Ethanol treatment for sterilization, concentration, and stabilization of a biodegradable plastic–degrading enzyme from Pseudozyma antarctica culture supernatant

Biodegradable plastics must be sufficiently stable to maintain functionality during use but need to be able to degrade rapidly after use. We previously reported that treatment with an enzyme named PaE, secreted by the basidiomycete yeast Pseudozyma antarctica can speed up this degradation. To facilitate the production of large quantities of PaE, here, we aimed to elucidate the optimal conditions of ethanol treatment for sterilization of the culture supernatant and for concentration and stabilization of PaE. The results showed that Pseudozyma antarctica completely lost its proliferating ability when incubated in ≥20% (v/v) ethanol. When the ethanol concentration was raised to 90% (v/v), PaE formed a precipitate; however, its activity was restored completely when the precipitate was dissolved in water. To reduce ethanol use, PaE was successfully concentrated and recovered by sequential ammonium sulfate precipitation and ethanol precipitation steps. Over 90% of the activity in the original culture supernatant was recovered and the specific activity was increased 3.4-fold. By preparing the enzyme solution at a final concentration of 20% (v/v) ethanol, about 60% of the initial activity was maintained at ambient temperature for over 6 months without growth of microbes. We conclude that ethanol treatment is effective for sterilization, concentration, and stabilization of PaE, and that concentrating PaE by sequential ammonium sulfate precipitation and ethanol precipitation substantially increases the PaE purity and decreases ethanol use.

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Radioimmunoassay for yeast killer toxin from Saccharomyces cerevisiae

Canadian Journal of Microbiology ◽

10.1139/m81-132 ◽

1981 ◽

Vol 27 (8) ◽

pp. 847-849

Author(s):

Farooq A. Siddiqui ◽

Howard Bussey

Keyword(s):

Saccharomyces Cerevisiae ◽

Ammonium Sulfate ◽

Column Chromatography ◽

Specific Activity ◽

Killer Toxin ◽

Ammonium Sulfate Precipitation ◽

Antibody Preparation ◽

Precipitation Band ◽

Yeast Killer ◽

Yeast Killer Toxin

Aradioimmunoassay was developed for the K1 killer toxin from strain T158C/S14a of Saccharomyces cerevisiae. 125I-labeled toxin was made to a specific activity of 100 μCi/mg of protein (1 μCi = 37 kBq). Antibody to purified toxin was prepared in rabbits using toxin cross-linked to itself. These antibodies, partially purified by 50% ammonium sulfate precipitation and Sepharose CL-6B column chromatography, produced one precipitation band with killer toxin and bound 125I-labeled toxin in a radioimmunoassay. The antibody preparation also bound with the toxins from another K1 killer, A364A, and three chromosomal superkiller mutants derived from it.

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THE ISOLATION AND PURIFICATION OF A TRYPSIN INHIBITOR FROM WHOLEWHEAT FLOUR

Canadian Journal of Biochemistry ◽

10.1139/o64-194 ◽

1964 ◽

Vol 42 (12) ◽

pp. 1825-1832 ◽

Cited By ~ 25

Author(s):

G. Shyamala ◽

R. L. Lyman

Keyword(s):

Ammonium Sulfate ◽

Trypsin Inhibitor ◽

Carboxymethyl Cellulose ◽

Moving Boundary ◽

Specific Activity ◽

Trypsin Inhibitors ◽

Ammonium Sulfate Precipitation ◽

Commercial Sample ◽

Isolation And Purification ◽

Single Protein

An impure concentrate of a trypsin inhibitor was prepared from a commercial sample of wholewheat flour by ammonium sulfate precipitation. The impure inhibitor was heat-labile and did not inhibit pepsin or α-chymotrypsin activity. When the crude inhibitor was chromatographed on carboxymethyl cellulose, a single protein peak that was about 20 times more active than the original impure inhibitor preparation was obtained.Uultracentrifugal sedimentation patterns and moving-boundary electrophoresis indicated that the material isolated on the carboxymethyl cellulose was nearly homogeneous. In comparison with other known trypsin inhibitors that have been isolated in nearly pure form, wholewheat trypsin inhibitor appears to have a low specific activity.

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PURIFICATION AND SOME PROPERTIES OF ALPHA-AMYLASE OF PEAR FRUIT

HortScience ◽

10.21273/hortsci.26.6.723e ◽

1991 ◽

Vol 26 (6) ◽

pp. 723E-723

Author(s):

Shiao J. Li ◽

Tim Facteau ◽

Paul M. Chen

Keyword(s):

Ammonium Sulfate ◽

Time Course ◽

Specific Activity ◽

Alpha Amylase ◽

Acetate Buffer ◽

Pear Fruit ◽

Ammonium Sulfate Precipitation ◽

Freeze Dried ◽

Optimum Ph ◽

The Difference

Alpha-amylase was purified from freeze-dried pear fruit by extraction at pH 7.40 with Tris, Acetate and Imidazole buffer followed by differential ammonium sulfate precipitation and desalting column. The specific activity of the enzyme was increased 5.68 fold during purification. The optimum pH was 5.64 in Acetate buffer. The difference in the time course of alpha-amylase was observed between freeze-dried and fresh samples.

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Detection and Partial Purification of a Natural Heparin Inhibitor from Hog Small Intestine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646809 ◽

1979 ◽

Vol 41 (03) ◽

pp. 567-575 ◽

Cited By ~ 3

Author(s):

Menard M Gertler ◽

Mingjien Chien ◽

Robert H Yue

Keyword(s):

Small Intestine ◽

Ammonium Sulfate ◽

Zinc Sulfate ◽

Specific Activity ◽

Partial Purification ◽

Neutralizing Activity ◽

Ethanol Precipitation ◽

Ammonium Sulfate Fractionation ◽

Sulfate Fractionation

SummaryA natural occurring heparin inhibitor was detected and was partially purified from the mucosa of hog small intestine. The mucosa was homogenized and was extracted overnight in 0.15 M NaCl, 0.01 M imidazole, 0.001 M EDTA, pH 6.5. When the extract was made to 85% saturation in ammonium sulfate, a large quantity of heparin neutralizing activity was detected in the precipitate. Each small intestine contains approximately 35,000 units of heparin neutralizing activity. This heparin inhibitor was further purified by the procedures of zinc sulfate precipitation, ammonium sulfate fractionation, ethanol precipitation and heparin-sepharose chromatography. A 37 fold partial purification with 15% overall recovery was achieved to yield heparin inhibitor with specific activity of 50-65 units per mg of protein.

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New Procedure for Purifying and Crystallizing Alkaline Phosphatase from Human Intestinal Mucosa

Clinical Chemistry ◽

10.1093/clinchem/18.6.548 ◽

1972 ◽

Vol 18 (6) ◽

pp. 548-553 ◽

Cited By ~ 4

Author(s):

Sheshadri Narayanan ◽

Harold D Appleton

Keyword(s):

Alkaline Phosphatase ◽

Enzyme Activity ◽

Ammonium Sulfate ◽

Intestinal Mucosa ◽

Specific Activity ◽

Active Enzyme ◽

Purification Procedure ◽

Ammonium Sulfate Precipitation ◽

Extraction And Purification ◽

Butanol Extraction

Abstract A new procedure is described for extracting, purifying, and crystallizing alkaline phosphatase orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from human intestinal mucosa. Active enzyme, extracted with trichlorotrifluoroethane, is subsequently purified by chromatography on Sephadex G-200 and DEAE Sephadex. The acetone and ammonium sulfate precipitation steps that are part of the conventional butanol extraction and purification procedure are eliminated. Enzyme activity is concentrated at each stage of the purification procedure, yielding a preparation with a specific activity three times greater than that obtained with the conventional butanol procedure.

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

Scientific Research Journal ◽

10.24191/srj.v10i2.9403 ◽

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.

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A set of simple methods for detection and extraction of laminarinase

Scientific Reports ◽

10.1038/s41598-021-81807-2 ◽

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.

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