Assays amylase activity

When assaying amylase activity in bleach-containing detergents, sodium sulfite should be added to the reaction medium to reduce the perborate and avoid interference with the color development. 

Groves and Teng (1992) investigated the effect of compactional pressure on biologically active proteinaceous enzymes such as a-amylase, P-glucuronidase, lipase, and urease. Assaying the activity of these enzymes before and after the compaction... 

Enzymes were immobilized onto silica gel by covalent crosslinking with glutaraldehyde in a procedure similar to that of Kondo et al. (3). Briefly, 1-4 g of silica gel was incubated in 1-13 wt% glutaraldehyde and in 100-1000 mL of enzyme solution for up to 48 h. The immobilized enzyme was recovered by filtration and washed to remove loosely bound enzyme. Samples of the soluble enzyme were collected before and after immobilization and assayed for activity, to provide an estimate of enzyme uptake onto the support. The a-amylases studied were Spezyme Fred (Genencor), Allyzme (Alltech), and Liquozyme (Novozymes). The cellulases studied were Spezyme CP and Spezyme CE (Genencor). 

It is difficult to compare directly the immobilized amylase activities (i.e., Vmax and Km) observed in these studies with values obtained by other researchers, owing to the fact that we used industrial corn flour as the substrate, whereas others used an activity assay based on soluble starch (5-9). Moreover, the substrate concentrations used in our studies (100-300 g/L) are high enough to facilitate gelatinization, and the reaction takes place with the substrate present as a slurry. By contrast, the starch assays are typically based on a substrate concentration of 10-20 g/L (5-7,9), low enough to completely dissolve the substrate. 

Application and Principle This procedure is used to determine the a-amylase activity of enzyme preparations derived from Aspergillus niger var. Aspergillus oryzae var. Rhizopus oryzae var. and barley malt. The assay is based on the time required to obtain a standard degree of hydrolysis of a starch solution at 30° 0.1°. The degree of hydrolysis is determined by comparing the iodine color of the hydrolysate with that of a standard. 

Assay Preparation For Pancreatin having about the same amylase activity as the USP Pancreatin Reference Standard, weigh accurately about 40 mg of Pancreatin into a suitable mortar. 

Note For Pancreatin having a different amylase activity, weigh accurately the amount necessary to obtain an Assay Preparation having amylase activity per mL corresponding approximately to that of the Standard Preparation. 

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

Of the schemes which have been proposed for the assay of the unique amylase activity of B. macerans, the simplest and most direct is the Tilden and Hudson microscopic test. ... 

Amylase activity was measured using 1% (w/v) soluble starch dissolved in acetate buffer (0.2 M, pH 4.8) as the substrate. Reaction mixtures were incubated for 10 min at 40 °C and the reducing sugars released were measured using the DNS assay with maltose as the standard sugar. 

Rinderknecht et al. (14) designed a liquid assay for measuring a-amylase activity using amylose that had been covalently linked to a dye, Remazol Brilliant Blue R (RBB). In principle, this dye can be coupled to every soluble polysaccharide. RBB was coupled to... 

Amylase synthesis in soil was measmed by adding 1 g of cornstarch to 100 g of moist soil and incubating at 30° C. Samples were removed periodically and assayed for amylase activity (7). 

The increase in absorbance at 405 nm is proportional to the amylase activity. It might be pointed out that many different assay procedures and units of amylase activity are used in the literature. 

A study of the kinetics of the Beckman Enzymatic Amylase Method, for determination of human serum and urinary amylase, by use of the centrifugal analyser showed that the rate of reaction is not linear. It showed a pronounced initial lag period of 10 min, after which the lag was diminished but never completely abolished. Furthermore, the function relating rate of reaction to enzyme activity was not linear but increased disproportionately with increase in enzyme activity. When glycogen was substituted for soluble starch in the Beckman reagent, the initial lag period was less than 3 minutes and subsequently the reaction became linear. When mixed soluble starch-glycogen substrate was used, the a-amylase activity of serum and urine specimens correlated well with the activity determined by the Phadebas method. A dialysable factor present in a urine specimen from a kidney transplant patient inhibited the Beckman assay but not the Phadebas assay. This inhibition was simulated by the addition of pyruvate and reversed by the addition of NADH. 

Since the macerans amylase was produced in the cell culture following primary growth, the culture was incubated for 5 days at 37 C. At the end of this time the cells were removed by centrifugation and the clear filtrate was collected. The filtrate was assayed for macerans amylase activity and units of enzyme were calculated by the Tilden-Hudson method ( ). Cultures grown under the above conditions yielded supernatant solutions which contained from 1 to 2 units of enzyme activity per ml which were generally suitable for use in most types of experiments. If solutions containing higher enzyme activities are required, the initial supernatant can be concentrated by lyophilization. 

They argued that if the extracted a-amylase contained (and was consequently heavier) it must have been synthesized from the amino acids. This would be proof of de novo synthesis of the enzyme which could be separated from light ( O) enzyme on the basis of its density. In order to obtain a convenient label for the light enzyme, Filner and Varner first incubated aleurone layers in GA3, plus H-lysine. The H-labelled a-amylase-produced was collected and placed on an equilibrium density gradient (cesium chloride). An initial experiment showed a coincidence between the radioactive ( H) peak and that of assayable a-amylase activity (Fig. 7.2A). The fact that a-amylase could incorporate H-lysine was, of course, good evidence for its de novo synthesis, but the final proof came when it was shown that assayed a-amylase synthesized in GA3-treated aleurone layers incubated in H2 0 was heavier than the radioactive marker ( H) peak (Fig. 7.2B). 

Fig. 7.2. (A) Equilibrium distribution of radioactivity ( ) and a-amylase activity (o) after centrifugation of a mixture of purified a-amylase-and crude a-amylase induced in H2 0 (i.e. a-amylase- O). Density of the CsCl gradient increases to the left. Radioactivity measures a-amylase- 0- H, and enzyme assay a-amylase-The enzyme activity contributed by the radioactive a-amylase-is negligible with respect to that contributed by the crude a-amylase-(B) Equilibrium distribution of radioactivity ( ) and a-amylase activity (o) of a mixture of purified a-amylase-and crude a-amylase induced in H2 0 (i.e. a-amylase- 0). Density of the CsCl gradient increases to the left. Radioactivity measures a-amylase-and enzyme assay a-amylase-After Filner and Varner, 1967 [36]...

The Falling Number test (cf. 15.4.1.1.1) and an amylographic assay are the most important tests to assess the baking properties of rye flour. These tests depend to a great extent on gelatinization properties of starches and the presence of a-amyl-ase. The higher the a-amylase activity, the lower the Falling Number. 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

The diastase activity was traditionally determined according to the Schade method in the earlier years (Schade et al., 1958). One unit of diastase activity (or more specifically, a-amylase), DN, is defined as that amoimt of enz)nne that converts 0.01 g of starch to the prescribed endpoint in 1 h at 37 °C under the experimental conditions. In this assay, a standard solution of starch, which reacts with iodine to produce a color solution, is used as a substrate for honey enzymes under the standard conditions (Rendleman, 2003). A recently developed procedure uses an insoluble, dyed starch substrate (Persano Oddo and Pulcini, 1999). As this substrate is hydrolyzed by ot-amylase, soluble dyed starch fragments are released into solution. After reaction termination and insoluble substrate removal by centrifugation, absorbance of the supernatant solution (at 620 nm) is measured. The absorbance is proportional to the diastase activity. This procedure has been widely adopted in the honey industry due to the convenience of a commercially available substrate and the simple assay format. 

Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and >galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and -galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and -galactosidase, respectively, were preincubatcd at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

CGTase Activity Assay. Aaivity was measured by the Pharmacia Phadebas Amylase Assay at pH 6.0, 60 C in O.IM sodium acetate (100 ppm Ca+ " ) for 15 minutes using B, stearothermophilus alpha-amylase as a standard. Alpha-amylase preparations were assayed under the same conditions. One Phadebas unit is defined as the amount of enzyme that will catalyze the hydrolysis of 1.0 micromole of glucosidic linkages of Lintner starch per minute at 6OOC, pH 6.0. 

Table-TV shows the effect ot fhe LMW fraction on the activity of some of these enzymes in vitro. Maltase, lactase and invertase were competitively inhibited at a concentration of 10 mg/ ml. When the effectsof a range of concentrations (2.5-20 mg/ml) of the LMW fraction were studied, it was revealed that the inhibition was not of the pure competitive type. Table V shows the effect of the HMW fraction. Low concentrations had to be used in the assays, as the intense brown color of this fraction interfered with the spectrophotometric measurements. In spite of this a strong competitive inhibition of lactase and of invertase was found. Maltase was also inhibited, and, to a lesser extent, even trehalase. a-Amylase from saliva was not affected at the concentration tested.

Roles of Metabolites of Abscisic Acid. Nothing is known about the physiological role of PA and DPA in plants, although these two metabolites of ABA have been tested in several bioassays recently. In the cotton explant abscission assay PA had one-tenth of the activity of ABA (19). PA and DPA were equally effective in inhibiting a-amylase secretion by barley aleurone layers treated with glbberellin A3 DPA had approximately one-tenth of the activity of ABA in this system (74). The effect of PA on growth of bean embryos was negligible (75). 

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