Amount of enzyme required

Various authors have considered the amount of enzyme required for an end point assay (15-17). While it is advisable not to be too wasteful, most common coupling enzymes are not too expensive and a few preliminary experiments will soon determine the minimum amount needed to give reUable data in a reasonable time. Putting the problem quantitatively, our ideal amount of enzyme should lead to the reaction being virtually complete (from 100% to 1% of substrate remaining) in a reasonable time (say, lO min). Since the enzyme will slow down as the substrate is used up, we caimot just calculate the amount of enzyme needed from its given activity (V) divided into the substrate available instead we need to consider the integrated rate equation for the process. Remember that V is a measure of the amount of enzyme added (V = kcat o) stid so when we choose the amount of enzyme to add, we choose a V value. 

Integration gives us the total time for given change in s 

For a decrease in s from 100% to 1%, in a time of 10 min (i.e. values we considered reasonable above), the following expression must hold 

This equation tells us that, for a reaction to go to completion in 10 min, the amount of enz3mie added should be an amount able to change the concentration of substrate in the assay by 0.46Km per minute. If Kn, = 1 him, for example, for 99% completion in 10 min 

The concentration of enzyme is conventionally given in U/ml, where 1 U is the amount of enz3mae that vnll convert 1 pmol substrate/min at saturation. If we add 1 U of enzyme to an assay volume of 3 ml, fiar example, then V = 1/3 (tmol/ml/min = 0.33 mmol Utre min . Thus, if IC = 1 mi, for V s 0.46 mmol litre min , we need to add s 0.46/0.33 Units of enzyme if the assay voltune is 3 ml. 

---

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

A great savings in enzyme consumption can be achieved by immobilizing the enzyme in the reactor (Fig. 12). In addition to the smaller amount of enzyme required, immobilization often increases the stability of the enzyme. Several designs of immobiliz-ed-enzyme reactors (lERs) have been reported, with open-tubular and packed-bed being the most popular. Open-tubular reactors offer low dispersion but have a relatively small surface area for enzyme attachment. Packed-bed reactors provide extremely high surface areas and improved mass transport at the cost of more dispersion. 

Most enzyme treatments of wool are carried out at about 50 °C for 30-60 minutes. The amount of enzyme required depends on the specific enzyme type and its commercial... 

Viscosimetric determination of activity is used only with pectic enzymes displaying the random-action pattern. The activity is mostly expressed as the time required for attaining a 50% decrease of viscosity (in s) or as the amount of enzyme required for attaining a certain decrease of viscosity per unit time.223... 

Assays for endo-l,4- -glucanase [EC 3.2.1.4] (i.e., CMCase) and saccharifying cellulase (i.e., international filter paper U, IFPU) activities partially followed the methods recommended in the 1987 lUPAC report (65). When even undiluted enzyme samples fail to give the required glucose yield under prescribed assay conditions, the lUPAC committee recommends a less precise method. In the current study, cellulase activities in digester extracts were so low that the CMCU could only be defined as follows one CMC unit of activity was that amount of enzyme required to liberate one Hg glucose from CMC in 60 min. 

Activity Assays. The standard activity assay mixture of 3 ml contained about 0.1 U/ml lignin peroxidase, 0.4 mM veratryl alcohol (Fluka, purum >97%) and 0.1M sodium tartrate, pH 3.0. The reaction was started by adding 15 fil of 54 mM H2O2 to make a final concentration of 0.28 mM in the reaction. The production of veratraldehyde was followed by recording the change of absorbance for 12 seconds at 310 nm in a cuvette which was thermostated to 37°C. The reaction was started 24 seconds before the recording. One unit of lignin peroxidase is defined as the amount of enzyme required to oxidize one imol of veratryl alcohol to veratraldehyde in one minute. 

Determine the activity units, where a unit is defined as that amount of enzyme required to produce an absorbance change of 1.0 per min in a 1-cm cuvette. 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

Compound Relative amount of enzymes required for hydrolysis... 

Enzyme concentration may be expressed in mass unit instead of molar unit. However, the amount of enzyme is not well quantified in mass unit because actual contents of an enzyme can differ widely depending on its purity. Therefore, it is common to express enzyme concentration as an arbitrarily defined unit based on its catalytic ability. For example, one unit of an enzyme, cellobiose, can be defined as the amount of enzyme required to hydrolyze cellobiose to produce 1 /imol of glucose per minute. Whatever unit is adopted for CEq, the unit for k3CEQ should be the same as r, that is, kmole/m3s. Care should be taken for the consistency of unit when enzyme concentration is not expressed in molar unit. 

Protein phosphatase 2A (PP2A) (Upstate Biotechnology, USA) the activity of the stock solution is 1900 U mL 1, 1 unit being defined as the amount of enzyme required to hydrolyse 1 nmol of p-nitrophe-nyl phosphate (p-NPP) in 1 min at room temperature. 

The emulsified water-insoluble substrate is usually incubated with a buffered aqueous enzyme preparation. Lipase activity can be determined by continuous measurement of the reaction products or by incubating for a set time and determining the total amount of product formed, or substrate used. One unit (U) of lipase activity is usually defined as the amount of enzyme required to liberate a certain quantity (e.g., 1 pmol) of product in a certain time (e.g., 1 min) under given conditions. 

One unit of enzyme activity is defined as the amount of enzyme required to form 1 u.mol of ADP-glucose/min at 37°C (assay in the direction of synthesis). 

The variation of the enzyme loading is a means of determining the minimum amount of enzyme required for maximum sensitivity. Furthermore, this test reveals the magnitude of the enzyme reserve of diffusion controlled sensors. 

Assay of Enzyme Activity. Activity of this enzyme was determined by the increase in ninhydrin color after hydrolysis of Carbobenzoxy-L-glutamyl-L-tyrosine (Z-Glu-Tyr) as substrate at 30, pH 3.1. One katal of acid carboxypeptidase activity was defined as the amount of enzyme required to liberate 1 mol of C-terminal amino acid per second. 

Enzyme loading test. The minimum amount of enzyme required for maximum sensitivity is determined by varying the enzyme loading (figure 17.2). This test also, reveals the enzyme limit of diffusion-controlled sensors. Owing to differences in /Ka/ values and the layer thickness, the transition from the kinetic to diffusion control of different enzyme electrodes takes place at rather... 

Methods involving in vitro polyketide and nonribosonal peptide production involve a similar set of considerations. High turnover numbers are essential to increase product yields and minimize the amount of enzyme required. It is important that proteins used in these experiments be readily expressible in practical quantities and exhibit broad substrate tolerance. The latter is imperative to minimize laborious purification of numerous proteins for library construction. 

Proteolytic activity was assayed as described by Kembhavi et al. [9], with modification. The reaction mixture was made up of 0.4 mL of casein (Sigma) 0.5% (w/v) in distilled water and 0.4 mL 0.2 M acetate buffer, pH 5.0, to whieh 0.2 mL of the crude enzyme solution was added. The reaction was carried out at 60°C and stopped after 30 min with 1 mL of 10% trichloroacetic acid (TCA). Test tubes were centrifuged at 5,000 rpm/5 min, and the absorbance of the supernatant was measured at 280 nm. An appropriate control was prepared in which the TCA was added before the enzymatic solution. One unit of enzyme activity (U) was arbitrarily defined as the amount of enzyme required to cause an increase of 0.01 in absorbance at 280 nm under the assay conditions. 

Glycosidases can be coaxed to synthesize saccharides. Similar attempts have been made with proteases for the peptide synthesis. The equilibrium of a glycosidase-catalyzed reaction normally lies on the side of the thermodynamically more stable cleavage products. The large amount of enzyme required and the low reaction rate are drawbacks of this process. The saccharide competes with the water molecule as the acceptor for the glycosyl moiety. Different manipulations are possible to shift the equilibrium in favor of... 

Enzyme activity is expressed as units/g fresh weight, where one unit is defined as the amount of enzyme required to produce 1 nmol of /3-aspartylphosphate/min under the conditions of the individual experiments. 

One unit of DNA topoisomerase VI is defined as the amount of enzyme required to decatenate 0.2 p,g of k-DNA into open circular or covalently closed minicircles under standard reaction conditions. 

The nicking reactions are usually carried out under standard conditions described by the commercial suppliers. The amount of enzyme required to produce nicked DNA ranges from 3 to 17 units/yg of dsDNA with reaction times typically between 1.5 and 3 hr. It is important to ensure that all RF IV DNA has been destroyed, i.e., converted to nicked DNA (RF II), so that all the heteroduplex molecules can be substrates for the gapping reaction. Should any heteroduplex DNA be transfected a lowering of the mutational efficiency will result. 

---