starch xanthide styrene-butadiene rubbers. effect of humidity and outdoor weathering
TRANSCRIPT
[ l l ] Szejtli, J . , und I?. Ddsu: Acta chim. Acad. Sci. Hung. 91 (1977),
[12] Richter, M. , und J . Szejtli: Starke 18 (1966), 95. [13] Hall& J . , und J . Szejtlic Starke 8 (1956). 133. [14] Hoild, J . , und J . Szcjtli: “The Reaction of Starch with Iodine”,
Chapter VII in: Radley, J . A , : “Starch and Its Derivatives”, Chapman and Hall, London 1968, p. 203-246.
[I51 Anrju, R., und P . G . Hurries: Deutsche 0. S. 2241 869 (1973).
[16] Szejtli, J . , und J . Auguhtut: Starke 18 (1966), 38. [17] Holld, J . , und J . Szejt l i : Kolloidnij Zhurnal 20 (1958), 229.
Anschrift der Verfasser: Dr. J . Szejtli und Dr. E. Banky-Hod, Biochemisches Forschungslaboratorium der Chinoin Pharmazeuti- sche und Chemische Werke, Endrodi S. u. 38-40, 1026 Budapest (Ungarn). (Eingegangen: 21. April 1977)
433.
Starch Xanthide Styrene-Butadiene Rubbers. Effect of Humidity and Outdoor Weathering
By R. A. Buchanan, J. McBrien, F. H. Otey and C. R. Russell, Peoria, Illinois
Conventional and starch xanthide (SX) reinforced styrene-butadiene (SBR) rubbers from several molding compounds and a tire tread compound were compared during 90 days’ storage at various controlled humidities and during 1 year’s outdoor weathering. For practical compounds, where SX served as a replacement for silica or as a partial replacement for carbon black, there was little difference between conventional and corresponding SX reinforced SBR rubbers. Properly formulated SX reinforced rubbers would prove satisfactory even in demanding applications.
Starke-Xanthid-Styrol-Butadiengummi. Wirkung von Feuchtigkeit und Witterungseinfliissen. Im Rahmen eines neunzigtagigen Lage- rungsversuches bei verschiedenen bestimmten Feuchtigkeiten und eines einjahrigen Verwitterungsversuches wurden mit konventionel- len Xanthiden und mit Starkexanthiden (SX) verstirkte Styrol- Butadien-(SBR)-Gummis aus verschiedenen PreBmassen und aus einer Verbindung fur Reifenlaufflachen miteinander verglichen. Bei praktisch verwendbaren Verbindungen, in denen SX als Ersatz fur Siliciumdioxid oder als teilweiser Ersatz fur RUB diente, waren nur geringe Unterschiede zwischen den mit konventionellen Xanthiden und mit SX verstarkten SBR-Gummis zu beobachten. Exakt ausgedruckt erwiesen sich SX-verstirkte Gummis auch gegenuber anspruchsvollen Anforderungen als zufriedenstellend.
1 Introduction
Previously, we studied the effect of prolonged water im- mersion on starch xanthide (SX) reinforced styrene-bu- tadiene (SBR) rubbers [I]. Compounding studies resulted in economical and practical methods for providing sufficient water resistance for most applications [l, 21. Prolonged exposure to high humidity in air was not studied previously, nor was actual outdoor weathering that involves the complex interaction of intermittent water immersion, high humidity, freeze-thaw cycles, actinic light and other factors. We have now evaluated the effect of prolonged exposure to high humidity and of 1 year’s outdoor weathering on practical SX reinforced SBR rubbers and are reporting our results here.
2 Experimental
2.1 Materials
The SX-SBR masterbatch for these studies was the EP-1 S type made in our pilot plant [l]. The geometric-mean diameter of the SX particles was about 200 nm and about 38% of the particles had complex shapes [3]. The SBR was type 1502 [4 (Appendix)] ; starch content was 45 parts per 100 parts SBR in the masterbatch. Technical grades of resorcinol and paraformaldehyde and Union Carbide’s A1102 aminosilane were used in some compounds. Sulfur, accelerators, antioxidants, fillers and other rubber compounding ingredients were all regular commercial products.
2.2 Compounding, Curing and Specimen Preparation
Nine test compounds were formulated (Table 1). These are representative of general-purpose molding compounds (low- pigmented for bright colors, 1A and 1 B ; highly filled for white or opaque pastel colors, 2A and 2B; soft black, 3B; and highly loaded black, 3A and 3C) and of a premium tire tread compound (4A and 4B). The conventional compounds without SX (lA, 2A, 3A and 4A) served as controls. SX was employed as a replacement for semi-reinforcing silica (IB),
.highly reinforcing silica (2B) and carbon blacks (3B, 3C and 4B). Certain protective agents which usually would have been employed, i. e., antiozonants and protective waxes, were omitted to avoid masking possible relevant activity of SX.
Resorcinal- formaldehyde and aminosilane were incorporated to improve water resistance as previously described [2] , otherwise usual processing was employed. Curing characteris- tics were measured with an oscillating disc rheometer operated at 1.67 Hz frequency, 3” amplitude and 150 “C per ASTM D2084-71T [4]. Test specimens were press-cured at 150°C and the optimum time. Coupons for swelling measurements were die cut 50 mm by 25 mm by 2 mm, ASTM D471-72 [4]. “Dumbells” for tensile measurements were 6 mm by 2 mm in cross-section at the narrow part, ASTM D412-72 [4]. Theeffect of humidity and other weather factors is strongly dependent on specimen shape and these standard “dumbell” tensile test pieces provide a sensitive test since the surface tovolume ratioofthenarrow section is about 14 cmpl [l].
Starch/Starke 30 (1978) Nr. 3, S. 91 -96 ’Pi Verlag Chemle, GmbH, D-6940 Weinheim, 1978 91
Table 1. Formulation of Test Compounds.
Ingredient
Low-pigmented Highly filled Black molding molding white molding -
1A Control
SBR, SX masterbatch" SBR 1606, black masterbatch') SBR 1502l) Stearic acid Zinc oxide Resorcinol Para formaldehyde Plasticizer, processing aid Light paraffinic oil Aromatic extender oil Paraffin wax Polyethylene glycol Coumarone-idene resin High-styrene resin Titanium dioxide Hard clay Semi-reinforcing silica Reinforcing silica N990 Carbon black N330 Carbon black Antioxidant, phenol type Aminosilane Sulfur N-tert-butyl-2-benzothiazole-sulfenamide Benzothiazyl disulfide N-oxydiethylene- benzot hiazole-2-sulfenamide Tetramethylthuiram disulfide Zinc diethyldithiocarbarnate Diphenyl guanidine
Total parts
100 1.5 3.0
2.0
60
1.5
1.75 1.25
1B 2A 2B 3A SX') Control SX Control
145
100 1.5 1 .o 3.0 3.0 0.55 0.45
2.0
1 .o 1.0 20 15 15 100
25
1 .o 1 .o 0.5 1 .o 3.0 1.25
0.5
0.45
145
1 .o 3.0 0.55 0.45 2.0 5.0
1 .o 20 15 15 100
1.8 0.5 2.2
0.5
0.45
100 2.0 5.0
2.0
120 30 2.0
2.0
1 .o 0.15
3B sx
145
1.5 3.0 0.55 0.45
20
20 1 .o 0.5 1 .o 1.25
__ 3c sx
__ 145
3.0 5.0 0.55 0.45 2.0
5.0
100
2.0 0.5 1.3
1 .o 0.3
Premium tire tread
4A 4B Control SX
48.3 162 108
1.5 1.5 3.0 3.0
0.18 0.15
3.3
1.8 1.8
1.75 1.5 1.0 1 .o
0.17
0.25 0.25
171.0 154.25 287.95 313.45 244.15 194.25 266.1 171.3 169.15
1) SX = starch xanthide; SBR types 1502 and 1606 are described in reference [4] appendix, SBR 1606 contains 52 parts N330 black and 10 parts oil per 100 parts elastomer. Each SX compound was formulated to give vulcanizates of about the same compression set, hardness and tensile strength as the conventional (control) compound; thus, the total volume of filler and reinforcing agent varies from the control in compounds 2B and 3C. There is no control for 3B, but it may be compared with 1B and 3A.
2.3 Testing
Except for the outdoor and controlled humidity aging, all testing was by ASTM methods [4]. Each SX containing compoupd had acceptable processing characteristics and gave vulcanized rubbers similar enough to its control to meet the same basic ASTM D 2000 (SAE J 200) line-call-out specification [4]. A detailed match in properties could have been obtained by using more than one type of SX-SBR masterbatch and making more extensive compounding changes. However, little additional information on the effect of weathering would have been obtained. No control was provided for the soft black compound, 3B, but it is compared with compound 3A in the tables (Table 2).
2.5 Outdoor Exposure
Specimens were spread out flat on 0.6 cm mesh wire 2.5 cm above floor level from May 22,1975, until May 24,1976, in a sheltered location and on a rooftop at Peoria, Illinois, USA. Specimens were not turned over during aging so that the down-side was not exposed to direct sunlight. The sheltered location was a concrete walk under an overhanging roof at the west end of a building. This location was protected from most precipitation and direct sunlight but exposed to driving rains and snows from the west, afternoon sunlight and the full variation of temperature and humidity. The rooftop was on a flat building about 10 m tall. Specimens at this location were exposed to full sunlight, precipitation and wind and were covered by snow for periods in winter.
2.4 Exposure at Constant Humidity 3 Results and Discussion
Glass hygrometric test chambers located in a 22.2 "C constant temperature room were maintained at various constant humidities by saturated aqueous salt solutions. Relative humidity levels were 0, 32, 43, 50, 66, 79, 89, 95 and 100%. Test pieces were stored in these chambers edgewise on a wire rack allowing free circulation of air around each one.
3.1 Effect of High Humidity
Tensile properties of all but the low-pigmented and the soft black rubbers were insensitive to humidity and the swelling at high humidity was low (Table 3).
92 Starch/Stiirke 30 (1978) Nr. 3, S. 91 -96
Table 2. Properties of Test Compounds.
Low-pigmented Highly filled Black molding Premium molding white molding tire tread
Measurement
Mooney viscosity, ML1+4 (100°C)
ODR Cure Characteristics (150°C) Minimum torque (J) Scorch time (min) Cure time (min)
Vulcanizate Properties Density (kg/m3) Hardness (IRHD) Rebound resilience (YO) Tear strength (die C, KN/m) Compression set (B, YO) Modulus, 100% elongation (MPa) Modulus, 300% elongation (MPa) Elongation at break (%) Tensile strength (MPa) Energy to break' ' Set at break, %
1A 1B Control SX
84 89
2.3 2.7 11.5 9.0 32.5 22
1194 1096 69 65 46 52 37 36 20.8 22.3 2.2 3.3 5.4 11.9
550 400 11.9 14.6
720 680 25 30
2A 2B 3A Control SX Control
3B sx
3C 4A 4B SX Control SX
27
1.7 7.7
100
1427 91 25 48 50.2 3.6 6.3
11.8 570
845 100
36
1.9 3.5
59
1397 91 25 48 35.2 7.3
11.8 315
12.1 49 5
55
26
2.0 3.8
25.5
1321 75 46 34 16.9 4.2 -
280 10.6
365 5
59.5
1.7 7.8
22.5
1129 58 44 43 25.9
2.4 10.1
520 16.4
995 30
86 29 68
2.7 2.3 2.2 2.8 6.4 8.5
14.5 23.5 23.5
1300 80 38 39 19.0 6.2 -
270 12.1
395 20
1149 61 41 53 18.9 2.1
12.1 525 25.6
1520 15
1149 58 47 47 25.7 2.1
10.2 580 23.8
1565 25
1) Area under the tensile stress-strain curve in arbitrary units.
Table 3. Effect of High Humidity on Rubber Properties.
Low-pigmented Highly filled white
Black Tire tread
Measurement 1A 1B 2A Control SX Control
2B 3A SX Control
Original Properties Modulus, 100% elongation (MPa) Elongation at break (Yo) Tensile strength (MPa)
After 90 Days at 50% Relative Humidity Volume increase (Yo) Modulus, 100% elongation (MPa) Elongation at break (Yo) Tensile strength (MPa) after 90 Days at 79% Relative Humidity Volume increase (Yo) Modulus, 100% elongation (MPa) Elongation at break (%) Tensile strength (MPa)
After 90 Days at 89% Relative Humidity Volume increase (%) Modulus, 100% elongation (MPa) Elongation at break ("4) Tensile stregth (MPa)
After 90 Days at 95% Relative Humidity Volume increase (%) Modulus, 100% elongation (MPa) Elongation at break (%) Tensile strength (MPa)
After 90 Days at 100% Relative Humidity Volume increase (Yo) Modulus, 100% elongation (MPa) Elongation at break (Yo) Tensile strength (MPa)
2.2
11.9
0.1 2.0
7.9
1.1 1.9
10.1
2.8 1.9
8.6
8.2 1.9
8.3
12.7 1 ,8
8.6
550
440
480
41 0
450
480
3.3
14.6
0.7 4.1
11.9
3.7 3.0
8.5
5.5 2.2
6.0
400
280
270
290
10.4 1.5
4.6
18.1 1.4
4.2
300
290
3.6
11.8
0.2 6.3
10.8
0.4 3.5
7.9
1.4 3.0
9.8
3.4 2.5
9.5
6.9 2.3
9.4
570
280
480
540
560
610
7.3
12.1
0 8.3
190 10.9
2.0 6.8
10.7
3.3 5.2
9.3
6.0 4.1
8.9
7.9 4.0
8.6
315
250
290
340
330
4.2
10.6
0 4.2
280 10.8
0.4 3.9
10.7
1.2 4.2
10.9
280
290
290
2.8 4.2
10.8
3.5 4.2
10.6
280
260
3B sx
2.4
16.4 520
0.6 2.8
17.2
3.1 2.3
13.7
4.5 1.9
13.2
8.4 1.2
8.0
460
450
490
420
15.1 1.2
7.9 430
3c 4A 4B SX Control SX
~
6.2
12.1
0.1 6.9
12.4
2.1 5.5
11.8
3.3 4.0
11.4
270
230
280
330
7.8 2.8
11.2
11.3 2.8
12.0
410
460
2.1 2.1
25.6 23.8 525 580
0.4 0.3 2.2 2.5
26.3 24.0 525 540
0.4 1.4 2.3 2.2
25.9 25.0 530 560
0.5 2.2 2.1 1.9
24.6 20.3 530 500
1.0 4.8 2.2 1.7
26.1 20.8 540 530
1.6 6.3- 2.1 1.7
24.7 22.6 540 550
~ -
Starch/Starke 30 (1978) Nr. 3, S. 91 -96 93
The low-pigmented rubbers swelled much more at high humidity than the other compositions (Table 3) and the SX reinforced rubber (1B) swelled appreciably more than the silica filled rubber (1A) (Fig. 1). Their response to high humidity was similar to their response to water immersion but much less severe depending on humidity level. Rubbers never reach an equilibrium swelling during prolonged storage at high humidity [2, 51, and for SX reinforced rubbers, the swelling is fully reversible [I].
1 Compound 11, Semi.reinforcing silica I Compound 1B. starch xanlhide 1
20 251
-0 20 40 60 80 0 20 40 60 80 Storage at 22.2"C ldaysl
.Figure 1. Swelling behavior of the low-pigmented rubbers.
Storage at high humidity also had a deleterious effect on tensile properties of the low-pigmented and soft black rubbers (Table 3). Strength and modulus of each fell rapidly upon exposure to high humidity, but the effect was much greater for the SX reinforced low-pigmented rubber than for any other (Fig. 2). Also, there was an interaction between time and humidity such that long exposure to a relative humidity as low at 70% had as great an effect as shorter exposures at higher humidities (Fig. 3).
m Tensile strength, I A
Modulus 1A
Modulus 1B 0
0' " " " " " 0 20 40 60 80 100
Storage a t 100% relative humidity and 2 2 . 2 " C [days]
Figure 2. Effect of prolonged storage in moisture saturated air on tensile properties of the low-pigmented rubbers. = semi- reinforcing silica filled, compound IA; @ = SX reinforced, compound 1B.
3.2 Outdoor Weathering
Only the low-pigmented rubbers (IA and 1B) deteriorated during the year in the sheltered location ; they deteriorated rapidly (Fig. 4). Within 2 months, both these rubbers
94
developed a hard skin and surface cracks, typical effects of sunlight on poorly protected diene elastomers. However, it is possible that swelling or other weather effect contributed to the predominant effect of actinic light degradation. SX offered no more protection than silica, but both are nearly transparent fillers in rubber and unable to screen the elastomer from light. For outdoor use, translucent bright- colored rubber articles would require a more suitable and adequately protected elastomer. In our experience, bright- colored articles from SX reinforced SBR have a good service life in indoor applications. ;;; 20, I
L I
I I I I I I 50 60 10 80 90 100
Relative humidity ( % I
Figure 3. Interactionoftimeand humidityon tensile properties ofthe SX reinforced low-pigmented rubber.
Outdoor e i p o s u r e (months)
Figure 4. Effect of outdoor exposure in a sheltered location on the low-pigmented rubbers. o = semi-reinforcing silica filled, com- pound I A ; 0 = SX reinforced, compound IB.
The rooftop exposure was slightly more severe toward rubber properties than the sheltered location, and the difference might have increased with a longer time. Properties of both highly filled white SBR rubbers (2A and 2B) deteriorated somewhat during 1 year on the rooftop (Fig. 5) . As with the low-pigmented rubbers, these changes were apparently entirely due to the action ofsunlight at the rubber surface. However, the opaque fillers in these compositions were more effective in screening the elastomer from light so that deterioration was slower. Stabilization of these com- pounds could have been obtained by including appropriate antidegradants. SX was a desirable substitute for reinforcing silica (Hi-Sil) in this rubber compound, compare 2A and 2B, Tables 1 and 2.
StarchlStarke 30 (1978) Nr. 3, S. 91 -96
= Outdoor e x p o s u r e Imonthsl
Figure 5. Effect of rooftop exposure on the highly filled white rubbers. o = silica reinforced, compound 2A; = SX reinforced, compound 2B.
Properties of black rubbers (3A, 3B and 3C) also changed during the year on the rooftop (Fig. 6). The largest reduction in tensile properties occurred with the black and oil-extended soft rubber where SX was the main reinforcing agent (3B). However, properties did not fall below an acceptable level for this rubber and remained at about the level for highly loaded black rubbers. Changes in tensile properties of the soft black rubber did not appear to result from humidity or wetting, since there was a concurrent slight increase in modulus indicating that swelling of SX was not the factor. Probably, the action of sunlight was again reponsible. SX replaced a substantial part of the carbon black in general-purpose molding compounds without seriously affecting age and weather resistance.
Figure 6. Effect of rooftop exposure on the black rubbers. 0 = highly loaded hard black control, compound 3A; 0 = soft carbon black-oil extended SX reinforced, compound 3B; = highly loaded SX reinforced, compound 3C.
The control tread rubber (4A) remained unchanged through 8 months of rooftop exposure, then deteriorated with surface cracking (Fig. 7). The tread rubber with SX gave rather erratic tensile strength values possibly reflecting some influence of rain or other weather variable. However, its properties never fell below a satisfactory level and it did not surface crack as the control did. SX has been demonstrated in actual service to be satisfactory in some tire treads, replacing 50% of the carbon black [6].
Starch/Starke 30 (1978) Nr. 3, S. 91 -96
Outdoor expQSUte l m o n l h s i
Figure 7. Effect of rooftop exposure on the tire tread rubbers. 0 = conventional premium tire tread, compound 4A; 0 = premium tire tread with one-third replacement of carbon black by SX, compound 4B.
There was no sign of microbiological attack on any of the specimens, either outdoors or in controlled humidity.
4 Conclusions
Where the level of SX was high and the level ofauxiliary fillers low, SX reinforced rubbers swelled and softened somewhat more than comparable conventional rubbers under the influence of prolonged exposure to high humidity. However, the effect of high humidity on SX reinforced rubber was less severe than the effect of water immersion, dependent on the size and shape of the test specimen and fully reversible [I]. Thus, high humidity acting alone would not be likely to reduce the service life of well-designed rubber articles from practical compounds containing SX and auxiliary fillers. In outdoor weathering of practical SX reinforced rubbers, sunlight and ozone were more important in causing degradation than humidity or wetting. In low-pigmented rubbers, SX and silica were alike in that both lacked ability to screen the elastomer from light and allowed rapid deteriora- tion. In highly filled white rubbers, SX was satisfactory as a replacement for reinforcing silica. In black general-purpose molding compounds, SX served well as a partial replacement for carbon black with little effect on humidity or weather resistance. Formulated for replacement of one-third of the black with SX, a premium tire tread had slightly better outdoor weather resistance than its control. These results confirm our previous observations implying that well-designed rubber articles from properly formulated SX reinforced SBR would provide satisfactory service in demanding applications involving exposure to high humidity, water immersion or outdoor weathering. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.
References
[ I ] Buchanan, R. A , : Stiirke 26 (1974), 165. [2] Buchanan, R. A, , W. M . Dome, C. R. Russelland W . F . Kwolek: J.
[3] Buchanan, R. A. , H . L. Seckinger, W.F. Kwolek, W. M . Doaneand Elast. Plast. 7 (1975), 97.
C. R. Russell: J . Elast. Plast. 8 (1976), 82.
95
[4] American Society for Testing and Materials: Annual Book of ASTM Standards Part 28, Philadelphia, Pennsylvania (USA)
from the authors, Independence Technical Center, B. F. Goodrich Chemical Company, Independence, Ohio 44131.
1972. [ 5 ] Rayuskc , p'. G,, E , A . zhiuoua, R, M , p'a/asenin and p', ye, Gul':
Polym. sci. USSR 12 (1970), 9; Vysokomol, soyed, ~ 1 2 (1970), 10.
[6] Mule, F . J . , R. W. Hullrnann and D. W. Wright : "Production and Evaluation of Starch-Reinforced Rubbers", manuscript available
Address of authors: R. A . Buchanan, F . H . Otey and C . R. Russell, Northern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 61604 (USA); John McBrien, 306 Capri Street, Pekin, Illinois, 61 554 (USA). (Received: July 12, 1977)
Rate of Starch-Hydrolysis by Bacterial Amylase By H. M. El-Saied, Y. Ghali and S. Gabr, Cairo
The rates of starch-hydrolysis by different bacterial amylases were compared using the digital oscillator densimeter technique. Reaction rates of amylase determined by this method were evaluated using the standard method for amylase activity.
Geschwindigkeit der Starkehydrolyse durch Bakterien-Amylase. Die Geschwindigkeiten der Starkehydrolyse durch verschiedene Bakte- rien-Amylasen wurden unter Verwendung des Digital-Oszillator- Densimeterverfahrens miteinander verglichen. Die durch diese Methode ermittelten Reaktionsgeschwindigkeiten wurden unter Einbeziehung der Standardmethode fur die Amylaseaktivitat ausgewertet.
1 Introduction Amylases are enzymes which catalyze the hydrolysis of 1.4-a- glucosidic links in polysaccharides such as starch, glycogen, amylose and amylopectin, or their partially hydrolyzed degradation products. Amylases are produced by a number of different microorganisms, but the most important sources are Bacillus subtilis and Aspergillus oryzae. B. subtilis amylases are relatively heat stable and can be used at high temperature [I]. Amylases are useful in theindustrial applications in which the hydrolysis of starch is carried out [2,3,4] such as baking, cereals, cocoa, paper, textiles, and pharmaceuticals. Glucose is now produced by enzymatic breakdown of starch using amylase plus amyloglucosidase [5, 61. As it is well known, the reaction rate of an enzyme-catalyzed reaction is determined by analysis either for the amount of product formed or for the amount of suhtrate which reacted in a given time period [7]. In this work, the reaction rate of bacterial amylase catalyzed reaction was measured by determination of the rate of starch hydrolysis using the digital oscillator densimeter technique which was an easily measurable quantity.
2 Experimental
2.1 Materials
Rhozyme H-39, a thermostable bacterial amylase produced as powder from B. subtilis, which is used as food grade for starch liquefaction at high temperature and was obtained from Rohm and Haas Company, Philadelphia (USA), Thermamyl L-60, a thermostable bacterial amylase produced as liquid (31.22% total solids) from B. licheiziformis, which is used for starch liquefaction at high temperature, and was obtained from Novo Industry A/S, Bagsvaed (Denmark), BAN L-120, a bacterial amylase produced as liquid (36.61 % total solids) from B. subtilis, which is used for starch liquefaction and was obtained from Novo Industry A/S, Bagsvaed (Denmark), Nasr Amylase produced as powder
from B. subtilis, which is used for textiles and was obtained from El-Nasr Pharmaceutical Company Abo-Zabaal, Cairo (Egypt). Soluble starch was supplied from E. Merck Company (FRG).
2.2 Methods
The determination of the rate of amylase-catalyzed reaction was based on the change of density of the starch-enzyme mixture which was measured by digital densimeter DMA 02C manufactured by A. Paar, Graz (Austria) [8c, 121. The principle of the technique lies in the fact ,that hydrolysis of starch solution by amylase takes place in the presence ofwater and the volume of the starch-amylase mixture decreases, Thus, density increases according to the reaction :
nC6H1 2'6 +"KO
(C6H1005)n Amylase + Amyloglucosidase> . -
(Starch) (Glucose)
The starch used was dispersed in boiled distilled water (of pH value 7.0), to a concentration of 5% and preincubated at 40°C. The enzyme preparation was dissolved in distilled water (of pH value 7.0) by using a magnetic stirrer for 30 min [13] and preincubated at 40 "C. Distilled water was adjusted to pH value 7 with O.~-N NaOH. The dried oscillator was filled with a mixture of 5 ml starch solution and 1 ml enzyme dilution. The first density measurement was carried out after 8 min which was required for thermal equilibrium. The increase in density was measured periodically at intervals of 0.5 min for 30 min. The density was calculated according to the equation : d = At2 + B, where t was the period in seconds of the oscillor filled with the sample. A and B were constants determined by two calibration measurements using air and distilled water which are the best choice standards of known density [Sb]. Accurate data on the densities of these two substrates are available over a wide rangeoftemperature [l 11. The dependence of density on the time of the enzymic reaction using different enzyme concentrations was plotted by means
96 Starch/Starke 30 (1978) Nr. 3, S. 96 -98 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1978