Big Chemical Encyclopedia

Chemical substances, components, reactions, process design ...

Articles Figures Tables About

Centrifugation enzymes

Homogenization with extraction buffer (phosphate buffer with sodium ascorbate and 2-mercaptoethanol) boiled for 12min centrifugation enzyme digestion centrifugation SPE (SAX cartridge)... [Pg.624]

Fig. 5. Effect of HCOj -free medium on inactivation and phosphorylation of acetyl-CoA carboxylase. Enzyme preparation was placed in the main compartment of a Warburg flask, capped with a serum stopper, containing a small piece of Whatman filter paper (2x3 cm) plus 0.1 ml of Hyamine 10-X in the central well. Vessels were evacuated and refilled with COj-free Nj several times. This procedure was repeated several times during a 3-hour period at room temperature to completely remove dissolved HCOs firom the medium. Enzjmie preparation was equilibrated at 37°C for 10 minutes prior to the addition of [y- PlATP (specific activity, 28 iiCi/iimole at 0.7 miW final concentration) which was also treated in a similar way to remove COi. AT indicated times, P incorporation was terminated by addition of a cold solution containing ATP and EDTA at 5 times the concentrations of [y- PlATP and MgCl2, respectively. The labeled en me was purified by DE AE-cellulose chromatography as described before, and immunoprecipitation of the enzyme was carried out by incubation with rabbit antibody to the carboxylase for 15 minutes and with the membrane preparation fromS. aureus (18) for another 15 minutes. Enzyme-antibody precipitates were collected and immediately washed by centrifugation. Enzyme activity-control enzyme -ATP, O +ATP, . COj-free enzyme —ATP, A -H ATP, . Radioactivily-control enzyme, C02-free enzyme, . From Lent et al. (71). Fig. 5. Effect of HCOj -free medium on inactivation and phosphorylation of acetyl-CoA carboxylase. Enzyme preparation was placed in the main compartment of a Warburg flask, capped with a serum stopper, containing a small piece of Whatman filter paper (2x3 cm) plus 0.1 ml of Hyamine 10-X in the central well. Vessels were evacuated and refilled with COj-free Nj several times. This procedure was repeated several times during a 3-hour period at room temperature to completely remove dissolved HCOs firom the medium. Enzjmie preparation was equilibrated at 37°C for 10 minutes prior to the addition of [y- PlATP (specific activity, 28 iiCi/iimole at 0.7 miW final concentration) which was also treated in a similar way to remove COi. AT indicated times, P incorporation was terminated by addition of a cold solution containing ATP and EDTA at 5 times the concentrations of [y- PlATP and MgCl2, respectively. The labeled en me was purified by DE AE-cellulose chromatography as described before, and immunoprecipitation of the enzyme was carried out by incubation with rabbit antibody to the carboxylase for 15 minutes and with the membrane preparation fromS. aureus (18) for another 15 minutes. Enzyme-antibody precipitates were collected and immediately washed by centrifugation. Enzyme activity-control enzyme -ATP, O +ATP, . COj-free enzyme —ATP, A -H ATP, . Radioactivily-control enzyme, C02-free enzyme, . From Lent et al. (71).
Urease is one of the enzymes which have been obtained in the crystalline state. This has been done by stirring jack bean meal with 30°o aqueous acetone, filtering and allowing the filtrate to remain at o for several hours. The urease which crystallises out is separated by centrifuging and is then recrystallised. Like crystalline pepsin and trypsin, it is a protein. [Pg.519]

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). [Pg.290]

The economics of an immobilised cell process depend on the lifetime of the microorganism and a continued level of clean product delivered by the fixed cells. It is important to eliminate the free cells from the downstream product without the use of any units such as centrifuge or filtration processes. Since the cells are retained in the ICR, the activity of intracellular enzymes may play a major role. It is assumed that the deactivation of the enzyme at constant temperature follows a first-order equation as shown below 17... [Pg.218]

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. [Pg.277]

At present, several of the instruments which are being utilized for enzyme analysis, such as the centrifugal analyzers (15), have been measuring samples of the order of 5 pi. [Pg.105]

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. [Pg.190]

White blood cells, red blood cells and cultured fibroblasts are commonly used to measure enzyme activities, especially for the diagnosis of inherited enzyme abnormalities. Leukocytes may be collected by sedimentation in viscous media such as Fycol. The collection of red cells presents no problem following centrifugation of anticoagulated blood. The assay of enzymes and fibroblasts requires appropriate tissue culture facilities and extensive experience in dealing with cultured human cells. [Pg.192]

Azospmlhtm strains were grown on LB medium containing 1% (w/v) pectin at 30°C for seven days, and Escherichia coli transformants for two days. Culture media were centrifuged at 10.000 g for 10 minutes. The supernatant constituted the enzyme preparation. [Pg.380]

In liquid medium, the thiobarbuturic acid test was used to determine polygalacturonase and pectate lyase activity (Sherwood, 1965). 1 ml of the crude enzyme preparation was added to 2 ml of 0.5 N HCl in a test tube. 4 ml of 0.01 M thiobarbuturic acid, dissolved in distilled water, were added. The tubes were heated in a boiling water for Ih and centrifuged. The absorption of the supernatant was determined in the spectrophotometer over the range 480-580 nm. Reaction mixtures without enzyme, which showed no reaction with thiobarbuturic acid, were used as controls. [Pg.380]

Apples (Red Belle de Boskoop, Jonagold or Mutzu) were cut and milled (1.5 mm) and 5% (w/w) of a 2% ascorbic acid solution was added immediately. Enzyme preparations (25 mg enzyme protein / kg mash) were added and the mash was incubated for 2 hours at 20°C whereafter it was pressed. The resulting apple juice was pasteurised at 85°C to discontinue further enzyme degradation. The cloud was measured as turbidity in EF/F units [15]. The cloud stability was determined by a centrifugation test as the amount of turbidity remaining after centrifugation at 4,200 x g for 15 minutes [15]. [Pg.465]

Soy cell wall material (Soy CWM) was isolated by Alcalase treatment and jet cooking (115°C, 4 minutes) of soy meal followed by centrifugation and recovery of insolubles. Aliqots of 1% suspensions of soy CWM in O.IM acetate buffer pH 5.0 were incubated with enzymes (40pg of each to 1.5 ml of substrate) at 30°C for 24 hours and the solubilized material was analyzed by HPSEC and HPAEC. [Pg.466]

Defatted soy flour was suspended in water to 8% (v/w) protein, pH was adjusted to 6 by HCl before 640 mg/L Novozyme 415 (a-galactosidase), 10 mg/L Phytase L (Novo Nordisk) and 53 mg enzyme protein/L rhamnogalacturonase B were added. After 4 hours at 50 C the pH was adjusted to 8 by NaOH, and the slurry was centrifuged. The supernatant was pasteurized (85°C, 5 minutes) and freezedried. Protein was measured as 6.25 x Kjeldahl N. Phytate was measured as described in [16], and dietary fibres were analysed as described in [17]. [Pg.466]

Carrots (Boleo) were peeled by 2% NaOH at 88-96°C for 4 minutes, minced by a meat mincer (2 mm) and homogenised for 2 minutes by an ULTRA-TURRAX T25 homogenizer (from Jahne Kunkel). The carrot mash was preheated to 45°C (20 minutes) before the enzyme preparations, 25 mg enzyme protein/kg mash, were added. The enzymes were dissolved in water to give a dilution of 5% (v/v) of the carrot mash. The mash was incubated at 45°C under stirring (60 rpm) for 2 hours, before the enzymes were inactivated at 86°C for 5 minutes in a microwave oven. Finally the purees were homogenised for 1 minute by ULTRA TURRAX. The viscosity of the puree was measured by a BROOKFIELD viscosimeter Model DV-n + with spindle A from HELIPATH SPINDLE SET at 2.5 rpm thermosta-ted at 50°C. The stability of the puree was measured as the sediment (in %) after centrifugation in 10 ml tube a 1660 x g for 10 minutes. [Pg.466]

Enzyme Turbidity before centrifugation Increase in turbidity relative to untreated control % Cloud stability %... [Pg.467]


See other pages where Centrifugation enzymes is mentioned: [Pg.73]    [Pg.166]    [Pg.53]    [Pg.73]    [Pg.166]    [Pg.53]    [Pg.2816]    [Pg.178]    [Pg.232]    [Pg.295]    [Pg.394]    [Pg.1874]    [Pg.2058]    [Pg.504]    [Pg.532]    [Pg.538]    [Pg.540]    [Pg.556]    [Pg.572]    [Pg.350]    [Pg.184]    [Pg.1160]    [Pg.1160]    [Pg.181]    [Pg.335]    [Pg.409]    [Pg.292]    [Pg.195]    [Pg.178]    [Pg.178]    [Pg.179]    [Pg.182]    [Pg.209]    [Pg.119]    [Pg.457]    [Pg.458]    [Pg.473]   


SEARCH



© 2024 chempedia.info