Amylase activity, determination

Ceska, M. Birath, R. and Brown, B. A new and rapid method for the clinical determination of amylase activities in human serum and urine. Optimum conditions. 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

Figure 5.4 Determination of the optimum temperature of amylase activity.

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. 

Application and Principle This procedure is used to determine maltogenic amylase activity in preparations derived from Bacillus subtilis containing a Bacillus stearothermophilus amylase gene. The test is based on a 30-min hydrolysis of malto-triose under controlled conditions and measurement of the glucose formed by high-performance liquid chromatography (HPLC). 

The amylolytic activity of A. oryzae a-amylase is determined by comparison of the amount of glycosidic linkages broken in the starch to the amount released by a reference amylase under the same experimental conditions. It is expressed in terms of micromoles of glycosidic linkages broken per minute. 

C2. Close, J. R., and Street, H. V., An ultramicro method for the determination of amylase activity in blood serum. Clin. Chim. Acta 3, 476-479 (1958). 

Dougherty, T.M. Determination of a-amylase activity in flue-cured tobacco using a chromogeiric substrate Tob. 

However, the main goal was to study the behavior and performance of a novel bioartificial material (obtained with an innovative method involving prefreezing and inversion steps) as a suitable matrix for the entrapment of proteins or enzymes in a stable manner. The tests performed (determination of enzyme activity, determination of and V ax kinetic parameters, repeatability test) confirmed both that the enzyme immobilised in the bioartificial polymeric matrix maintained its catalytic activity unchanged and that the catalytic reaction rate was comparable with that of the free a-amylase reaction (used as control). 

The a-amylase activities of semm, liver, pancreas, submaxillary and parotid glands, saliva, duodenum, colon, lung, heart, spleen, kidney, and skeletal muscle of the domestic cat Felix catus) have been determined.As in most mammals, the highest a-amylase content was found in the pancreas. Levels in saliva and salivary glands were very low. a-Amylase activities of the serum, liver, and pancreas has a pH optimum of 7.0-7.5, and were reversibly inactivated by removal of chloride ion. 

The determination of a-amylase activity from cotton leaves with amylopectin azure has been described. 

A semi-automated nephylometric determination of a-amylase activity in sprouted wheat kernels is dependent upon the decrease in turbidity of an amylopectin suspension when acted upon by a-amylase.The rate of nephlos change is used as a measure of a-amylase activity. 

The acid-stable a-amylase of Aspergillus niger and the acid-labile a-amylase of A. oryzae have been studied.It was shown that in addition to the more stable acid-resistant properties, the a-amylase of A. niger possesses increased thermal stability in comparison with the a-amylase of A. oryzae. The molecular weight of the acid-stable a-amylase is 5.8 x 10 The amino-acid composition, as well as the C- and A -terminal amino-acids of both forms of a-amylases were determined. It was shown that the enzymes each contain one thiol group, which, being bound to Ca ", plays an important role in maintaining the catalytically active conformation of the enzyme. 

Amylases are responsible for hydrolysis of starch to oligosaccharides. a-Amylase hydrolyzes the 1,4-a-glucoside bonds in compounds involving three or more molecules of glucose. ff-Amylase liberates (mainly) S-maltose from starch and other compounds. The simplest way to determine amylase activity requires the determination of the time required to change the starch iodine color from blue... 

Two different kinetic methods are available for the determination of amylase activity used in clinical diagnostics. These methods can be applied for the analysis of industrial products. 

Free-hanging vines of Cuscuta reflexa were obtained and segments from the desired regions were treated with hormones or inhibitors, and incubated as described earlier [ 19,21 ]. Procedures for the determination of PAL or a-amylase activity and lignin content are given in Tables 1, 6 and 5. Lignification was microscopically visualized as described in Fig. 2. 

The kinetics of the hydrolysis of starch by a mixture of 3- and a-amylases in a membrane reactor have been examined. Cross-linked amylose has been used to determine the p- and oe-amylase activities of an amylolytic preparation (see Scheme 3). ... 

The textural (stickiness and consistency on cooking) and physiochemical properties of rice could not be explained on the basis of the total amylose content but these properties correlated well with the insoluble amylose content. The protein content had no effect on these properties. A cross-linked amylose has been used as a substrate to determine the a- and )3-amylase activities of amylo-lytic preparations. ... 

A method reported for the quantitative determination of a- and j3-amylase activities present together in amylolytic preparations has been based on the use of non-modified amylase and cross-linked amylose substrates. The former is cleaved by both enzymes whereas the latter is cleaved by a-amylase and not by /3-amylase (see Scheme 3). 

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. 

A method reported for differential determination of jS- and -amylase activities in mixtures of the two enzymes relies on the non-susceptibility of cross-linked amylase to 8-amylase (see p. 427). ... 

Factors affecting the Phadebas amylase test as performed on samples of plasma, urine, and duodenal juice have been discussed and the importance of performing suitable blank determinations emphasized. A new method for the quantitative determination of a- and i3-amylase activities in amylolytic preparations involves the use of unmodified amylose as substrate for the determination of total (a + /3) activity, and the use of amylose cross-linked with epichlorohydrin for the determination of a-amylolytic activity alone. In each case the maltose produced is measured using 2,4-dinitrosalicylate rea nt. 

Cell concentration OD550 reading of sample broth diluted three times was converted to dry cell weight per ml as described previously [2]. (11) a-Amylase activity a-Amylase activity was determined with the entire sample broth as saccharolytlc power by the method of Bernfeld [5]. One unit was defined as the enzyme quantity that releases 1 mg of reducing sugar (as maltose) at 60°C for 3 min. 

Analytical procedures Protein was determined with the Biorad-method. Glucose was determined by the hexokinase-method (Boehringer), amylase activity by the method of Manning and Campbell (1961) at 60 C. One unit of amylase is defined as the amount of protein which will hydrolyse 10 mg of starch per minute. 

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