ZbI. Bakt. II. Abt., Bd. 133, S. 115-120 (1978) [Agricultural Microbiology Dept., College of Agriculture, Ain Shams University, Cairo, Egypt] Utilization of Industrial and Agricultural By-Products for Fungal Amylase Production S. A. Z. Mahmoud, A. M. Abdel-Hafez, W. A. Mashhoor and A. A. A. Refaat Summary Attempts were made for using industrial and agricultural by-products and wastes as carbon and nitrogen sources in fermentation medium for (X-amylase production by Aspergillus niger NRRL-337. The original carbon source of the basal medium was replaced by one of the following materials: rice bran, wheat bran, corn bran, corn starch, cane molasses, and glucose syrup. Rice bran proved to be the best carbon source that secured the highest amylase activity. The nitrogen source of the basal medium was then replaced by different cheap materials, viz: dried yeast, corn steep liquor, gluten-30, gluten-50, and corn steep precipitate. Corn steep precipitate proved to be superior in amylase produc- tion. In consideration of these results an economical medium that secured high activity, containing the following ingredients, was suggested: 2.5 % corn steep precipitate, 7.2 % rice bran, 0.1 % MgS0 4 , 0.1 % KH 2 P0 4 , and 0.1 % CaC0 3 • From this medium fungal amylase was precipitated and purified. The pure enZj'me gave the highest activity at 40°C and pH 4.3. Zusammenfassung Es wurde die Verwendung von Riickstanden aus Industrie und Landwirtschaft als Kohlen- und Stickstoffquelle zur Produktion von (X-Amylase durch niger NRRL-337 gepriift. Die Kohlenstoffquelle der urspriinglichen Nahrlosung wurde durch folgende Materialien ersetzt: Reis- kleie, Maiskleie, Maisstarke, Zuckerrohrmelasse und Glukose-Sirup. Reiskleie envies sich als giinstigste C-Quelle und erbrachte die hochste Amylaseaktivitat. Die Stickstoffquelle der urspriinglichen Nahr- losung wurde ersetzt durch: Trockenhefe, Maisextrakt, Gluten-30, Gluten-50 und Maisextrakt- Prazipitat. Letzteres ergab die beste Amylaseproduktion. Die besten Ergebnisse wurden mit folgender Nahrlosung erzielt: 2,5 % Maisextrakt-Prazipitat, 7,2 % Reiskleie, 0,1 % MgS0 4 , 0,1 % KH 2 P0 4 und 0,1 % CaC0 3 • Das reine Enzym ergab die hochste Aktivitat bis 40°C und bei pH 4,3. Although purified ingredients are readily fermentable, they are too expensive when economical enzyme yield was sought. From the economical point of view, the enzyme production necessitates the use of cheap carbon and nitrogen sources, such as agricul- tural and industrial waste products. FENIKSOVA and DVADTSATOVA (1960), YAMADA and (1966), and VOINO et al. (1969) recommended the utilization of different cheap nutritional sources, such as wheat bran, corn steep liquor, soybean flour, and corn flour for the production of fungal amylases. However, it is obvious that the major qualifications in selecting raw materials have to be loca.l products, cheapness, few impurities, and small troubles with ma.nufacturing processes. S'
Transcript
ZbI. Bakt. II. Abt., Bd. 133, S. 115-120 (1978)
[Agricultural Microbiology Dept., College of Agriculture, Ain Shams University, Cairo, Egypt]
Utilization of Industrial and Agricultural By-Productsfor Fungal Amylase Production
S. A. Z. Mahmoud, A. M. Abdel-Hafez, W. A. Mashhoor and A. A. A. Refaat
Summary
Attempts were made for using industrial and agricultural by-products and wastes as carbon andnitrogen sources in fermentation medium for (X-amylase production by Aspergillus niger NRRL-337.The original carbon source of the basal medium was replaced by one of the following materials: ricebran, wheat bran, corn bran, corn starch, cane molasses, and glucose syrup. Rice bran proved to bethe best carbon source that secured the highest amylase activity. The nitrogen source of the basalmedium was then replaced by different cheap materials, viz: dried yeast, corn steep liquor, gluten-30,gluten-50, and corn steep precipitate. Corn steep precipitate proved to be superior in amylase production.
In consideration of these results an economical medium that secured high activity, containingthe following ingredients, was suggested: 2.5 % corn steep precipitate, 7.2 % rice bran, 0.1 % MgS04 ,
0.1 % KH2P04, and 0.1 % CaC03•
From this medium fungal amylase was precipitated and purified. The pure enZj'me gave thehighest activity at 40°C and pH 4.3.
Zusammenfassung
Es wurde die Verwendung von Riickstanden aus Industrie und Landwirtschaft als Kohlen- undStickstoffquelle zur Produktion von (X-Amylase durch Asper~illus niger NRRL-337 gepriift. DieKohlenstoffquelle der urspriinglichen Nahrlosung wurde durch folgende Materialien ersetzt: Reiskleie, Maiskleie, Maisstarke, Zuckerrohrmelasse und Glukose-Sirup. Reiskleie envies sich als giinstigsteC-Quelle und erbrachte die hochste Amylaseaktivitat. Die Stickstoffquelle der urspriinglichen Nahrlosung wurde ersetzt durch: Trockenhefe, Maisextrakt, Gluten-30, Gluten-50 und MaisextraktPrazipitat. Letzteres ergab die beste Amylaseproduktion.
Die besten Ergebnisse wurden mit folgender Nahrlosung erzielt: 2,5 % Maisextrakt-Prazipitat,7,2 % Reiskleie, 0,1 % MgS04 , 0,1 % KH2P04 und 0,1 % CaC03•
Das reine Enzym ergab die hochste Aktivitat bis 40°C und bei pH 4,3.
Although purified ingredients are readily fermentable, they are too expensive wheneconomical enzyme yield was sought. From the economical point of view, the enzymeproduction necessitates the use of cheap carbon and nitrogen sources, such as agricultural and industrial waste products.
FENIKSOVA and DVADTSATOVA (1960), YAMADA and TO~IODA (1966), and VOINOet al. (1969) recommended the utilization of different cheap nutritional sources, suchas wheat bran, corn steep liquor, soybean flour, and corn flour for the production offungal amylases. However, it is obvious that the major qualifications in selecting rawmaterials have to be loca.l products, cheapness, few impurities, and small troubles withma.nufacturing processes.
S'
116 S. A. Z. MAHMOUD, A. M. ABDEL-HAFEZ, W. A. MASHHOOR and A. A. A. REFAAT
Therefore, the present work was focused on the utilization of different industrialand agricultural waste materials, locally produced in Egypt, as carbon and nitrogensources for the production of fungal lX-amylase by Aspergillus niger NRRL-337.
Materials and Methods
Culture
The organism used was A8pergillus niger NRRL-337. The cultures were maintained in a refrigera.tor on Czapek's agar medium after incubation at 30°C for 7 days. Fermentation was carried out in250 ml Erlenmeyer flasks. Each flask contained 50 ml of the basal medium (MAHMOUD et al., underpublication), unless otherwise stated. Two per cent of fungal spore suspension were used as an inoculum. The fermentation flasks were shaken on a reciprocating shaker at 30°C for 5 days. All experiments were performed in duplicate. At the end of the incubation period, the contents of each flaskwas centrifuged at 4000 rpm for 15 minutes to obtain a clear enzyme liquor.
Media
1. Czapek's agar medium for maintenance of cultures is after AINSWORTH and BISBY (1945). Itconsisted of the following composition: NaN03, 0.3; K 2HP04, 0.1; KCl, 0.05; MgS04 • 0.05; FeS04,
0.001; sucrose, 3.0; agar, 1.5; pH 5.5.2. Basal medium. Its composition was as follows: dextrin, 3.0; yeast extract, 2.0; MgS04, 0.1;
CaC03 , 0.1; KH2P04 , 0.1.
Amylase activity
The activity of fungal amylase was estimated according to the method of SKB (SANDESTEDT et aI.1939) after having been modified by Novo (1967).
Fractional precipitation of the enzyme
To known aliquots of the clear filtrate, ammonium sulfate was added in different concentrationsi.e. 10, 20, 30, 40, 45, 50, and 60 % (w/v) by dripping and continuous stirring, using an electro-mecha·nical stirrer. Each treatment was centrifuged (4000 rpm/20 min) and the precipitate was dissolvedin distilled water to a standard volume to give a fair comparison between each treatment.
Purification of the enzyme
The purification of the fungal amylase was carried out according to TAHA et al. (1967).
Results and Discussion
Carbon source
The carbon source of the basal medium (dextrin) was replaced by different cheapraw materials, namely rice bran, wheat bran, corn bran, corn starch, cane molasses,and glucose syrup. The amount of each carbon compound, added to the basal medium,was calculated to give a final concentration of 1.32 % (on carbon base). Thus, theamount of carbon was fixed for the test organism regardless of the source to eliminateerrors that might occur as a result of differences in carbon concentration of each individual source.
Results recorded in Table 1 clearly show that rice bran gave the highest yield ofenzyme, being 528.3 Nu/ml (Novo unit per millilitre). This was followed in decreasingorder by the other tested sources. It should be noticed that the fungal amylase activityin the rice bran medium was slightly higher than that obtained in the basal mediumwhich contained pure dextrin as carbon source. Therefore, the addition of rice bran
Utilization of Industrial and Agricultural By-Products for Fungal Amylase Production 117
can be recommended as an economical carbon source for higher amylase activity byAspergillus niger NRRL 337. However, the results confirm the findings of TARA et al.(1968) who found that rice bran gave the highest dextrogenic and saccharogenic en.zymes.
Nitrogen source
The original nitrogen source in the basal medium (yeast extract) was replaced bydifferent cheap industrial and agricultural wastes and by-products, viz: dried yeast,corn steep liquor, gluten-30, gluten-50, and corn steep precipitate. These materialswere added individually to the medium at a concentration of 0.2 % of nitrogen.
Results in Table 2 show that there is no significant difference between the basalmedium and corn steep precipitate medium for amylase formation. Both have securedthe highest yield of amylolytic activities, being 511.8 and 504.7 Nujml, respectively.This was followed in decreasing order by corn steep liquor (352.2 Nujml). Other testedsources of nitrogen gave varying degrees of enzyme activities, but generally lowerthan the above sources. Such results can be attributed to the chemical compositionof corn steep precipitate which may cover all requirements of the tested organism;beside the presence of growth factors that stimulate fungal amylase formation. Whileother tested materials may lack these essential ingredients in available forms.
From the above-mentioned data, an experiment was conducted for investigatingthe effect on amylase activity by using a modified medium containing cheap materialsthat had proved to be the most suitable carbon and nitrogen sources. It consisted of2.5 % corn steep precipitate, 7.2 % rice bran, 0.1 % CaCOa, 0.1 % MgS04, and 0.1 %KH2P04'
The enzyme activity was determined periodically up to 6 days. Results recordedin Table 3 clearly show that the maximum activity of amylase reached 475.5 Nujmlafter five days of incubation.
Table 1. Effect of replacing original carbon source by different industrial and agricultural by.products on the production of amylase by A. niger-337
Table 2. Effect of replacing original nitrogen source by different industrial and agricultural by·products on the production of amylase by A. niger-337
118 S. A. Z. MAHMOUD, A. M. ABDEL-HAFEZ, W. A. MASHHOOR and A. A. A. REFAAT
Since the utilization of industrial and agricultural wastes (corn steep precipitateand rice bran) gave relatively higher yields of amylolytic enzymes, i.e., almost similarto those produced when utling pure sources, the utilization of these cheap sources canbe recommended in fungal amylase production.
Table 3. Effect of rice bran - corn steep precipitate medium on amylase production at differentincubation periods
Incubation period(days)
23456
Amylase activity(Nujml)
105.7317.0440.3475.5297.2
Fractional precipitation of the enzyme
Ammonium sulfate was used at different concentrations for exploring the mostsuitable concentration that secures highest enzyme recovery and activity. Resultsrecorded in Table 4 show that amylase activities increase with the increase of ammonium sulfate concentration, reaching a peak at 45 %(NH4)2S04' after which the activities of the enzyme were stable.
Table 4. Fractional precipitation of fungal amylase by (NH4)2S04
The extraction and purification of the fungal amylase may be summarized as follows:
1. From the above-mentioned experimentation, it was found that 45 % of ammonium sulfate, used for precipitating the enzyme, was the best concentration. Therefore that concentration was used for the precipitation of the enzyme. Ammoniumsulfate was added at a concentration of 20 %while stirring for 15 minutes. The precipitate was discarded by centrifugation at 4000 rpm for 30 minutes. The clear solution,vas then adjusted to pH 7.0.
2. Ammonium sulfate was then added to raise the concentration to 45 % withmechanical stirring for 30 minutes. The precipitate was dissolved in 0.07 M of sodiumacetate.
3. The cold-dried acetone was added to the afore-mentioned enzyme solution togive a final concentration of 2.5 volume. Then the precipitate was collected by centrifugation and re-dissolved in distilled water.
Utilization of Industrial and Agricultural By-Products for Fungal Amylase Production 119
4. To avoid the interference of sulfate ions in the chemical analysis of the enzyme,they were replaced by acetate ions by passing the enzyme solution through a columncontaining anion exchange resin Amberlite IRA-400 that had been previously chargedwith acetate and washed with distilled water. The resin was maintained in a glass tube(6 inches long and 3/4inch inside diameter) between two cotton plugs.
5. In order to concentrate the enzyme solution and simultaneously to remove thesalt, acetone was added dropwise, using an electromagnetic stirrer (15 minutes). Theenzyme was centrifuged and the supernatant fluid was discarded.
6. The enzyme was dried on phosphorus pentoxide under vaccum at 40 °e.
Results recorded in Table 5 clearly show the above mentioned steps.
Table 5. Purification of amylolytic enzyme
Procedure Volume Enzyme Totalactivity (Nu/ml)
Yield %
Initial extractClarification(NH4)zS04 (ppt.)Acetone (ppt.) IAcetone (ppt.) II
10001000
1005050
510.4510.4
5080.08560.07564.0
510.4510.4508.0428.0378.2
100.0100.099.583.874.1
By applying these steps of purification, amylase was obtained in highly purifiedstate.
The final product was used for further studies, i.e., effect of temperature and pHon the enzyme activity.
Effect of temperature
The effect of temperature on the reaction velocity of the purified enzyme wascarried out by the addition of a known quantity of the enzyme to aliquots of the sub·strate and subjecting to different temperatures, ranging from 20 °0 to 70 °0as can beseen in Table 6. A gradual increase in the activity of the enzyme can be observed. Theactivity reached its maximum at 40 °0, then it began to drop.
Table 6. Effect of temperature on pure amylase activity
Temperature °C 20
Amylase activity (Nu/ml) 264.1
30
317.0
40
528.3
50
352.2
60
55.3
70
0.0
Effect of pH
In this experiment a range of pH values from 2.21 to 10.35 was used. The bufferedsubstrates were prepared by mixing 10 m! of 1.4 % soluble starch (Merck) solutionwith 10 ml of buffer solution, according to DELONY and KING (1945).
Table 7 shows the activity of enzyme reaction at different pH values. It can gener·ally be seen that the maximum activity was observed at pH 4.3.
120 S. A. Z. MAHMOUD et. aI., Utilization of Industrial and Agricultural By-products
Table 7. Effect of pH value on the amylase activity
pH value
Enzyme activity(Nu/ml)
References
2.21 3.13
0.0 99.3
3.80 4.30
317.0 452.8
5.30 6.10 7.20
270.8 201.4 79.2
8.12
19.6
9.30 10.35
0.0 0.0
AINSWORTH, G. C., and BISBY, G. R.: A dictionary of the fungi. The Imperial Mycological Institute,2nd, Kew, Surrey, England, 1945.
DELONY, G. E., and KING, E. J.: Carbonate and bicarbonate buffer. Biochem. J. 39 (1945), 245.FENIKSOVA, R. V., and DVADTSATOVA, E. A.: Selection of fermentation media for high yields of amylo
lytic enzymes. Trudy Tsentral. Nauch. Issled. 9 (1960), 3-6.MAHMOUD, S. A. Z., ABDEL-HAFEZ, A. M., ATTIA, R. M., MASHHOOR, W. A., and REFAAT, A. A. A.: A
suggested medium for higher amylase production by Aspergillus niger. (under publication).Novo INDUSTRIALS: Modified Lohlein Volhard Method, Copenhagen, Denmark, 1967.SANDESTEDT, R. M., KNEEN, E., and BLISH, M. J.: A standardized Wohlgemuth procedure for alpha
amylase activity. Cereal Chemistry 16 (1939), 712-723.TARA, S. M., MAHMOUD, S. A. Z., and A=IA, R. M.: Purification and chemical composition of alpha
amylase from B. subtilis (s-3217), Egyptian strain. J. Bot. U.A.R. 10 (1967), 25- 32.- - and AT'J'IA, R.A.: Factors influencing amylase production by local strain of B. subtilis. J. Bot.
U.A.R. 11 (1968), 49-58.VOINO, L. 1., VESELOV, 1. YA., GRACHEVA, I. M., and LEVCHIK, L. A.: Enzyme biosynthesis by A.
oryzae 8f during submerged cultivation. Prikl. Biochim. Mikrobiol. 516 (1969), 668-672.YAMADA, N., and TOMODA, K.: Alpha amylase production. U.S. 3 (1966),293; c.r. Chern. Abstr. 66
(1967), 54264v.
Author's address:
Prof. Dr. S. A. Z. MAHMOUD, Agricultural Microbiology Dept., College of Agriculture, Ain ShamsUniversity, Cairo, Egypt.
