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Bacterial Alpha-Amylase Inhibitors
ZbI. Mi kro biol. 138 (1983), 145-150
[Agr icultural Micr obi ology Dep t ., F a cult y of Agri cultur e, Mini a U niv ., Mini a , Fac, of Agri cultur e, Ain Sha ms U niv ., Cairo, and Enzyme Un it , Agriculture R esearch Center, Giza, E gypt]
Bacterial Alpha-Amyla se Inhibitors M. S. A. S AFW AT , S.
A.
Z.
M AH~IO UD ,
R . M. A TTI A, M. ABD.EL-NASSER , and F. S.
ALI
Summary Th e effect of 5 growth r egula t or s at con cen t rat ions of 50, 100, a nd 1000 ppm on the activit y of B . su btilis-37 alpha-amylase wa s studied. Indol butyric ac id (lEA) did not inhibit alpha-amylase ac t iv ity at con cen t rat ion s of 50 t o 1000 ppm. The other 4 growth r eg ula t ors, G-furfurylamino. p urine, gi bbere llic acid, n a p hthalene ace t ic acid, and aton ik -G, a t a ll concen t rat ions inhi bited a lp ha -a m y la se activ ity. Th e effect of orga nic ac ids and for m al deh y de a t 0.01 M, 0.001 M, an d 0.00051\1 was st udied . Aceti c acid . oxa lic ac id, la ctic aci d, and m a lic acid at 0.0 1 1\1 an d 0.001 1\1 ca use d 100 % in hibit ion of alp ha -amy lase, bu t at 0.0005 lVI the inhibitions were 14 %, 46 %, 12 %, a nd 28 %, respective ly . Tartaric ac id ind uced 100 % , 85 % , a nd 62 % in hib ition for al pha. amylase a t 0.0 1, 0.001, a nd 0.00051\1, resp ectively , hu t at t he same conce n t ra t ion formaldeh y de ca used 60 % . 53 %, a n d 51 % in hibitio n . At 0.0 1 il'1 conce ntra t ion isoprop a nol, glycer ol, and manni t ol did not inhibit a lp ha -a mylase activ ity. T he in h ibi t ion per centag es, caused by p heno l, arabinose, lact ose, maltose, gala cto se, et hanol. a nd sod iu m be nzoa te , were 13, 10, 8, 6, 6.5, ami 4. So rbi t ol, glucose, a nd fru ctose ca use d 3 % in hibi ti on, wh ile it was 2 % by sucro se 11t 0.0 1 ~l. But ammoni um citra t e di basic a t 0.0 1 ~I induced 60 % inhibi tion for alpha -amylas e a ctivi ty. It wa s found tha t Zn SO j a nd F e2(SOl h wore t he st r ongest inhib ito rs, followed by SnC I2 , I'b( C2H 30 2 ) 2 ' AgN0 3 , AlCla• Cd(C 2 H 30 2 ) , CuC I2 • F eSO I , H gCI2 , a nd Na 2- E DT A. in decr easi ng or de r . Mn S0 4 wa s a rno der a te in hibi t or. T he slig ht inh ibit ors in decr ea si ng ord er wer e E a CI2 , Co(N0 3h, Na 2 H P 0 4 • Ni::;Op a n d fi na lly MgCI2 •
Zusammenfassung E s wurde di e Wirkung von 5 Wu chsst offen in Konzen t r a ti onen von 50, 100 und 1000 pp m a uf die Akbivita - von B. su btilis. 37.A lpha.A m y lase untersu cht. I n dol.B uttersaure hemmte di e Alpha.A mylaseAktivite.t in eine r Kon zentration von 50 bis 1 000 ppm nicht . Die 4 anderon Wu ch sstoffe: 6-Furfurylamin opurin , Gibberellm sa ure. Naphthyl essigsuu ro und Atonik-G hem m ten die AlphaAm ylase -Aktivitut in a llen K onzentrationen. F erner wurde di e Wirkung v on or gan isc hen Saure n und v on F ormaldeh y d bei 0.01 lVI, 0.001 .M u nd 0.00 05 M untersucht. E ssig . sa ure, Ox a lsuure, Milchsau re un d Apfolsaure v er ursac hte n bei 0.01 M und 0.001 .M cine 100 % ige H emm un g del' Alp ha-A my las e,
Zbl. l Ii krobiol., Bd. 138
146
M. S. A. SHWAT et al .
A less kn own aspect of microbial amylase is t heir pr esence in starch-containing canned foods, such as fish balls in jui ce, sausage in brine, and meet balls in sauce. Sta rch break-down may completely change t he consiste ncy of t he pr odu ct s, resulting in great economical loss in food industry . Sprouting in the ear is also common , almost annua lly causing crop det eriorati on of wheat in areas with rather wet and cold Climate at harvest. A great part of th e crop is only slightly affect ed, but its tec hnological qua lit y, especially for industrial uses, is lowered . . Therefore, t he possibilit y to improve starch -containing canned foods and t he ba king qua lity of sprouted gra in by red ucing the. alp ha-amy lase activity seemed to be necessary . Th e aim of t he present work was to st udy some inhibiting fact ors for t he produ ct ion of bacteria l alpha -amylase .
Materials and Methods Enz ym e determination T he hydr oly sis of starch was measured as an estimation of a mylo lytic ac t iv it y according t o Soc. Arner. B a ct. (1957). The a my lase a ctivities of the cult u re su pernatant fluid s were d et ermin e d color ime t rically accor di ng to A TT I A' S and SAMI A ' S m eth od (1977 ).
E nzy me pr odu ction A newly suggested m ediu m .No. 4, wh ich wa s inves t iga t ed during t he present s t ud y, wa s used as fermentation m ed iu m to determine se ve ra l fa c t ors a ffecting enzy me production . It con t ains s % wh eat bran extract a nd 0.5 % peptone at pH 6.5.
Results and Discussion Gro w th reg u lato r s The effect of growt h regulators, such as ind ol butyric acid , 6-furfury l aminopurine , gibbe rellic acid , naphthalin acetic, and atonik-G, used at 50, 100, and 1,000 ppm, on t he activity of alpha-amylase was st udied . Table I shows t he effect of growt h regulators on t he acti vity of t he B subtilis- 37 alpha-amylase. It is clear t hat indol but yr ic acid (lBA) did not inhibit alpha -amylase activity at concent ratio ns of 50 t o 1,000 ppm. The inhibition by 6-furfurylaminopurine at concentrations of 1,000, 100, and 50 ppm was 14 %. Gibberellic acid cau sed 19 %, 16 %, and 16 % inhibition at the previous concentrati ons, respectively, while naphtalene acetic acid (NAA) caused 33 %, 31 %, and 20 % inhibition, respectively, at the same concentrations. Atonik -G caused 60 %, 13 %, and 9 % inhibition at concent rati ons of 1,000, 100, and 50 ppm , respectively. Table 1. Effect of gro wth regulators on t he a ct iv it y of a lp ha-amylase. The res ults rep resen t the percen tage of inhibit ion observed Growth regulator
I n dol but yric a cid (I B A) 6-Furfurylamino purine Gibberell ic a cid N aphtalene ac etic a cid (N AA ) Aton ik -G
% I n hi bi ti on at
a concentrat ion of
1,000 p pm
100 p pm
50 ppm
o
o
o
14 19
14
14
10 31 13
16 20
33 60
9
Bacterial Alpha-Amyl.u« Inhibitors
147
On the other hand, growth regulators had no stimulating effect on synthesis of alpha-amylase by organisms. SRIVASTAVA et al. (1962), PUZINA (1976), GOLENIOWSKI and CORREA (1976), ONCKELEN et al. (1976), BIELENSKA and BIALEK (1977), CHAI and CHUNG (1977), and MACChtEGOR (1978a). Organic a c i d s and formaldehyde The effect of seven organic acids and formaldehyde at concentrations of 0.01, 0.001, and 0.0005 M on the activity of alpha-amylase was studied. Table 2 shows the effect of organic acids and formaldehyde on the activity of B. subtilis-37 alpha-amylase. Acetic acid, oxalic acid, lactie acid, and malic acid at 0.01 M and 0.001 M caused 100 % inhibition for alpha-amylase, but at 0.0005 M the inhibition was 14 %, 46 %' 12 % and 28 %, respectively. However, tartaric acid eaused 100 %, 85 %, and 62 % inhibition for alpha-amylase at concentrations of 0.01, 0.001 and 0.0005 M, respectively. On the other hand, formic acid caused 62 %, 56 % and 55 % inhibition for alpha-amylase at concentrations of 0.01, 0.001, and 0.0005 M, respectively, while benzoic acid caused 83 %, 69 %, and 62 % inhibition, respectively. Formaldehyde caused 60 %, 53 %, and 51 % inhibition for alpha-amylase at the previous concentration, respectively. These results are in accord with those by HAGIHARA et al. (1956a), who found that B. subiilis alpha-amylase treated with 0.01 M acetic acid (10 °C, 10 minutes) resulted in 58.6 % denaturation, and CHRISTINA et al. (1979) who found that lactic acid inhibited wheat alpha-amylase activity. The effect of alcohols, polyhydric alcohols, monosaccharides, disaecharides, :-;0dium benzoate, and ammonium citrate dibasic on the activity of alpha-amylase was studied (Table 3). Methanol, iso-propanol, glycerol, and mannitol did not inhibit alpha-amylase, while 2 % inhibition of alpha-amylase was recorded when using suerose. Sorbitol, glucose, and fructose caused 3 % inhibition for alpha-amylase and sodium benzoate caused 4 %, ethanol 5 %, galactose and maltose 6 %, lactose 8 %, arabinose 10 %, and phenol 1:~ %, while ammonium citrate dibasio caused 60 % inhibition for alpha-amylase activity. These results are in line with those by WHITAKER et al. (1962) who found that alcohols inhibited alpha-amylase activity in the decreasing order of ethanol, iso-propanol, glyceroL and methanol. Inhibition by carbo. hydrates decreased in the order disaccharides, sugar alcohols, and monosaccharides. Maltose was the most inhibitory of the tested carbohydrates. MCWETHY and HARTMAN (1977) also found that there was a slight inhibitory effect caused by fructose. Table 2. Effect of organic acids and formaldehyde on the activity of alpha-amylase. The results represent the percentage of inhibition observed Organic acids
Inhibition at the concentration of
0.01 M Formic acid Acetic acid Oxalic acid Lactic acid MaliC' acid Tartaric acid Benzoic acid Formaldehyde 10'
0.001 M
(i2
5(i
100 100 100
100 100 100 100 85 69 53
100
100 83 (i0
0.0005 M 55 14
46 12 28 62 62
51
148
M.
S.
A.
SAFWAT
et a!.
Table 3. Effect of alcohols, polyhydric alcohols, monosaccharides, disaccharides, sodium benzoate, and ammonium citrate dibasic on the activity of alpha-amylase
% Inhibition at the concentration of 0.01 M
Substance
o
Methanol Ethanol Iso-propanol Glycerol Mannitol Sorbitol Phenol Arabinose Glucose Galactose Fructose Maltose Lactose Sucrose Sodium benzoate Ammonium citrate dibasic
5
o o ()
3 13 10 3 6 3 6 8 2
4 60
Table 4. Effect of metal ions and EDTA on the activity of alpha-amylase Ions
% Inhibition at 10mM
1mM
the concentration of 0.5mM
O.lmM
0.01 mM
NaCl
0
0
0
0
0
NaF
0
0
0
0
0
NaH 2P04 Na 2HP0 4 Na 2-EDTA
0
0
0
0
0
5
0
0
0
0
21
20
12
0
0
3 9 25 100 100 100 5 37 100 100 100 100 7 100 100
0
0
0
0
0
0
0
0
13
10
0
0
82
58
33
MgCl 2 BaC12 HgC~
CuC12 SnCl 2 AIC13 NiS0 4 MnS04 ZnS04 FeS04 Fe 2(S04h AgN0 3 CO(N0 3 ) 2 Cd(C 2H302) 2 Pb(C 2H302 ) 2
100
74
0
0
100
31
0
0 0
0
0
0
23
0
0
0
100
100
7
6
62 100
56
3
100
8
0 0
100
62
8
0
0
0
0
0
100
22
7
0
100
65
0
0
Bacterial Alpha-Amylase Inhibitors
149
Effect of different metal ions and Na 2-EDTA Table 4 shows the effect of metal ions and Na 2-EDTA on the activity of the B. subtilis-37 alpha-amylase. NaCl, NaF, NaH 2P0 4 had no effect on alpha-amylase activity at 10 mM. These results are in line with those by Roy (1955a) and MOULIN and GALZY (1978), who found that sodium had no effect on alpha-amylase. Na 2HP0 4, MgC1 2 , BaC12 , NiS0 4, and CO(N03 ) 2 showed no effect on alpha-amylase activity at low concentrations, but were inhibitory at 10 mM, being 5 %, a %, 9 %, 5 %, and 7 % inhibition, respectively. These results, too, were in line with those by Roy (1955b) who found that sodium arsenite did not inhibit fungal alpha-amylase, Roy (1955a), who found that Mg had no effect on fungal alpha-amylase, FISCHER and TOVAREK (1976), who stopped the reaction between alpha-amylase and a chromogenic substrate (colored starch) by addition of an aqueous solution of MgS0 4 or BaCI 2 , which gave an insoluble complex with residual starch, MOULIN and GALZY (1978), who found that Ba2 + and Mg 2+ were strong inhibitors at concentrations of 100 mMfl, and CHIANG et al. (1979), who found that C0 2+ was moderately inhibitory. MnS0 4 had no effect on alpha-amylase activity at low concentration, but was inhibitory at 1 mM and 10 mM by 23 % and 37 % inhibition, respectively. This result corresponds with MOULIN and GALZY (1978), who found that Mn 2 + was a strong inhibitor at a concentration of 100 mMfI. Na 2-EDTA and HgC12 inhibited alpha-amylase at concentrations of 10 mM, I mM, and 0.5 mM, by 21 %' 20 %, and 13 % inhibition for Na 2-EDTA and 25 %, 13 %, and 10 % inhibition for HgCI 2 , respectively. These results are corresponding to Roy (1955b), MCWETHY and HARTMAN (1977), MOULIN and GALZY (1978), CHIANG et al. (1979), and CHRISTINA et al. (1979). They found that EDTA inhibited alpha-amylase activity at different concentrations. The inhibition with SnCI2 , AICI 3 , AgN0 3 , Cd(C2H302 )2 and Pb(C 2H302 )2 was 100 % at concentrations of 1 mM and 10 mM, but was 74 %,31 %, 62 %,22 %, and 65 %, respectively, at a concentration of 0.5 mM. The inhibitory effect of AgN0 3 and Cd(C2H302 ) 2 was 8 % and 7 % at a concentration of 0.1 mM. These results are in accord with Roy (1955a), COLE (1977), CHIANG et al. (1979), and CHRIS'fINA et al. (1979), who found that such metal ions inhibited alphaamylase activity. The inhibition with CuCl2 and FeS0 4 was 100 % at a concentration of 10 mM, while at 1,0.5, and 0.1 mM inhibition by CuCl2 was 82 %,58 %, and 33 %, respectively, but by FeS04 was 62 %, .56 %, and :~ %, respectively. These results are in accord with Roy (I955a) and MOULIN and GALZY (1978). The inhibition by ZnS0 4 and ]'e2(S04h was 100 % at concentrations of 10, 1 and 0.5 mM, but ZnS0 4 caused 7 % and 6 % inhibition at concentrations of 0.1 and 0.01 mM, respectively, while Fe 2(S04h caused 8 % at 0.1 mM. These results correspond with MOULIN and GALZY (1978) and CHIANG (1979) et al., who found that Zn 2 + was a strong inhibitor for alphaamylase. References ATT[A, R. M., and SAMIA, A. A.: Simple and rapid colorimetric method for the micro-determination of alpha-amylase. Zb!. Bakt. II 132 (1977), 19:1195. Bn;LJNSKA, C. M., and BIALEK, K.: Amylase and acid phosphatase activity in potato tubers, treated with gibberellic acid and stored at 2 DC and 8 DC. Aeta Agrobot. 30 (I) (1977), 95~ 102. CHAI, 1., and CHUNG, Y. S.: Studies on gibberellic-acid-promoted and indole-3-acetic-acid-repressed amylase synthesis by barley seeds. Korean J. Bot. 20 (2) (1977), 91-102. CHIANG, .r.r., ALTAR,.r. E., and ST.F:RBERG, M.: Purification and characterization of a thermostable alpha-amylase from Bacillus licheniforrnis. Starch 31 (:l) (1979), 86-92. CHRISTINA, "V., LEENA, .T., and PERRA, K.: Efforts to improve the baking properties of sproutdamaged wheat by reagent-reducing amylase activity. 1. Sereening tests by the falling number method. Lebensm.-Wiss. u. Techno!. 12 (1979), :121-:324.
150
M. S. A. SAFWAT et al., Bacterial Alpha-Amylase Inhibitors
COLE, M. A.: Lead inhibition of enzymes synthesis in soil. Appl. Environ. MicrobioI. 33 (2) (1977), 262-268. FISCHE;R, D., and TOVA;REK, J.: Detorrnination of alpha-amylase activity. Chern. Abstr. 86 (1976)'10254A W. GOLENIOWSKI, M. E., and CORREA, N. S.: Effect of abscisic acid on germination of Eragostis curvuta: Interaction with other growth regulators and its effects on different enzymes. Phyt. Rev. Int. Bot. Exper. 34 (2). [Cited from BioI. Abstr. 64 (2) (1976), 10948.] HAGIHARA, B., 'l'AKEYOSHI, N., and KAZUO, 0.: Denaturation and inactivation of enzyme proteins. II. Denaturation and inactivation of bacterial amalyse. J. Biochem. 43 (4) (1956a), 469~-481. (Cited from BioI. Abstr. 31, 14419.) MAC GREGOR, A. W.: Alpha-amylase I from malted barley - physical properties and action pattern on amylase. Cereal Chern. 55 (5) (1978a), 754-765. MCWETHY, S. ,T., and HARTMAN, P. A.: Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus. J. Bact. 129 (3) (1977), 1537-1544. MOULIN, G., and GALZY, P.: Etude de I amylase de la paroi de Pichia burtonii Boidin. Z. Allg. MikrobioI. 18 (4) (1978), 269-274. ONCKELEN, V., HENRI, A., RITA, V., and DEGREE]), .T. A.: Estimation of gibberellin and inhibitors of alpha-amylase synthesis in Phaseolus vulgaris L., using a modified barley endosperm bioassay. Bull. Soc. R. Bot. Belg. 109 (2) (1976), :335-346. PUZINA, T. 1.: Effect of gibberellin on some physiological and biochemical processes of germinating potato tubers. BioI. Nauki (Moscow) 19 (3) (1976), 81-85. [Cited from BioI. Abstr. 63 (6), 35303.] RoY, D. K.: Effect of some salts and vitamins on the activity and stabilization of fungal alphaamylase. Ann. Biochem. and Expel'. Med. 15 (I) (1955a), 63-64. BioI. Abstr. 30, 20579. - Inhibitory effect of some compounds on fungal alpha-amylase. Ann. Biochem. and Exper. Med. 15 (I) (1955), 65-68. BioI. Abstr. 30, 20580. Society of American Bacteriologists: Manual of Microbiological Methods (1957). McGraw-Hill Book Co., Inc., N.Y. SRIVASTAVA, B. 1., SAHAI, and MEREDITH, W. O. S.: Mechanism of action of gibberellic acid. Inhibition of alpha-amylase development during germination and barley by chloramphenicol and its reversal by gibberellic acid. Canad. J. Bot. 40 (9) (1962), 1257-1265. (Cited from BioI. Abstr. 41, 11951.) WHITAKER ,J. R., TAPPEL, A. L., and EVA, 'V.: Modification of enzymic activity. I. Effect of organic additives on alpha-amylase activity. Biochim. Biophys, Acta 62 (2) (1962), 300-309. Eingegangen am 3. 2. 1982 Authors' addresses: M. S. A. SAFWAT, M. ABD-EL-NASSER, and F. S. ALI, Fac, of Agriculture, Minia University, Minia; Prof. S. A. Z. MAHMOUD, Fac, of Agriculture, Ain Shams University Cairo; R. M. ATTIA, Enzyme Unit, Agric. Research Centre, Giza, Egypt.
[Agr icultural Micr obi ology Dep t ., F a cult y of Agri cultur e, Mini a U niv ., Mini a , Fac, of Agri cultur e, Ain Sha ms U niv ., Cairo, and Enzyme Un it , Agriculture R esearch Center, Giza, E gypt]
Bacterial Alpha-Amyla se Inhibitors M. S. A. S AFW AT , S.
A.
Z.
M AH~IO UD ,
R . M. A TTI A, M. ABD.EL-NASSER , and F. S.
ALI
Summary Th e effect of 5 growth r egula t or s at con cen t rat ions of 50, 100, a nd 1000 ppm on the activit y of B . su btilis-37 alpha-amylase wa s studied. Indol butyric ac id (lEA) did not inhibit alpha-amylase ac t iv ity at con cen t rat ion s of 50 t o 1000 ppm. The other 4 growth r eg ula t ors, G-furfurylamino. p urine, gi bbere llic acid, n a p hthalene ace t ic acid, and aton ik -G, a t a ll concen t rat ions inhi bited a lp ha -a m y la se activ ity. Th e effect of orga nic ac ids and for m al deh y de a t 0.01 M, 0.001 M, an d 0.00051\1 was st udied . Aceti c acid . oxa lic ac id, la ctic aci d, and m a lic acid at 0.0 1 1\1 an d 0.001 1\1 ca use d 100 % in hibit ion of alp ha -amy lase, bu t at 0.0005 lVI the inhibitions were 14 %, 46 %, 12 %, a nd 28 %, respective ly . Tartaric ac id ind uced 100 % , 85 % , a nd 62 % in hib ition for al pha. amylase a t 0.0 1, 0.001, a nd 0.00051\1, resp ectively , hu t at t he same conce n t ra t ion formaldeh y de ca used 60 % . 53 %, a n d 51 % in hibitio n . At 0.0 1 il'1 conce ntra t ion isoprop a nol, glycer ol, and manni t ol did not inhibit a lp ha -a mylase activ ity. T he in h ibi t ion per centag es, caused by p heno l, arabinose, lact ose, maltose, gala cto se, et hanol. a nd sod iu m be nzoa te , were 13, 10, 8, 6, 6.5, ami 4. So rbi t ol, glucose, a nd fru ctose ca use d 3 % in hibi ti on, wh ile it was 2 % by sucro se 11t 0.0 1 ~l. But ammoni um citra t e di basic a t 0.0 1 ~I induced 60 % inhibi tion for alpha -amylas e a ctivi ty. It wa s found tha t Zn SO j a nd F e2(SOl h wore t he st r ongest inhib ito rs, followed by SnC I2 , I'b( C2H 30 2 ) 2 ' AgN0 3 , AlCla• Cd(C 2 H 30 2 ) , CuC I2 • F eSO I , H gCI2 , a nd Na 2- E DT A. in decr easi ng or de r . Mn S0 4 wa s a rno der a te in hibi t or. T he slig ht inh ibit ors in decr ea si ng ord er wer e E a CI2 , Co(N0 3h, Na 2 H P 0 4 • Ni::;Op a n d fi na lly MgCI2 •
Zusammenfassung E s wurde di e Wirkung von 5 Wu chsst offen in Konzen t r a ti onen von 50, 100 und 1000 pp m a uf die Akbivita - von B. su btilis. 37.A lpha.A m y lase untersu cht. I n dol.B uttersaure hemmte di e Alpha.A mylaseAktivite.t in eine r Kon zentration von 50 bis 1 000 ppm nicht . Die 4 anderon Wu ch sstoffe: 6-Furfurylamin opurin , Gibberellm sa ure. Naphthyl essigsuu ro und Atonik-G hem m ten die AlphaAm ylase -Aktivitut in a llen K onzentrationen. F erner wurde di e Wirkung v on or gan isc hen Saure n und v on F ormaldeh y d bei 0.01 lVI, 0.001 .M u nd 0.00 05 M untersucht. E ssig . sa ure, Ox a lsuure, Milchsau re un d Apfolsaure v er ursac hte n bei 0.01 M und 0.001 .M cine 100 % ige H emm un g del' Alp ha-A my las e,
Zbl. l Ii krobiol., Bd. 138
146
M. S. A. SHWAT et al .
A less kn own aspect of microbial amylase is t heir pr esence in starch-containing canned foods, such as fish balls in jui ce, sausage in brine, and meet balls in sauce. Sta rch break-down may completely change t he consiste ncy of t he pr odu ct s, resulting in great economical loss in food industry . Sprouting in the ear is also common , almost annua lly causing crop det eriorati on of wheat in areas with rather wet and cold Climate at harvest. A great part of th e crop is only slightly affect ed, but its tec hnological qua lit y, especially for industrial uses, is lowered . . Therefore, t he possibilit y to improve starch -containing canned foods and t he ba king qua lity of sprouted gra in by red ucing the. alp ha-amy lase activity seemed to be necessary . Th e aim of t he present work was to st udy some inhibiting fact ors for t he produ ct ion of bacteria l alpha -amylase .
Materials and Methods Enz ym e determination T he hydr oly sis of starch was measured as an estimation of a mylo lytic ac t iv it y according t o Soc. Arner. B a ct. (1957). The a my lase a ctivities of the cult u re su pernatant fluid s were d et ermin e d color ime t rically accor di ng to A TT I A' S and SAMI A ' S m eth od (1977 ).
E nzy me pr odu ction A newly suggested m ediu m .No. 4, wh ich wa s inves t iga t ed during t he present s t ud y, wa s used as fermentation m ed iu m to determine se ve ra l fa c t ors a ffecting enzy me production . It con t ains s % wh eat bran extract a nd 0.5 % peptone at pH 6.5.
Results and Discussion Gro w th reg u lato r s The effect of growt h regulators, such as ind ol butyric acid , 6-furfury l aminopurine , gibbe rellic acid , naphthalin acetic, and atonik-G, used at 50, 100, and 1,000 ppm, on t he activity of alpha-amylase was st udied . Table I shows t he effect of growt h regulators on t he acti vity of t he B subtilis- 37 alpha-amylase. It is clear t hat indol but yr ic acid (lBA) did not inhibit alpha -amylase activity at concent ratio ns of 50 t o 1,000 ppm. The inhibition by 6-furfurylaminopurine at concentrations of 1,000, 100, and 50 ppm was 14 %. Gibberellic acid cau sed 19 %, 16 %, and 16 % inhibition at the previous concentrati ons, respectively, while naphtalene acetic acid (NAA) caused 33 %, 31 %, and 20 % inhibition, respectively, at the same concentrations. Atonik -G caused 60 %, 13 %, and 9 % inhibition at concent rati ons of 1,000, 100, and 50 ppm , respectively. Table 1. Effect of gro wth regulators on t he a ct iv it y of a lp ha-amylase. The res ults rep resen t the percen tage of inhibit ion observed Growth regulator
I n dol but yric a cid (I B A) 6-Furfurylamino purine Gibberell ic a cid N aphtalene ac etic a cid (N AA ) Aton ik -G
% I n hi bi ti on at
a concentrat ion of
1,000 p pm
100 p pm
50 ppm
o
o
o
14 19
14
14
10 31 13
16 20
33 60
9
Bacterial Alpha-Amyl.u« Inhibitors
147
On the other hand, growth regulators had no stimulating effect on synthesis of alpha-amylase by organisms. SRIVASTAVA et al. (1962), PUZINA (1976), GOLENIOWSKI and CORREA (1976), ONCKELEN et al. (1976), BIELENSKA and BIALEK (1977), CHAI and CHUNG (1977), and MACChtEGOR (1978a). Organic a c i d s and formaldehyde The effect of seven organic acids and formaldehyde at concentrations of 0.01, 0.001, and 0.0005 M on the activity of alpha-amylase was studied. Table 2 shows the effect of organic acids and formaldehyde on the activity of B. subtilis-37 alpha-amylase. Acetic acid, oxalic acid, lactie acid, and malic acid at 0.01 M and 0.001 M caused 100 % inhibition for alpha-amylase, but at 0.0005 M the inhibition was 14 %, 46 %' 12 % and 28 %, respectively. However, tartaric acid eaused 100 %, 85 %, and 62 % inhibition for alpha-amylase at concentrations of 0.01, 0.001 and 0.0005 M, respectively. On the other hand, formic acid caused 62 %, 56 % and 55 % inhibition for alpha-amylase at concentrations of 0.01, 0.001, and 0.0005 M, respectively, while benzoic acid caused 83 %, 69 %, and 62 % inhibition, respectively. Formaldehyde caused 60 %, 53 %, and 51 % inhibition for alpha-amylase at the previous concentration, respectively. These results are in accord with those by HAGIHARA et al. (1956a), who found that B. subiilis alpha-amylase treated with 0.01 M acetic acid (10 °C, 10 minutes) resulted in 58.6 % denaturation, and CHRISTINA et al. (1979) who found that lactic acid inhibited wheat alpha-amylase activity. The effect of alcohols, polyhydric alcohols, monosaccharides, disaecharides, :-;0dium benzoate, and ammonium citrate dibasic on the activity of alpha-amylase was studied (Table 3). Methanol, iso-propanol, glycerol, and mannitol did not inhibit alpha-amylase, while 2 % inhibition of alpha-amylase was recorded when using suerose. Sorbitol, glucose, and fructose caused 3 % inhibition for alpha-amylase and sodium benzoate caused 4 %, ethanol 5 %, galactose and maltose 6 %, lactose 8 %, arabinose 10 %, and phenol 1:~ %, while ammonium citrate dibasio caused 60 % inhibition for alpha-amylase activity. These results are in line with those by WHITAKER et al. (1962) who found that alcohols inhibited alpha-amylase activity in the decreasing order of ethanol, iso-propanol, glyceroL and methanol. Inhibition by carbo. hydrates decreased in the order disaccharides, sugar alcohols, and monosaccharides. Maltose was the most inhibitory of the tested carbohydrates. MCWETHY and HARTMAN (1977) also found that there was a slight inhibitory effect caused by fructose. Table 2. Effect of organic acids and formaldehyde on the activity of alpha-amylase. The results represent the percentage of inhibition observed Organic acids
Inhibition at the concentration of
0.01 M Formic acid Acetic acid Oxalic acid Lactic acid MaliC' acid Tartaric acid Benzoic acid Formaldehyde 10'
0.001 M
(i2
5(i
100 100 100
100 100 100 100 85 69 53
100
100 83 (i0
0.0005 M 55 14
46 12 28 62 62
51
148
M.
S.
A.
SAFWAT
et a!.
Table 3. Effect of alcohols, polyhydric alcohols, monosaccharides, disaccharides, sodium benzoate, and ammonium citrate dibasic on the activity of alpha-amylase
% Inhibition at the concentration of 0.01 M
Substance
o
Methanol Ethanol Iso-propanol Glycerol Mannitol Sorbitol Phenol Arabinose Glucose Galactose Fructose Maltose Lactose Sucrose Sodium benzoate Ammonium citrate dibasic
5
o o ()
3 13 10 3 6 3 6 8 2
4 60
Table 4. Effect of metal ions and EDTA on the activity of alpha-amylase Ions
% Inhibition at 10mM
1mM
the concentration of 0.5mM
O.lmM
0.01 mM
NaCl
0
0
0
0
0
NaF
0
0
0
0
0
NaH 2P04 Na 2HP0 4 Na 2-EDTA
0
0
0
0
0
5
0
0
0
0
21
20
12
0
0
3 9 25 100 100 100 5 37 100 100 100 100 7 100 100
0
0
0
0
0
0
0
0
13
10
0
0
82
58
33
MgCl 2 BaC12 HgC~
CuC12 SnCl 2 AIC13 NiS0 4 MnS04 ZnS04 FeS04 Fe 2(S04h AgN0 3 CO(N0 3 ) 2 Cd(C 2H302) 2 Pb(C 2H302 ) 2
100
74
0
0
100
31
0
0 0
0
0
0
23
0
0
0
100
100
7
6
62 100
56
3
100
8
0 0
100
62
8
0
0
0
0
0
100
22
7
0
100
65
0
0
Bacterial Alpha-Amylase Inhibitors
149
Effect of different metal ions and Na 2-EDTA Table 4 shows the effect of metal ions and Na 2-EDTA on the activity of the B. subtilis-37 alpha-amylase. NaCl, NaF, NaH 2P0 4 had no effect on alpha-amylase activity at 10 mM. These results are in line with those by Roy (1955a) and MOULIN and GALZY (1978), who found that sodium had no effect on alpha-amylase. Na 2HP0 4, MgC1 2 , BaC12 , NiS0 4, and CO(N03 ) 2 showed no effect on alpha-amylase activity at low concentrations, but were inhibitory at 10 mM, being 5 %, a %, 9 %, 5 %, and 7 % inhibition, respectively. These results, too, were in line with those by Roy (1955b) who found that sodium arsenite did not inhibit fungal alpha-amylase, Roy (1955a), who found that Mg had no effect on fungal alpha-amylase, FISCHER and TOVAREK (1976), who stopped the reaction between alpha-amylase and a chromogenic substrate (colored starch) by addition of an aqueous solution of MgS0 4 or BaCI 2 , which gave an insoluble complex with residual starch, MOULIN and GALZY (1978), who found that Ba2 + and Mg 2+ were strong inhibitors at concentrations of 100 mMfl, and CHIANG et al. (1979), who found that C0 2+ was moderately inhibitory. MnS0 4 had no effect on alpha-amylase activity at low concentration, but was inhibitory at 1 mM and 10 mM by 23 % and 37 % inhibition, respectively. This result corresponds with MOULIN and GALZY (1978), who found that Mn 2 + was a strong inhibitor at a concentration of 100 mMfI. Na 2-EDTA and HgC12 inhibited alpha-amylase at concentrations of 10 mM, I mM, and 0.5 mM, by 21 %' 20 %, and 13 % inhibition for Na 2-EDTA and 25 %, 13 %, and 10 % inhibition for HgCI 2 , respectively. These results are corresponding to Roy (1955b), MCWETHY and HARTMAN (1977), MOULIN and GALZY (1978), CHIANG et al. (1979), and CHRISTINA et al. (1979). They found that EDTA inhibited alpha-amylase activity at different concentrations. The inhibition with SnCI2 , AICI 3 , AgN0 3 , Cd(C2H302 )2 and Pb(C 2H302 )2 was 100 % at concentrations of 1 mM and 10 mM, but was 74 %,31 %, 62 %,22 %, and 65 %, respectively, at a concentration of 0.5 mM. The inhibitory effect of AgN0 3 and Cd(C2H302 ) 2 was 8 % and 7 % at a concentration of 0.1 mM. These results are in accord with Roy (1955a), COLE (1977), CHIANG et al. (1979), and CHRIS'fINA et al. (1979), who found that such metal ions inhibited alphaamylase activity. The inhibition with CuCl2 and FeS0 4 was 100 % at a concentration of 10 mM, while at 1,0.5, and 0.1 mM inhibition by CuCl2 was 82 %,58 %, and 33 %, respectively, but by FeS04 was 62 %, .56 %, and :~ %, respectively. These results are in accord with Roy (I955a) and MOULIN and GALZY (1978). The inhibition by ZnS0 4 and ]'e2(S04h was 100 % at concentrations of 10, 1 and 0.5 mM, but ZnS0 4 caused 7 % and 6 % inhibition at concentrations of 0.1 and 0.01 mM, respectively, while Fe 2(S04h caused 8 % at 0.1 mM. These results correspond with MOULIN and GALZY (1978) and CHIANG (1979) et al., who found that Zn 2 + was a strong inhibitor for alphaamylase. References ATT[A, R. M., and SAMIA, A. A.: Simple and rapid colorimetric method for the micro-determination of alpha-amylase. Zb!. Bakt. II 132 (1977), 19:1195. Bn;LJNSKA, C. M., and BIALEK, K.: Amylase and acid phosphatase activity in potato tubers, treated with gibberellic acid and stored at 2 DC and 8 DC. Aeta Agrobot. 30 (I) (1977), 95~ 102. CHAI, 1., and CHUNG, Y. S.: Studies on gibberellic-acid-promoted and indole-3-acetic-acid-repressed amylase synthesis by barley seeds. Korean J. Bot. 20 (2) (1977), 91-102. CHIANG, .r.r., ALTAR,.r. E., and ST.F:RBERG, M.: Purification and characterization of a thermostable alpha-amylase from Bacillus licheniforrnis. Starch 31 (:l) (1979), 86-92. CHRISTINA, "V., LEENA, .T., and PERRA, K.: Efforts to improve the baking properties of sproutdamaged wheat by reagent-reducing amylase activity. 1. Sereening tests by the falling number method. Lebensm.-Wiss. u. Techno!. 12 (1979), :121-:324.
150
M. S. A. SAFWAT et al., Bacterial Alpha-Amylase Inhibitors
COLE, M. A.: Lead inhibition of enzymes synthesis in soil. Appl. Environ. MicrobioI. 33 (2) (1977), 262-268. FISCHE;R, D., and TOVA;REK, J.: Detorrnination of alpha-amylase activity. Chern. Abstr. 86 (1976)'10254A W. GOLENIOWSKI, M. E., and CORREA, N. S.: Effect of abscisic acid on germination of Eragostis curvuta: Interaction with other growth regulators and its effects on different enzymes. Phyt. Rev. Int. Bot. Exper. 34 (2). [Cited from BioI. Abstr. 64 (2) (1976), 10948.] HAGIHARA, B., 'l'AKEYOSHI, N., and KAZUO, 0.: Denaturation and inactivation of enzyme proteins. II. Denaturation and inactivation of bacterial amalyse. J. Biochem. 43 (4) (1956a), 469~-481. (Cited from BioI. Abstr. 31, 14419.) MAC GREGOR, A. W.: Alpha-amylase I from malted barley - physical properties and action pattern on amylase. Cereal Chern. 55 (5) (1978a), 754-765. MCWETHY, S. ,T., and HARTMAN, P. A.: Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus. J. Bact. 129 (3) (1977), 1537-1544. MOULIN, G., and GALZY, P.: Etude de I amylase de la paroi de Pichia burtonii Boidin. Z. Allg. MikrobioI. 18 (4) (1978), 269-274. ONCKELEN, V., HENRI, A., RITA, V., and DEGREE]), .T. A.: Estimation of gibberellin and inhibitors of alpha-amylase synthesis in Phaseolus vulgaris L., using a modified barley endosperm bioassay. Bull. Soc. R. Bot. Belg. 109 (2) (1976), :335-346. PUZINA, T. 1.: Effect of gibberellin on some physiological and biochemical processes of germinating potato tubers. BioI. Nauki (Moscow) 19 (3) (1976), 81-85. [Cited from BioI. Abstr. 63 (6), 35303.] RoY, D. K.: Effect of some salts and vitamins on the activity and stabilization of fungal alphaamylase. Ann. Biochem. and Expel'. Med. 15 (I) (1955a), 63-64. BioI. Abstr. 30, 20579. - Inhibitory effect of some compounds on fungal alpha-amylase. Ann. Biochem. and Exper. Med. 15 (I) (1955), 65-68. BioI. Abstr. 30, 20580. Society of American Bacteriologists: Manual of Microbiological Methods (1957). McGraw-Hill Book Co., Inc., N.Y. SRIVASTAVA, B. 1., SAHAI, and MEREDITH, W. O. S.: Mechanism of action of gibberellic acid. Inhibition of alpha-amylase development during germination and barley by chloramphenicol and its reversal by gibberellic acid. Canad. J. Bot. 40 (9) (1962), 1257-1265. (Cited from BioI. Abstr. 41, 11951.) WHITAKER ,J. R., TAPPEL, A. L., and EVA, 'V.: Modification of enzymic activity. I. Effect of organic additives on alpha-amylase activity. Biochim. Biophys, Acta 62 (2) (1962), 300-309. Eingegangen am 3. 2. 1982 Authors' addresses: M. S. A. SAFWAT, M. ABD-EL-NASSER, and F. S. ALI, Fac, of Agriculture, Minia University, Minia; Prof. S. A. Z. MAHMOUD, Fac, of Agriculture, Ain Shams University Cairo; R. M. ATTIA, Enzyme Unit, Agric. Research Centre, Giza, Egypt.