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Determination of fungal α-amylase by flow injection analysis
Analytica Chimica Acta, 168 (1984)
376-377
Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
DETERMINATION ANALYSIS
OF FUNGAL
a-AMYLASE
BY FLOW INJECTION
PREBEN W. HANSEN
Enzyme Microbiological Labomtory, (Denmark)
Novo Industri A/S, Novo All& 2880 Bagsvaerd
(Received 21st November 1983)
Summary. A manual iodine-starch method is adapted to analyse broths from Aspergillus oryzae fermentations. The method is based on degradation of starch by the enzyme, and measurement of the residual starch/iodine complex at 670 nm. Calibration graphs are linear for the range 0.01-0.1 amylase units ml-‘. Eighty samples per hour can be processed.
Flow injection analysis (f.i.a.) [l] has many advantages such as high sampling rates, fast response, flexibility and simple apparatus. Several manual analytical methods are laborious as well as time-consuming and could with advantage be adapted to f.i.a. In this laboratory, a manual method for determination of acid cw-amylase activity in culture broth from Aspergillus oryzae is used daily. It is based on the degradation of starch at 37°C and pH 4.7 by the enzyme. Use is made of the colour formed when iodine is added to the starch solution. Initially it is black-blue but with progressive hydrolysis of the starch it changes to red-brown. The enzyme activity is based on the time necessary to reach a certain colour evaluated by a coloured glass standard (details are available on request). As this method suffers from the disadvantages mentioned above, it was decided to adapt it for f.i.a. Experimental Flow injection system. The basic units are the Bifok FIA 05 unit with a 30-~1 injection valve, Bifok FIA 08 peristaltic pump, Bifok FIA 06 photometer with an l&p1 flow cell and a 570-nm filter, and an Omniscribe pen recorder (Houston Instruments). The manifold modules are of Plexiglas and the tubing and coils of teflon (0.5 mm i.d.). The f.i.a. manifold is shown in Fig. 1. The coils and flow cell are thermostatted at 37°C. Reagents. The starch solution, pH 4.7, was prepared by dissolving 0.270 g of dry starch (Merck) by gently boiling for 1 min in ca. 200 ml of acetate buffer (6.0 g of sodium acetate and 2 ml of anhydrous acetic acid per litre). After slow cooling to room temperature, acetate buffer was added to give a final volume of 400 ml. A solution of 40.0 g of potassium iodide and 4.0 g of iodine in deionized water was prepared and diluted to 1 1; it was kept 0003-2670/84/$03.00
o 1984 Elsevier Science Publishers B.V.
316
Flow
rate -1
ml mtn Iodine
0.92
Starch
0.92
InJector Pump
37w
nm FAU
ml-'
Fig. 1. Flow injection system. Coil lengths are given in cm; samples are introduced at the injector. Fig. 2. Calibration graphs for cy-amylase with 0.067% starch solution and 5 ml of iodine reagent diluted to: (a) 250 ml;(b) 180 ml;(c) 150 ml. R is the peak height.
in brown bottles in the dark. Before use, 5 ml of this solution was diluted to 180 ml with deionized water. Fungamyl 800 L (Novo) with an activity of 800 FAU g-l (fungal aamylase unit; 1 FAU is the amount of enzyme which breaks down 5.26 g of starch h-l using the above-mentioned manual standard method) was dissolved in deionized water to give a stock solution containing 0.2 FAU ml-‘. This was diluted to obtain standards in the range 0.01-0.10 FAU ml-‘. Procedure. As a decrease in absorbance is measured, the photometer was calibrated in the following way. With the photometer calibration knob, the factor was adjusted to ca. 95%. While all reagents were pumped through the flow injection system, the photometer display was adjusted to 50-55 with the zero knob. This represents the highest reading that can be obtained when there is no degradation of starch, and therefore represents the baseline. Any drift in the baseline was corrected for with the zero knob only. In the flow system, flow rates were as indicated in Fig. 1, and samples were allowed to react with starch in a 200~cm coil, before the iodine was added. The absorbance was measured at 570 nm. Results and discussion The reaction temperature and pH used were those normal to the manual method, 37°C and 4.7, respectively. From initial test-tube experiments, the appropriate composition of reagents was estimated, as well as the mixing proportions. It was further found that with a reaction time of about 20 s the reaction had proceeded sufficiently for a reasonable sensitivity to be achieved. Several reaction coil lengths were evaluated as were the reagent flow rates. The recommended conditions are summarized in Fig. 1. The effect of reagent compositions (starch/iodine) on the determination of a-amylase were also investigated. With 0.09 or 0.054% starch solution, a linear calibration graph could not be obtained even with a wide variation in
377 TABLE 1 Comparison of results for the determination of amylase in diluted culture broth Sample no.
or-Amylase found (FAU ml-l) F.i.a. method
R.s.d? (%)
Manual method
1 2 3 4 5 6 7 8 9 10 11 12
0.0161 0.0697 0.0635 0.0673 0.0668 0.0731 0.0170 0.0671 0.0592 0.0671 0.0680 0.0762
4.3 0.0 1.1 0.6 1.5 1.0 4.1 0.9 0.7 1.0 0.6 0.0
0.0158 0.0692 0.0679 0.0675 0.0647 0.0710 0.0160 0.0669 0.0556 0.0678 0.0678 0.0717
Difference (%)
t1.9 +0.7 t8.8 -0.3 t1.7 t 2.9 +5.9 to.3 +6.1 -1.0 to.3 + 5.9
% = 3.
the concentration of the iodine solution. With 0.067% starch solution, the highest sensitivity and a linear calibration graph (up to 0.1 FAU ml-‘) were obtained when 5 ml of the iodine solution was diluted to 180 ml (Fig. 2). The method was compared with the manual method for determination of a-amylase activity in culture broth from fermentations with Aspergillus oryzae. The 5-point calibration graph was prepared from standards which contained 0.010-0.10 FAU ml-‘. Samples were diluted with deionized water to contain amylase activity within this range. All samples and standards were injected in triplicate. The results for 12 diluted culture broth samples are compared in Table 1. There is generally good agreement between the two methods, The f.i.a. method is well suited for the rapid, accurate and precise measurement of acid a-amylase activity in culture broths. Compared with the manual method, the f.i.a. method with a sampling rate of 80 h-l allows five times more samples to be examined in a working day. This is slightly less than the recirculating procedure of Nikolelis and Mottola [ 21, but the procedure is much simpler and cheaper. REFERENCES 1 J. RliziEka and E. H. Hansen, Flow Injection Analysis, Wiley-Interscience, NY, 1981. 2 D. P. Nikolelis and H. A. Mottola, Anal. Chem., 50 (1978) 1665.
New York,
376-377
Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Short Communication
DETERMINATION ANALYSIS
OF FUNGAL
a-AMYLASE
BY FLOW INJECTION
PREBEN W. HANSEN
Enzyme Microbiological Labomtory, (Denmark)
Novo Industri A/S, Novo All& 2880 Bagsvaerd
(Received 21st November 1983)
Summary. A manual iodine-starch method is adapted to analyse broths from Aspergillus oryzae fermentations. The method is based on degradation of starch by the enzyme, and measurement of the residual starch/iodine complex at 670 nm. Calibration graphs are linear for the range 0.01-0.1 amylase units ml-‘. Eighty samples per hour can be processed.
Flow injection analysis (f.i.a.) [l] has many advantages such as high sampling rates, fast response, flexibility and simple apparatus. Several manual analytical methods are laborious as well as time-consuming and could with advantage be adapted to f.i.a. In this laboratory, a manual method for determination of acid cw-amylase activity in culture broth from Aspergillus oryzae is used daily. It is based on the degradation of starch at 37°C and pH 4.7 by the enzyme. Use is made of the colour formed when iodine is added to the starch solution. Initially it is black-blue but with progressive hydrolysis of the starch it changes to red-brown. The enzyme activity is based on the time necessary to reach a certain colour evaluated by a coloured glass standard (details are available on request). As this method suffers from the disadvantages mentioned above, it was decided to adapt it for f.i.a. Experimental Flow injection system. The basic units are the Bifok FIA 05 unit with a 30-~1 injection valve, Bifok FIA 08 peristaltic pump, Bifok FIA 06 photometer with an l&p1 flow cell and a 570-nm filter, and an Omniscribe pen recorder (Houston Instruments). The manifold modules are of Plexiglas and the tubing and coils of teflon (0.5 mm i.d.). The f.i.a. manifold is shown in Fig. 1. The coils and flow cell are thermostatted at 37°C. Reagents. The starch solution, pH 4.7, was prepared by dissolving 0.270 g of dry starch (Merck) by gently boiling for 1 min in ca. 200 ml of acetate buffer (6.0 g of sodium acetate and 2 ml of anhydrous acetic acid per litre). After slow cooling to room temperature, acetate buffer was added to give a final volume of 400 ml. A solution of 40.0 g of potassium iodide and 4.0 g of iodine in deionized water was prepared and diluted to 1 1; it was kept 0003-2670/84/$03.00
o 1984 Elsevier Science Publishers B.V.
316
Flow
rate -1
ml mtn Iodine
0.92
Starch
0.92
InJector Pump
37w
nm FAU
ml-'
Fig. 1. Flow injection system. Coil lengths are given in cm; samples are introduced at the injector. Fig. 2. Calibration graphs for cy-amylase with 0.067% starch solution and 5 ml of iodine reagent diluted to: (a) 250 ml;(b) 180 ml;(c) 150 ml. R is the peak height.
in brown bottles in the dark. Before use, 5 ml of this solution was diluted to 180 ml with deionized water. Fungamyl 800 L (Novo) with an activity of 800 FAU g-l (fungal aamylase unit; 1 FAU is the amount of enzyme which breaks down 5.26 g of starch h-l using the above-mentioned manual standard method) was dissolved in deionized water to give a stock solution containing 0.2 FAU ml-‘. This was diluted to obtain standards in the range 0.01-0.10 FAU ml-‘. Procedure. As a decrease in absorbance is measured, the photometer was calibrated in the following way. With the photometer calibration knob, the factor was adjusted to ca. 95%. While all reagents were pumped through the flow injection system, the photometer display was adjusted to 50-55 with the zero knob. This represents the highest reading that can be obtained when there is no degradation of starch, and therefore represents the baseline. Any drift in the baseline was corrected for with the zero knob only. In the flow system, flow rates were as indicated in Fig. 1, and samples were allowed to react with starch in a 200~cm coil, before the iodine was added. The absorbance was measured at 570 nm. Results and discussion The reaction temperature and pH used were those normal to the manual method, 37°C and 4.7, respectively. From initial test-tube experiments, the appropriate composition of reagents was estimated, as well as the mixing proportions. It was further found that with a reaction time of about 20 s the reaction had proceeded sufficiently for a reasonable sensitivity to be achieved. Several reaction coil lengths were evaluated as were the reagent flow rates. The recommended conditions are summarized in Fig. 1. The effect of reagent compositions (starch/iodine) on the determination of a-amylase were also investigated. With 0.09 or 0.054% starch solution, a linear calibration graph could not be obtained even with a wide variation in
377 TABLE 1 Comparison of results for the determination of amylase in diluted culture broth Sample no.
or-Amylase found (FAU ml-l) F.i.a. method
R.s.d? (%)
Manual method
1 2 3 4 5 6 7 8 9 10 11 12
0.0161 0.0697 0.0635 0.0673 0.0668 0.0731 0.0170 0.0671 0.0592 0.0671 0.0680 0.0762
4.3 0.0 1.1 0.6 1.5 1.0 4.1 0.9 0.7 1.0 0.6 0.0
0.0158 0.0692 0.0679 0.0675 0.0647 0.0710 0.0160 0.0669 0.0556 0.0678 0.0678 0.0717
Difference (%)
t1.9 +0.7 t8.8 -0.3 t1.7 t 2.9 +5.9 to.3 +6.1 -1.0 to.3 + 5.9
% = 3.
the concentration of the iodine solution. With 0.067% starch solution, the highest sensitivity and a linear calibration graph (up to 0.1 FAU ml-‘) were obtained when 5 ml of the iodine solution was diluted to 180 ml (Fig. 2). The method was compared with the manual method for determination of a-amylase activity in culture broth from fermentations with Aspergillus oryzae. The 5-point calibration graph was prepared from standards which contained 0.010-0.10 FAU ml-‘. Samples were diluted with deionized water to contain amylase activity within this range. All samples and standards were injected in triplicate. The results for 12 diluted culture broth samples are compared in Table 1. There is generally good agreement between the two methods, The f.i.a. method is well suited for the rapid, accurate and precise measurement of acid a-amylase activity in culture broths. Compared with the manual method, the f.i.a. method with a sampling rate of 80 h-l allows five times more samples to be examined in a working day. This is slightly less than the recirculating procedure of Nikolelis and Mottola [ 21, but the procedure is much simpler and cheaper. REFERENCES 1 J. RliziEka and E. H. Hansen, Flow Injection Analysis, Wiley-Interscience, NY, 1981. 2 D. P. Nikolelis and H. A. Mottola, Anal. Chem., 50 (1978) 1665.
New York,