scholarly journals Study on Isolation and Partial Purification of Lactase (β-galactosidase) Enzyme from Lactobacillus Bacteria Isolated from Yogurt

2011 ◽  
Vol 4 (1) ◽  
pp. 239 ◽  
Author(s):  
N. H. M. R. Mozumder ◽  
M. Akhtaruzzaman ◽  
M. A. Bakr ◽  
F. Tuj Zohra
Keyword(s):  
Specific Activity ◽  
Culture Media ◽  
Deae Cellulose ◽  
Chemical Properties ◽  
Total Activity ◽  
Partial Purification ◽  
Anion Exchange Column ◽  
Ammonium Sulphate Precipitation ◽  
Agar Media ◽  
Modified Media

Lactase has many applications in dairy industry including for the treatment of lactose intolerance. The present study was conducted to identify the activity of lactase enzyme produced by Lactobacillus bacteria isolated from yogurts available in Dhaka city. The strains were identified to be gram positive, catalase negative, fermentative and lactase producer when cultured on selective MRS agar media by using standard bacteriological procedures and techniques. The study revealed that enzymes produced by lactobacilli were capable to produce glucose from substrate lactose in lactose modified media using lactase assay Kit Glu IB and their highest protein concentration (17.25 mg/ml) was observed in the supernatant of culture media isolated from L. lactis. Highest total activity (850.69 U/l) and specific activity (50.04 U/mg) of lactase enzyme was observed in the strain of L. bulgaricus. The crude extract which showed highest activity was further purified by ammonium sulphate precipitation followed by anion exchange column chromatography (DEAE cellulose). Final specific activity and fold purification of lactase enzyme reached to 62.80 U/mg and 1.47 respectively. The highest physic-chemical properties (Effect of pH and temperature) of lactase enzyme were observed at PH 6.0 which was 43.98 U/mg of protein and at 70°c temperature which was 111.11 U/mg of protein.Keywords: β-Galactosidase; Specific activity; DEAE cellulose; Fold purification; Yogurt.© 2012 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.doi: http://dx.doi.org/10.3329/jsr.v4i1.8478J. Sci. Res. 4 (1), 239-249 (2012)

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

Performance of Different Solid and Liquid Culture Media for the Improvement of Tuberculin Production

2020 ◽  
Keyword(s):  
Culture Media ◽  
Synthetic Medium ◽  
Chemical Properties ◽  
Economic Loss ◽  
Delayed Type Hypersensitivity ◽  
Basic Medium ◽  
Primary Methods ◽  
Lowenstein Jensen ◽  
Time Required ◽  
Modified Media

Tuberculosis (TB) is one of the most important zoonotic bacterial diseases. A huge economic loss which could be direct or indirect are associated with the disease. Currently, the primary methods used for detection of TB in humans and cattle include the measurement of a delayed type hypersensitivity to purified protein derivative (PPD). So, the need for preparation of purified PPD with adequate properties and increasing the final PPD yield with decreasing the time of tuberculin production has stimulated the interest in the development of its preparation. Our study was performed to compare between the standard and modified media for improving tuberculin production. Middle brook 7H10 agar medium was used as a modified basic medium for mycobacterial growth, followed by cultivation of mycobacteria on Middle brook 7H9 broth medium. For the production, strains were inoculated onto the culture medium (Dorest Henly synthetic medium). Other steps for tuberculin production was done according to standard Weighbridge protocol. The results demonstrated that the using of both Middle brook 7H10 agar and Middle brook 7H9 broth instead of Lowenstein-Jensen (LJ) and glycerin broth media which used in currently produced tuberculin, have better physical and chemical properties. In addition, reducing the time required for production by accelerating the time of microbial growth. Also, it was found that the tuberculin produced using modified media was slightly more potent or the same as currently tuberculin produced. So, both Middle brook 7H10 agar and Middle brook 7H9 broth media are recommended for production of tuberculin saving time and increasing potency of the product but more investigation was recommended for estimation types of protein present in both locally prepared and modified tuberculin.

Partial Purification and Some Properties of a Hydroxycinnamoyl Glucosyltransferase from Tomato Fruits

1980 ◽  
Vol 35 (11-12) ◽  
pp. 967-972 ◽  
Author(s):  
A. Fleuriet ◽  
J. J. Macheix ◽  
R. Suen ◽  
R. K. Ibrahim
Keyword(s):  
Ferulic Acid ◽  
Metal Ions ◽  
Deae Cellulose ◽  
Divalent Metal ◽  
Partial Purification ◽  
Divalent Metal Ions ◽  
Tomato Fruits ◽  
Ammonium Sulphate Precipitation ◽  
Sh Group ◽  
Cherry Tomatoes

A glucosyltransferase was isolated from immature “cherry” tomatoes and was partially purified (200-fold) by ammonium sulphate precipitation and successive chromatography on Sephadex G-100 and DEAE-cellulose columns. The enzyme utilised the free hydroxycinnamic acids and UDP-glucose in the formation of their respective glucosides (pH 8.0) and glucose esters (pH 7.0); but did not accept the CoA thiolesters of HCAs in the presence of glucose-1-phosphate. The constant glucoside/glucose ester ratio observed during purification suggests that both reactions are catalysed by the same enzyme. The Km values for ρ-coumaric, caffeic, ferulic and sinapic acids were 0.8, 1.5, 1.4 and 2.5 μᴍ, respectively. With ferulic acid as substrate, the Km value for UDPG was 10 μᴍ. The enzyme required an -SH group for activity and the reaction was strongly inhibited by EDTA, divalent metal ions and UDP.

scholarly journals Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

1980 ◽  
Vol 191 (1) ◽  
pp. 117-124 ◽  
Author(s):  
R Zecher ◽  
H U Wolf
Keyword(s):  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Half Maximum ◽  
Purification And Characterization ◽  
Dimeric Molecule ◽  
Maximum Inhibition ◽  
Optimum Ph ◽  
Triton X

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

scholarly journals Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7545
Author(s):  
Jianyou Zhang ◽  
Guangcheng Zhou ◽  
Lifeng Fei ◽  
Lifan Chen ◽  
Lei Sun ◽  
...  
Keyword(s):  
Polyphenol Oxidase ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Enzymatic Reactions ◽  
Inhibitory Effects ◽  
Purification And Characterization ◽  
Effective Prevention ◽  
Ammonium Sulphate Precipitation

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

scholarly journals PEMURNIAN PARSIAL DAN KRISTALISASI PAPAIN DARI GETAH Carica papaya

2019 ◽  
Vol 4 (2) ◽  
pp. 152
Author(s):  
Dwi Putri Mashfufatur Rohmah ◽  
Sofijan Hadi ◽  
Afaf Baktir
Keyword(s):  
Ammonium Sulphate ◽  
Carica Papaya ◽  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Purification Factor ◽  
Papaya Latex ◽  
Carica Papaya Latex

AbstractThis research has done partial purification by fractionation and optimization crystallization of papain from Carica papaya latex with the addition of ammonium sulphate. The enhancement of purification factor was determined by its specific activity. The fractionation results show that papain fraction of Carica papaya can be obtained by adding 40-80% saturated ammonium sulphate, with the highest specific activity, i.e. 2,0819 U/µg and the factor purification increase of 6,27 fold than papain extract. Meanwhile, the highest total activity, i.e. 10,7780 U of papain crystals can be obtained by presipitant agent addition of ammonium sulphate in the level / concentration 80% saturated at 15 °C. Microscopycally papain crystals profile in this condition have cube and tetragonal shape.Key words: crystallization, fractionation, ammonium sulphate, papain

Partial purification and characterization of a soluble acid phosphatase from the tapeworm, Hymenolepis diminuta

1991 ◽  
Vol 65 (2) ◽  
pp. 103-110
Author(s):  
Michael J. Bumbulis ◽  
Peter W. Pappas
Keyword(s):  
Acid Phosphatase ◽  
Cellular Localization ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Hymenolepis Diminuta ◽  
Partial Purification ◽  
Transferase Activity ◽  
Crude Enzyme Preparation ◽  
Ammonium Sulphate Precipitation ◽  
And Function

ABSTRACTAn acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130 000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44°C and pH 5·0. The enzyme appeared to be a monomer with a molecular weight of approximately 62 000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

Purification of a Highly Potent Heparin-Like Anticoagulant from Healthy Human Plasma

1979 ◽  
Author(s):  
Robert H. Yue ◽  
Toby Starr ◽  
Menard M. Gertler
Keyword(s):  
Human Plasma ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Chemical Properties ◽  
Healthy Human ◽  
Naturally Occurring ◽  
Salt Gradient ◽  
Physical And Chemical

A highly potent-heparin-like anticoagulant (accelerator) has been purified from citrated healthy human plasma. After heat defibrination and BaSO4. treatment on the plasma, the accelerator was adsorbed onto a DEAE-cellulose column and elution was achieved using a high ionic strength linear buffered salt gradient. The eluted accelerator was further purified by polyacrylamide gel electrophoresis. The purified accelerator can accelerate the inhibition of thrombin by antithrombin III and has a specific activity of 226 heparin units/mg of carbohydrate. The accelerator contains a small amount of protein-like material. The amount of accelerator present in healthy human blood is extremely small and can only be first detected in the concentrate after the DEAE-cellu-lose chromatography. A mere 0.5 mg of the purified accelerator is obtained from 100 ml of human plasma. Concomitant with this investigation, a second heparin-like substance also has been purified but has very low anticoagulant activity in terms of heparin units. The naturally occurring accelerator may function as heparin in the circulating blood and its level in blood may have a clinical significance in thrombotic vascular disease. Further work on its physical and chemical properties is now in progress.

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