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Purification of α-amylase by industrial autofocusing
J~WJUII qf Chmwurogruph~, Elsevier Science Publishers CHROM.
41 I (19x7) 486489 B.V.. Amsterdam ~
Printed
in The Netherlands
20 031
Note
Purification
of x-amylase
by industrial
autofocusing
T. DOBRANSKY
t. POLiVKA Slovak
and I. HAAS
Srarch Factories.
Trncrvu iC:erhoslo~~akia~
and 0. SOVA* and E. PETRVALSKL institute of Animal (Czechoslo vakia/ (Received
September
Physiology,
Slowk
Acadmq
of Sciences,
Dukel.sk,+ch Hrdinor
Ii B, 04001 Ko.Fice
7th, 1987)
For the production of chemically pure enzymes, economic processes are required which maximize the specific activity. This paper deals with the possibility of using industrial autofocusing’* as a technological purification technique to obtain chemically pure enzymes. The principles of this method have been reported elsewhere3. A pure enzyme r-amylase (1,4-II-glucan glucanhydrolase, E.C. 3.2.1 .l), which is produced by different strains of Bucillus subtili.F6, has been purified by industrial autofocusing. It is currently produced by the Western Slovakia Starch Factory in Trnava (Czechoslovakia) in crude technical purity. MATERIALS
AND
METHODS
Bacillus subtilis, strain A 32 was used to produce the enzyme. Bacterial cells were grown at 37°C and pH 6.9 in a fermentor for 16 h. The cultivation medium contained 0.5 g of starch, 1.8 g of corn steep. 2.5 g of ammonium hydrogenphosphate and 0.2 g of calcium chloride per litre of distilled water. After cultivation the cells were harvested by centrifugation at 10 000 g and the supernatant was condensed five times in a vacuum evaporator (Alpha Laval) and its conductivity adjusted to 560 PIS cm ~_’ by demineralization. A 10-I volume of the crude preparation was autofocused in an Autofocuser (Realizing Centre, Slovak Academy of Sciences, Kosice, Czechoslovakia) for 72 h at 4°C and at an electric field strength varying from 2.50 to 1000 V until the current decreased to zero. The autofocused medium was then divided into 20 equal fractions in which the pH, xc-amylase activity and protein concentration were determined. The fractions containing a-amylase activity were pooled and loaded onto a 80 cm x 5 cm I.D. Spheron P-40 column equilibrated with 0.01 M McIlvain buffer pH 6.07. The column was operated at 4°C at a flow-rate of 120 ml/h and 30-ml fractions were collected by an automatic fraction collector. All fractions were tested for their 0021..9673/87/$03.50
!c
1987 Elsevier
Science Publishers
B.V
487
NOTES
protein content and alpha-amylase activity and the active fractions were pooled for the final study of yield. Two methods were used to determine the cl-amylase activity. The first was a manometric determination of reducing saccharides arising from starch by means of the enzymatic reaction according to Kjeldahl-Bertrand (see ref. 8). The second, modified Sumners, method was also based on the determination of the increased reducing ability of starch arising from the enzyme cleavage. The amount of reducing compounds was determined after reaction with 3.5dinitrosalicylic acid by measuring the so-formed yellow-brown colour at 525 nm9 by spectrophotometry. The proteins in the individual fractions were determined by biuret reactionxO. RESULTS
Fig. 1 shows the results of the first step in the purification of a crude preparation of cx-amylase, involving autofocusing. The bulk proteins focused to pH 4.0, while the fractions containing amylase activity occurred between pH 5.82 and 6.18. Fig. 2 shows a purification of the focused preparation of a-amylase by gel chromatography. As many as five ballast fractions of proteins found by polyacrylamide gel electrophoresis (PAGE) were removed using gel chromatography on Spheron P-40, and the resulting had a protein activity of 3.5 Ujmg; 1 U catalyzes the liberation of 1 pmol of reducing groups from soluble starch (Zulkowsky) per minute II
“k?
PH
too0
1500
I2 -
800
70-
1000 bO0
b-
k
100 500
‘h
200
N
Fig. 1. Isolation of r-amylase tration in mg per fraction; e
by industrial autofocusing. iV = Fraction number; = pH gradient; A = activity of r-amylase in U.
0
= protein
concen-
NOTES
J 700
ffio
500
400
3(10
700
TOO
Fig. 2. Purification of n-amylase l = protein concentration; 0
after autofocusing by Spheron = activity of wamylase in U.
P 40 gel filtration.
_V = fraction
number;
at 40°C pH 6.0, calculated as maltose. The reducing groups are determined with 3,5-dinitrosalicylic acid. Table I shows a comparison of the balance of enzymatic activities and the amounts of protein before and after autofocusing. As can be seen, the specific activity increased 16 times; at the same time there was a slight decrease in total activity (5%) and a considerable defocusing of ballast proteins. The total specific activity of the purified enzyme increased 23 times during two steps. whereas the total activity decreased by 8%. After such a purification, only one protein fraction was found in the a-amylase obtained by electrophoresis. A part of the pure enzyme was lyophilized; a second part was divided into three parts and each of them was stabilized in a solution consisting of 0.1% calcium hydroxide, 0.1% sodium acetate or 0.1% calcium acetate at 4°C.
TABLE
I
PURIFICATION
OF r-AMYLASE
BY INDUSTRIAL
AUTOFOCUSING PuriJication
Step
Centrifugation Ultrafiltration Autofocusing Spheron pool
14 885 14 190 874 595
2250 2225 2128.4 2082.5
0.15 0.16 2.43 3.50
1
1.07 16.20 23.30
Recovery f%)
100 99 95 92
489
NOTES TABLE
II
DECREASE
IN a-AMYLASE
ACTIVITY
USING
VARIOUS
STABILIZING
MEDIA
Stabilization
Activity in 5 ml of sample
After 30 days
After 60 days
After 90 days
After 120 days
None 0.1% Calcium hydroxide 0.1% Sodium acetate 0. I % Calcium acetate
5.0 5.0 5.0 5.0
3.3 5.0 4.7 4.9
1.6 4.8 4.5 4.4
1.2 4.2 3.0 4.1
0.5 4.0 3.4 3.9
(66%) (100%) (94%) (98%)
(32%) (94%) (90%) (88%)
(24%) (84%) (72%) (82%)
Table II shows the decrease in stability using different stabilizing media. lyophilized sample the activity remained constant for as long as 4 months.
(10%) (80%) (68%) (78%)
In the
DISCUSSION
A natural pH gradient in autofocusing is usually created automatically by the purified supernatant. Irrespective of the undulations or steps in the pH gradient, the proteins reach an appropriate pZ along the pH gradient. The peaks are sufficiently separated and the void volume is far enough from the peak containing most of the r-amylase activity. In this step of the isolation, a large amounts of starting material (up to 1000 1 with a protein concentration of about 3 g per 100 ml) can be employed. It must be emphasized that this method should not be used in cases where the highest activity of the enzyme corresponds with the main peak of the proteins. Regarding the decrease in activity of the unstabilized preparation, we believe that this is due to proteolysis caused by proteases produced by Bacillus suhrilis together with the purified enzyme. For example, x-amylase from Aspergillus ovizae contains two known proteases: Aspergillus aspartic proteinase (E.C. 3.4.23.6) and Aspergillus acid carboxy peptidase (E.C. 3.4.16.1) which result in proteolysis. The method presented for isolation of pure a-amylase seems to be very effective and economically advantageous for large-scale industrial production. REFERENCES 1 2 3 4 5 6 7 8
0. Sova, J. Chromatogr., 320 (1985) 15. 0. Sova and K. Boda, Czech. Pat., 234 801 (1985). 0. SOW, J. Chromatogr., 320 (1985) 213. P. Chavalier and .I. de la Notte, Enzyme Microb. Tech?zol., 9 (19871 Jan. K. Toda and K. Sato, J. Ferment. Technoi., 19 (1984) 79. P. Adlercreutz, 0. Hoist and B. Mattiason, EnzJ’me Microb. Technol., 4 (1982) 395. V. Sjrkora, Chemicko-analytickP tabulky, SNTL, Prague, 1976, p. 153. J. Davidek, J. HrdliEka. M. KarvBnek. J. Pokornj, et al.. Laboratorni prirucku anuljq potravin, SNTL/ALFA, Prague, 1978, p. 652. 9 J. KBs, B. KrBlov9 and P. Rauch, Laboratorni cviceni z biochemie a radiochemie, SNTL, Prague, 1983, p. 175. 10 J. K&, B. KrBlovri and K. Moltas, Laboratorni cviceniz hiochemie a radiochemie, SNTL. Prague, 1986, p. 150. 11 S. Hanzdwa, A. Odogawa, H. Sakata, M. Takouchi and E. Ichishima, Curr. Microb., 14 (1985) 235.
41 I (19x7) 486489 B.V.. Amsterdam ~
Printed
in The Netherlands
20 031
Note
Purification
of x-amylase
by industrial
autofocusing
T. DOBRANSKY
t. POLiVKA Slovak
and I. HAAS
Srarch Factories.
Trncrvu iC:erhoslo~~akia~
and 0. SOVA* and E. PETRVALSKL institute of Animal (Czechoslo vakia/ (Received
September
Physiology,
Slowk
Acadmq
of Sciences,
Dukel.sk,+ch Hrdinor
Ii B, 04001 Ko.Fice
7th, 1987)
For the production of chemically pure enzymes, economic processes are required which maximize the specific activity. This paper deals with the possibility of using industrial autofocusing’* as a technological purification technique to obtain chemically pure enzymes. The principles of this method have been reported elsewhere3. A pure enzyme r-amylase (1,4-II-glucan glucanhydrolase, E.C. 3.2.1 .l), which is produced by different strains of Bucillus subtili.F6, has been purified by industrial autofocusing. It is currently produced by the Western Slovakia Starch Factory in Trnava (Czechoslovakia) in crude technical purity. MATERIALS
AND
METHODS
Bacillus subtilis, strain A 32 was used to produce the enzyme. Bacterial cells were grown at 37°C and pH 6.9 in a fermentor for 16 h. The cultivation medium contained 0.5 g of starch, 1.8 g of corn steep. 2.5 g of ammonium hydrogenphosphate and 0.2 g of calcium chloride per litre of distilled water. After cultivation the cells were harvested by centrifugation at 10 000 g and the supernatant was condensed five times in a vacuum evaporator (Alpha Laval) and its conductivity adjusted to 560 PIS cm ~_’ by demineralization. A 10-I volume of the crude preparation was autofocused in an Autofocuser (Realizing Centre, Slovak Academy of Sciences, Kosice, Czechoslovakia) for 72 h at 4°C and at an electric field strength varying from 2.50 to 1000 V until the current decreased to zero. The autofocused medium was then divided into 20 equal fractions in which the pH, xc-amylase activity and protein concentration were determined. The fractions containing a-amylase activity were pooled and loaded onto a 80 cm x 5 cm I.D. Spheron P-40 column equilibrated with 0.01 M McIlvain buffer pH 6.07. The column was operated at 4°C at a flow-rate of 120 ml/h and 30-ml fractions were collected by an automatic fraction collector. All fractions were tested for their 0021..9673/87/$03.50
!c
1987 Elsevier
Science Publishers
B.V
487
NOTES
protein content and alpha-amylase activity and the active fractions were pooled for the final study of yield. Two methods were used to determine the cl-amylase activity. The first was a manometric determination of reducing saccharides arising from starch by means of the enzymatic reaction according to Kjeldahl-Bertrand (see ref. 8). The second, modified Sumners, method was also based on the determination of the increased reducing ability of starch arising from the enzyme cleavage. The amount of reducing compounds was determined after reaction with 3.5dinitrosalicylic acid by measuring the so-formed yellow-brown colour at 525 nm9 by spectrophotometry. The proteins in the individual fractions were determined by biuret reactionxO. RESULTS
Fig. 1 shows the results of the first step in the purification of a crude preparation of cx-amylase, involving autofocusing. The bulk proteins focused to pH 4.0, while the fractions containing amylase activity occurred between pH 5.82 and 6.18. Fig. 2 shows a purification of the focused preparation of a-amylase by gel chromatography. As many as five ballast fractions of proteins found by polyacrylamide gel electrophoresis (PAGE) were removed using gel chromatography on Spheron P-40, and the resulting had a protein activity of 3.5 Ujmg; 1 U catalyzes the liberation of 1 pmol of reducing groups from soluble starch (Zulkowsky) per minute II
“k?
PH
too0
1500
I2 -
800
70-
1000 bO0
b-
k
100 500
‘h
200
N
Fig. 1. Isolation of r-amylase tration in mg per fraction; e
by industrial autofocusing. iV = Fraction number; = pH gradient; A = activity of r-amylase in U.
0
= protein
concen-
NOTES
J 700
ffio
500
400
3(10
700
TOO
Fig. 2. Purification of n-amylase l = protein concentration; 0
after autofocusing by Spheron = activity of wamylase in U.
P 40 gel filtration.
_V = fraction
number;
at 40°C pH 6.0, calculated as maltose. The reducing groups are determined with 3,5-dinitrosalicylic acid. Table I shows a comparison of the balance of enzymatic activities and the amounts of protein before and after autofocusing. As can be seen, the specific activity increased 16 times; at the same time there was a slight decrease in total activity (5%) and a considerable defocusing of ballast proteins. The total specific activity of the purified enzyme increased 23 times during two steps. whereas the total activity decreased by 8%. After such a purification, only one protein fraction was found in the a-amylase obtained by electrophoresis. A part of the pure enzyme was lyophilized; a second part was divided into three parts and each of them was stabilized in a solution consisting of 0.1% calcium hydroxide, 0.1% sodium acetate or 0.1% calcium acetate at 4°C.
TABLE
I
PURIFICATION
OF r-AMYLASE
BY INDUSTRIAL
AUTOFOCUSING PuriJication
Step
Centrifugation Ultrafiltration Autofocusing Spheron pool
14 885 14 190 874 595
2250 2225 2128.4 2082.5
0.15 0.16 2.43 3.50
1
1.07 16.20 23.30
Recovery f%)
100 99 95 92
489
NOTES TABLE
II
DECREASE
IN a-AMYLASE
ACTIVITY
USING
VARIOUS
STABILIZING
MEDIA
Stabilization
Activity in 5 ml of sample
After 30 days
After 60 days
After 90 days
After 120 days
None 0.1% Calcium hydroxide 0.1% Sodium acetate 0. I % Calcium acetate
5.0 5.0 5.0 5.0
3.3 5.0 4.7 4.9
1.6 4.8 4.5 4.4
1.2 4.2 3.0 4.1
0.5 4.0 3.4 3.9
(66%) (100%) (94%) (98%)
(32%) (94%) (90%) (88%)
(24%) (84%) (72%) (82%)
Table II shows the decrease in stability using different stabilizing media. lyophilized sample the activity remained constant for as long as 4 months.
(10%) (80%) (68%) (78%)
In the
DISCUSSION
A natural pH gradient in autofocusing is usually created automatically by the purified supernatant. Irrespective of the undulations or steps in the pH gradient, the proteins reach an appropriate pZ along the pH gradient. The peaks are sufficiently separated and the void volume is far enough from the peak containing most of the r-amylase activity. In this step of the isolation, a large amounts of starting material (up to 1000 1 with a protein concentration of about 3 g per 100 ml) can be employed. It must be emphasized that this method should not be used in cases where the highest activity of the enzyme corresponds with the main peak of the proteins. Regarding the decrease in activity of the unstabilized preparation, we believe that this is due to proteolysis caused by proteases produced by Bacillus suhrilis together with the purified enzyme. For example, x-amylase from Aspergillus ovizae contains two known proteases: Aspergillus aspartic proteinase (E.C. 3.4.23.6) and Aspergillus acid carboxy peptidase (E.C. 3.4.16.1) which result in proteolysis. The method presented for isolation of pure a-amylase seems to be very effective and economically advantageous for large-scale industrial production. REFERENCES 1 2 3 4 5 6 7 8
0. Sova, J. Chromatogr., 320 (1985) 15. 0. Sova and K. Boda, Czech. Pat., 234 801 (1985). 0. SOW, J. Chromatogr., 320 (1985) 213. P. Chavalier and .I. de la Notte, Enzyme Microb. Tech?zol., 9 (19871 Jan. K. Toda and K. Sato, J. Ferment. Technoi., 19 (1984) 79. P. Adlercreutz, 0. Hoist and B. Mattiason, EnzJ’me Microb. Technol., 4 (1982) 395. V. Sjrkora, Chemicko-analytickP tabulky, SNTL, Prague, 1976, p. 153. J. Davidek, J. HrdliEka. M. KarvBnek. J. Pokornj, et al.. Laboratorni prirucku anuljq potravin, SNTL/ALFA, Prague, 1978, p. 652. 9 J. KBs, B. KrBlov9 and P. Rauch, Laboratorni cviceni z biochemie a radiochemie, SNTL, Prague, 1983, p. 175. 10 J. K&, B. KrBlovri and K. Moltas, Laboratorni cviceniz hiochemie a radiochemie, SNTL. Prague, 1986, p. 150. 11 S. Hanzdwa, A. Odogawa, H. Sakata, M. Takouchi and E. Ichishima, Curr. Microb., 14 (1985) 235.