◽  
Plasma Protein ◽  
Protein A ◽  
Ammonium Sulfate Precipitation ◽  
Protein Depletion

scholarly journals Screening and Characterization of a Mutant Fungal Aspartic Proteinase from Mucor pusillus

2015 ◽  
Vol 9 (1) ◽  
pp. 119-126
Author(s):  
Li Yuqiu ◽  
Tan Hua ◽  
Li Da ◽  
Li Zhoulin ◽  
Chi Yanping ◽  
...  
Keyword(s):  
Cheddar Cheese ◽  
Aspartic Proteinase ◽  
Site Directed Mutagenesis ◽  
Soluble Nitrogen ◽  
Milk Clotting ◽  
Sds Page ◽  
Wide Range ◽  
Hydrolysis Of ◽  
Pepstatin A

In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutants showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60ºC. The enzyme was inhibited by metal ions (Fe2+, Fe3+, Cu+ and Zn2+), 1.10-Phenantrolin and pepstatin A. Further texture analysis of types of cheddar cheese made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without any significant change in flavor between these cheeses. The result implicated that, to some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably as a candidate for a useful milk coagulant.

Functional Characteristics of Extracellular Vesicles Produced by Pseudomonas fragi ATCC 4973

1990 ◽  
Vol 53 (10) ◽  
pp. 859-868
Author(s):  
ROBERT M. MYHARA ◽  
BRENT J. SKURA ◽  
THAKOR PATEL ◽  
M. NAKAJIMA
Keyword(s):  
Ammonium Sulfate ◽  
Proteolytic Activity ◽  
Extracellular Vesicles ◽  
Liquid Culture ◽  
Free Form ◽  
Ammonium Sulfate Precipitation ◽  
Functional Characteristics ◽  
Pseudomonas Fragi ◽  
Extracellular Proteinases

Extracellular vesicles, “blebs,” were isolated from Pseudomas fragi ATCC 4973 grown in liquid culture. Vesicles were isolated by ammonium sulfate precipitation, centrifugation, and dialysis of the supernatant of an 82 h culture of P. fragi in trypticase-soy broth. The vesicles displayed considerable proteolytic activity. The proteinase was similar to that found in a free form in the supernatant. The vesicles were not associated with bacteriocinogenicity. The vesicles may act in the physiological distribution of extracellular proteinases.

scholarly journals Characteristics of Curds With Milk Clotting Enzyme from Indonesian Local Isolate of Lactic Acid Bacteria

2020 ◽  
Vol 1 (2) ◽  
pp. 79-86
Author(s):  
Wendry Putranto ◽  
Apon Mustopa ◽  
Jendri Mamangkey ◽  
Netty Aritonang
Keyword(s):  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Observational Research ◽  
Ammonium Sulfate Precipitation ◽  
Fermentation Time ◽  
Milk Clotting ◽  
Milk Clotting Enzyme ◽  
Qualitative Observation ◽  
Milk Clotting Activity

To get the potential of lalcat acid bacteria isolate to produce Milk Clotting Enzyme (MCE), it is necessary to screen milk clotting activity both quantitatively and qualitatively. Through qualitative observation, the characteristics of the curd resulting from enzyme activity can be obtained. MCE is a protease that has the characteristics of milking. Based on the results of this observational research, the curd characteristic produced can be used as a benchmark to determine the length of time of fermentation and optimization of the determination of ammonium sulfate precipitation concentration. Isolate BAL shows the results of a compact curd at a fermentation time of 25 hours at 37 ℃ and the optimization results of the deposition of ammonium sulfate which shows the characteristics of a compact curd by 45% ammonium sulfate.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

scholarly journals A set of simple methods for detection and extraction of laminarinase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ananthamurthy Koteshwara ◽  
Nancy V. Philip ◽  
Jesil Mathew Aranjani ◽  
Raghu Chandrashekhar Hariharapura ◽  
Subrahmanyam Volety Mallikarjuna
Keyword(s):  
Ammonium Sulfate ◽  
Ammonium Sulphate ◽  
Gold Standard ◽  
Direct Detection ◽  
Low Cost ◽  
Direct Analysis ◽  
Reducing Sugar ◽  
Ammonium Sulfate Precipitation ◽  
Simple Method ◽  
Functional Step

AbstractA carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

Preparation of Human Immunoglobulin by Ammonium Sulfate Precipitation

Vox Sanguinis ◽  
1976 ◽  
Vol 31 (6) ◽  
pp. 423-434
Author(s):  
A.F.S.A. Habeeb ◽  
Robert D. Francis
Keyword(s):  
Ammonium Sulfate ◽  
Human Immunoglobulin ◽  
Ammonium Sulfate Precipitation
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