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Utilization of orange peels for the production of multienzyme complexes by some fungal strains
Process Biochemistry, Vol. 31, No. 7, pp. 645-650, 1996
Copyright © 1996Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00+0.00 ELSEVIER
PI I : S 0 0 3 2 - 9 5 9 2 ( 9 6 )
00012-X
Utilization of Orange Peels for the Production of Multienzyme Complexes by Some Fungal Strains Abdel-Mohsen Saber Ismail Natural and Microbial Products Chemistry Department, National Research Centre, Dokki, Cairo, Egypt (Received 13 October 1995; revised manuscript received 4 January 1996 and accepted 21 January 1996)
The production of multienzyme preparations containing pectinase, cellulase and xylanase enzymes was investigated using six fungal isolates grown on orange peels as the sole carbon source. Of the fungal isolates tested, Aspergillus niger A-20 proved to be the most potent and produced highly active multienzyme systems after 5 days in shaken cultures. These preparations included active induced pectinase, cellulase and xylanase enzymes with insignificant levels of amylase and lipase activities. This suggests potential uses in the extraction of the major components (starches and lipids) of plant materials. The optimum conditions for the activities of this enzyme complex were pH 4.0-5.0 and temperature 45-50°C. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
of the National Research Centre, Cairo, Egypt. Aspergillus oryzae 1911 and Penicillium chrysogenum 3486, were kindly provided by Dr S. W.
In Egypt, large amounts of fresh orange peels are produced annually as by-products from the orange canning industry. The disposal of the fresh peels remaining after orange manufacture is becoming a major problem to many factories. The present work therefore aims at the utilization of orange peels through its microbial biodegradation for the production of useful enzymes, including pectinases (EC 3.2.1.15, 3.2.1.67 and 3.2.1.82), cellulases (EC 3.2.1.4., 3.2.1.91, 3.2.1.74 and 3.2.1.21) and xylanases (EC 3.2.1.37 and 3.2.1.8). Some optimum conditions for enzyme activities are also investigated.
Peterson of the US Department of Agriculture and Development Division, Peoria, IL, USA (NRRL). Orange peels A number of similar orange fruits (Citrus sinensis var. Balady) were obtained from a local market in summer 1992. Pigmented peels were separated from some fruits while the thin pigmented skins from others were removed with a suitable grater and the unpigmented peels separated. Peels were cut into small pieces, washed in tap water several times, dried at 50°C and finally milled.
MATERIALS AND METHODS Pectin Pure citrus pectin was obtained from the EINasr Company for Preserved Foods (Kaha), Kalyubia, Egypt.
Microorganisms The fungi used in this work were obtained from the culture collection of the Centre of Cultures 645
646
A.-M. S. Ismail
Media
Czapek Dox's agar medium was used for culture maintenance and stock cultures. Growth enhancement medium was used for preparation of activated fungal inoculum and had the following composition (g/litre): glucose, 10; peptone, 5.0; yeast extract, 1; MgSO4, 0"5; KH2P04, l'0. The following culture media (g/litre) were used in the present investigation. Medium 1 (Abdel-Fattah et al. 1) contained (g/litre): citrus pectin, 15; KH2PO4 , 0"5; MgSO4, 0"5; KNO3, 2.5. Media 2 and 3 had the same composition as medium 1, but pectin, in equal basis, was substituted with dry grated peels (72-2, g/litre) and dry pigmented peels (61.04 g/litre), respectively. Moisture and ash content
A known weight of the sample (dry peels) was heated to 105°C to constant weight; the values were calculated on dry basis. Ashing was done in muffle furnace at 600°C. Preparation of corn cobs hemicellulose
This was carried out according to the method of Chen & Anderson. 2 Cultivation
Stock cultures were subcultured on Czapek Dox's agar slopes, incubated at 30°C for 7 days and then stored on a refrigerator at 4°C. Cultivation was carried out in 250 ml Erlenmeyer flasks, each containing 50 ml of sterile medium. 2.5 ml of mycelial suspension was used as inoculum. In some cases the culture flasks were incubated at 30°C on a rotary shaker at 200 rpm for different periods (3 and 5 days). In other cases, surface cultures were performed at 30°C for 4 and 7 days. Isolation of the crude multienzyme preparation
At the end of the incubation period, the culture broth was filtered off to separate mycelium from filtrate, this filtrate was centrifuged in a refrigerated centrifuge (1500g). The transparent culture filtrate was taken, as the multienzyme preparation, for different investigations. In shaken cultures with media 2 or 3 the mycelium and peel residue are associated together. Protein estimation and enzyme assays
Estimation of protein was carried out either by the Micro-Kjeldahl3 or Lowry method. 4
Pectinase activity was expressed as percentage reduction in viscosity of 0.5% buffered citrus pectin solution upon the enzyme action.1 Cellulase activity was carried out as filter paper-ase (FP-ase) activity according to the method of Mandels and Sternberg. 5 To 50 mg (1 × 6 cm) of Whatman No. 1 filter paper 1.0 ml of 0-05 M citrate-phosphate buffer (pH 4.8) and 1.0 ml diluted culture filtrate were added (the paper strip was coiled by touching the tube on to a vibratory mixer). The reaction mixture was incubated at 50°C for 60min. The released reducing sugars were determined by the method reported by Neish 6 and based on Somogyi7 and Nelson 8 methods. One enzyme activity unit was defined as the amount of enzyme that released 1/~mol of glucose per min under the assay conditions. Xylanase activity was determined according to the method described by Ismail et al. 9 One enzyme unit was defined as the amount of enzyme that released 1.0#mol xylose from xylan per min under assay conditions. a-Amylase activity was assayed by measuring the reducing sugars released during action on soluble starch according to the method of Bergmann et alJ ° T h e released reducing sugars (as glucose) were estimated with a Stanbio enzymatic glucose kit (GOD/POD) procedure No. 1075 and based on the method described by Trinder. t~ One enzyme unit was defined as the amount of enzyme which liberates 1.0 pmol of reducing sugars as glucose per min reaction under assay conditions. Lipase activity was assayed to the method of Parry et al. 12
RESULTS AND DISCUSSION
Chemical analysis of dry orange peels (grated and pigmented) revealed that the two samples were rich in pectin (21.5 and 25.6%), a-cellulose (16.98 and 21.71%) and hemicellulose (38.98 and 29.20%), while soluble materials comprised 20-7 and 22.2%, respectively. Low ash content was recorded (1-84 and 1.6%) in the same order. These values therefore justify the study of the utilization of peels by some fungal strains for the production of multienzyme complex containing pectinases, cellulases and xylanases. Some cellulose-rich plant residues including sugar cane bagasse (Abdel-Fattach et al. 13) and
Production of multienzyme complexes by fungi
wheat straw (Ismail et al. 9) were reported to be utilized by microorganisms for the production of cellulases and hemicellulases. The aforementioned authors also pointed out that these residues were devoid of pectin. The use of grated peels (medium 2) extensively favoured fungal growth, which reached maximal values in both static and shaken cultures. Pigmented peels (medium 2) also enhanced growth but only to a small extent. The synthesis of pectinases, cellulases and xylanases by all the fungal isolates tested depended on the presence of their substrates in the culture medium adopted. Using medium 1, none of the fungal isolates yielded cellulases, xylanases or amylases, indicating the inducible nature of these enzymes. The final pH of the culture depended on the fungal species and type of the culture. Culture filtrates of Aspergillus niger 2 and A. niger A-20, were acidic in all cases, while those of A. oryzae 1911 changed from acidic (pH 3.65) to alkaline (pH 8.1). The data also confirmed that the protein contents of the culture filtrates essentially depended upon the culture medium used. Media 2 and 3 afforded the highest protein level with all fungal strains tested. This may be attributed to biosynthesis of many enzymes (pectinases, cellulases, xylanases and sometimes amylases). It seemed that no consistent relationship existed between either the mycelial growth or the protein content of the culture filtrate and any of the investigated enzyme activities. The results recorded in Tables 1-4 indicated that the sequence of production of enzyme activities during incubation depended on the fungal strain, type and age of the culture and, above all, the culture medium used. Some
647
strains produced highly active pectinase in shaken cultures while static cultures were more suitable for the other strains. The alkalinity of the culture filtrate had the most adverse effect on pectinase activity and all alkaline filtrates possessed moderate or feeble pectinase activity. This is in accord with other reports on pectinase producing fungi (Abdel-Fattah et al.~). Of all the strains tested A. niger 2 and A. niger A-20 produced the most potent pectinases in shaken cultures with all media used. Pectinase activity amounted to 57 and 56% by the two strains, respectively. These values are similar to that of the potent pectinase producer Trichoderma lignorum (Abdel-Fattah et al.~). Table 2 shows the productivity of cellulases by the six fungal isolates. AspergiIlus niger A-20 in 5-day shaken cultures using medium 2 produced the highest cellulase activity (4.39 U/ml enzyme) and yielded the highest saccharifying activity (94.8%) after 60 min reaction at 50°C. On the other hand Memmoniella sp. 6 and Penicillium oxalicum 7 also yielded active cellulases in 4-day static culture using medium 2 (3-39 and 2.39 U/ml enzyme) and 73 and 51.6% saccharification under the assay conditions, respectively. The data also show that medium 3 was unsuitable and had an adverse effect on cellulase productivity by all the fungal strains used. This provides conclusive evidence of the necessity for the removal of pigments which are barriers for cellulase attack on cellulose. Similar findings have been reported by many authors with other agricultural wastes containing lignin which inhibits cellulase activity on cellulose thus needs to be removed (Ghose & Ghose; ~4 Ryu & Mandels; ~5 Chang et al.16). Table 3 shows xylanase activities in different
Table 1. Pectinase activities (% reduction in viscosity of 0"5% buffered pectin solution) in different static and shaken fungal cultures following different incubation periods a
Microorganism
Medium 1 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P chrysogenum 3486 P oxalicum 7
Shaken
Medium 2 Static
Medium 3 Shaken
Static
Shaken
4-day
7-day 3-day 5-day 4-day 7-day 3-day 5-day 4-day 7-day 3-day 5-day
4.33 34"90 13.90 5.30 51.10 44.40
38.70 40-10 36.40 8.37 28.70 44"60
5 7 " 0 5 55.29 21-07 43.00 5 2 " 5 4 53.54 36"70 44"60 3.85 24.78 2 9 " 0 0 31.50 1 2 " 7 1 11-00 4.76 10.80 5-25 1 4 . 4 5 54.70 26-70 1"08 3.76 14.90 48"20
~For media composition see Materials and Methods section.
47.80 39.58 5"00 36"99 28.02 20-93
55"90 56"00 6-49 42.34 23"75 21.45
53.92 35-99 52"56 6"48 41"27 11.15
53.72 51.05 53-26 18.58 24-99 47.19
47.59 46.69 4 0 " 3 6 51"51 4.97 3"05 2.26 7.23 0-00 0.00 4.52 3.01
A.-M. S. Ismail
648
Table 2. Cellulase activities (a) (U/ml enzyme) and saccharification (b) (%) in different static and shaken fungal cultures following different incubation periods a
Microorganism
Medium 2
Medium 3
Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P chrysogenum 3486 P. oxalicum 7
a b a b a b a b a b a b
Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
1.03 22.20 1.28 27.60 1-05 22-70 3.39 73.20 0.92 19.90 2.39 51.60
0.26 5.62 0.64 13.80 0"58 12.53 1-29 27.78 1.24 26.80 0.73 15.77
0.380 8.260 0.810 17-500 0.044 0.960 2.760 59.500 0.098 2-110 1.090 26.060
0.12 2.59 4.39 94-80 0.17 3.72 1.02 22.00 1.84 39.84 0.08 1.75
0.000 0.000 0.000 0.000 0.000 0.000 0.026 0-550 0.000 0.000 0.167 3.600
0.000 0.000 0.017 0-360 0"028 0.600 0.039 0.840 0.290 6.400 0.000 0.000
0.128 2.760 0.160 3.460 0.002 0.480 0.017 0.360 0.011 0.238 0.034 0.730
0.133 2.880 0.330 7.200 0.240 5.280 0"078 1"690 0.000 0.000 0.039 0.840
"No cellulases detected with culture medium 1.
funiculosum 258 (Ismail et al. TM) for utilization of sugar cane bagasse. Screening of a-amylase activities in the fungal cultures (Table 4) revealed moderate activities in static cultures only using media 2 or 3, by A. oryzae 1911. It is well known that the aforementioned fungus is a good producer of diastase. Other cultures showed feeble or no amylase activities and this may be attributed to the diminished content of starch in orange peels and to the inducible nature of a-amylases. The data recorded in Table 5 indicated that the optimum conditions for enzymic activities are pH 4-5 and reaction temperature 45-50°C.
culture media. Of all the tested strains, A. niger A-20 in 3- and 5-day shaken culture using medium 2 yielded the highest xylanase activity (3.27 and 3.33 U/ml enzyme) and 78.6 and 80% saccharification under the assay conditions, respectively. On the other hand, A. niger 2 and P oxalicum 7 in 3- and 5-day shaken cultures, respectively, yielded good xylanase activity using culture medium 2 or 3. Medium 3 with other fungal isolates afforded weak xylanase activities. In this respect, the xylanase of A. niger A-20 is superior to those yielded by Trichoderma viride 253-M16 (Abdel-Fattah et al.~3), AspergiUus terreus 603 (Abdel-Fattah et all 7) and Penicillium
Table 3. Xylanase activities (a) (U/ml enzyme) and saccharification (b) (%) in different static and shaken fungal cultures following different incubation periods"
Microorganism
Medium 2 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P. chrysogenum 3486 P. oxalicum 7
a b a b a b a b a b a b
Medium 3 Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
0.087 2.090 0.000 0-000 0.000 0.000 0.000 0.000 0.000 0.000 0.029 0.700
0-420 10.090 0.840 25"100 0"000 0.000 0"186 5.590 1.480 44.500 0.093 2.780
2"45 58"81 3"27 78-56 0"54 12.88 0-42 10"09 0.00 0.00 1.33 32"03
0"58 13.92 3.33 80-00 0.71 17.05 1-33 32.02 0.46 11.05 2-05 49.24
0.000 0'000 0.000 0"000 0.493 11.830 0"000 0.000 0.186 6"590 0"000 0"000
0.000 0.000 0-000 0.000 0-000 0.000 0.000 0.000 0-217 5"220 0-000 0"000
1"040 25.040 1.130 27.140 0"435 10"450 1.043 25"060 0.000 0.000 0.695 16-700
2"40 59"00 0"00 2"00 1.10 27"80 0"40 11"80 0"50 12"50 1"80 44'50
"No xylanases detected with culture medium 1.
Production of multienzyme complexes by fungi
649
Table 4. a-Amylase activities (U/ml enzyme) in different static and shaken fungal cultures following different incubation periods"
,rvIicroorganism
Medium 2 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P. chrysogenum 3486 P. oxalicum 7
Medium 3 Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
0"220 0.890 0"070 0.050 0.190 0.005
0.460 0.460 0.380 0.070 0.340 0-006
0"02 0.04 0.00 0.10 0"00 0' 15
0-030 0.010 0-000 0.004 0"000 0.000
0'000 0.097 1"300 0.000 0.250 0.000
0"36 0.29 1.24 0"00 0.00 0.00
0-000 0.396 0.430 0.340 0.210 0.000
0"160 0.000 0.190 0.290 0.030 0.000
"No a-amylase activities detected with culture medium 1.
Mabrouk et al. 19 reported the optimum pH and temperature for pectinase produced by Trichoderma lignorum to be pH 4.45 and 40°C, respectively. On the other hand, Abdel-Fattah et al. 13 found that the optimum pH and temperature were 5.5 and 45°C for both cellulase and hemicellulase produced by Trichoderma viride 253 M-16. The data collectively indicated that the local fungal strain A. niger A-20 was the most potent and produced a highly active multienzyme preparation containing pectinase, cellulase and xylanase, besides feeble amylase activity, in a 5-day shaken culture using grated orange peels instead of pure pectin. In unrecorded data, very weak lipase activity was also found in this enzyme preparation.
Table 5. Effect of temperature and pH value of the reaction on the different activities of A. niger A-20 enzyme preparation
Pectinase Cellulase Xylanase (% reduction (U/ml (U/ml in viscosity) enzyme) enzyme) Reaction temperature (°C) 35 40 45 50 55
45 56 58 59 58
3.00 4"20 4.80 4.39 4"00
2"5 3-5 4"0 4.2 4.0
42 58 54 50 40
3"00 4-40 4.80 5"00 2.00
3"2 4.0 4-5 4.0 3'8
Reaction pH" 3.40 4.05 4.45 4-99 5.89
"Enzymatic reactions were conducted at optimum temperatures.
The crude lyophilized multienzyme of A. niger A-20 was a deep yellow colour, readily soluble in water and stable following storage over 2 years at room temperature. Accordingly, this enzyme preparation is, to a considerable extent, superior to Novo Nordisk multienzyme complex product, Viscozyme L, which is produced from selected strains of the Aspergilli. The optimum conditions for this enzyme complex are pH 3.5-5.5 and a temperature of 40-50°C and a loss in its activity of 1-2% per month may occur in storage periods of more than 3 months (Novo Nordisk2°). REFERENCES 1. Abdel-Fattah, A. F., Mabrouk, S. S. & Ismail, A. S., Production of polygalacturonase and pectin methylesterase by fungi. Chem. Mikrobiol. Technol. Lebensm, 5 (1977) 38-41. 2. Chen, W. P. & Anderson, A. W., Extraction of hemicellulose from ryegrass straw for the production of glucose isomerase and use of the resulting straw residue for animal feed. Biotechnol. Bioeng., 20 (1980) 519-31. 3. A.O.A.C., Official Methods of Analysis of Association of Official Agricultural Chemists. Washington D.C., 1970. 4. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265-75. 5. Mandels, M. & Sternberg, D., Recent advances in cellulase technology. J. Ferment. Technol., 54 (1976) 267-86. 6. Neish, A. C., Analytical Methods for Bacterial Fermentations. Report No. 46-2-3, National Research Council of Canada, Second Revision 34, 1952. 7. Somogyi, M. J., Determination of blood sugar. J. Biol. Chem., 160 (1945) 61-8. 8. Nelson, N. A., A photometric adaptation of the Somogyi method for the determination of glucose. J. BioL Chem., 153 (1944) 375-80. 9. Ismail, A. S., Abdel-Naby, M. A. & Abdel-Fattah, A.
650
10.
11. 12. 13.
14. 15.
A.-M. S. Ismail F., Production of hemicellulytic enzymes by fungi. Agricultural Wastes, 18 (1986) 283-8. Bergmann, F. W., Abe, J. & Hizukurt, S., Selection of microorganisms which produce raw starch degrading amylases. Appl. Microbiol. Biotechnol., 27 (1988) 443-6. Trinder, P., Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J. Clin. Pathol., 22 (2) (1989) 158-61. Parry, R. M., Chandan, R. C. & Shahani, K. M., Rapid assay for milk-lipase. Dairy Science, 49 (4) (1966) 356-60. Abdel-Fattah, A. F. et al., Utilization of Cane-Sugar Bagasses. Final Technical Report, National Research Centre, Cairo/US Department of Agriculture, Grant No. FG-EG-180, 1981, pp. 388-415. Ghose, T. K. & Ghose, P., Bioconversion of cellulosic substances. J. Appl. Chem. Biotechnol., 28 (1978) 309-20. Ryu, D. Y. & Mandels, M., Cellulases: biosynthesis
16. 17.
18.
19.
20.
and application. Enz. Microb. Technol., 2 (1980) 91-102. Chang, M. M., Chou, T. Y. & Tsao, G. T., Structure, pretreatment and hydrolysis of cellulose. Adv. Biochem. Eng., 20 (1981) 15-42. Abdel-Fattah, A. F., Abdel-Naby, M. A. & Ismail, A. S., Purification and properties of xylan-degrading enzyme from Aspergillus terreus 603. Biological Wastes, 20 (1987) 143-51. Ismail, A. S., Sary, H. H. & Abdel-Fattah, A. F., Production of xylanase by Penicillium funiculosum 258. Bull. Fac. Pharm. Cairo Univ., 32 (2) (1994) 299-302. Mabrouk, S. S., Abdel-Fattah, A. F. & Ismail, A. S., Preparation and properties of pectic enzymes produced by Trichoderma lignorum. Zbl. Bakt. II. Abt., 134 (1979) 282-6. Novo-Nordisk Ferment Ltd, Viscozyme L. Novo Nordisk A/5 Enzyme process Division, Product Sheet, 4 pp. Novo Alle, 2880, Bagsvaerd, Denmark, 1994.
Copyright © 1996Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00+0.00 ELSEVIER
PI I : S 0 0 3 2 - 9 5 9 2 ( 9 6 )
00012-X
Utilization of Orange Peels for the Production of Multienzyme Complexes by Some Fungal Strains Abdel-Mohsen Saber Ismail Natural and Microbial Products Chemistry Department, National Research Centre, Dokki, Cairo, Egypt (Received 13 October 1995; revised manuscript received 4 January 1996 and accepted 21 January 1996)
The production of multienzyme preparations containing pectinase, cellulase and xylanase enzymes was investigated using six fungal isolates grown on orange peels as the sole carbon source. Of the fungal isolates tested, Aspergillus niger A-20 proved to be the most potent and produced highly active multienzyme systems after 5 days in shaken cultures. These preparations included active induced pectinase, cellulase and xylanase enzymes with insignificant levels of amylase and lipase activities. This suggests potential uses in the extraction of the major components (starches and lipids) of plant materials. The optimum conditions for the activities of this enzyme complex were pH 4.0-5.0 and temperature 45-50°C. Copyright © 1996 Elsevier Science Ltd
INTRODUCTION
of the National Research Centre, Cairo, Egypt. Aspergillus oryzae 1911 and Penicillium chrysogenum 3486, were kindly provided by Dr S. W.
In Egypt, large amounts of fresh orange peels are produced annually as by-products from the orange canning industry. The disposal of the fresh peels remaining after orange manufacture is becoming a major problem to many factories. The present work therefore aims at the utilization of orange peels through its microbial biodegradation for the production of useful enzymes, including pectinases (EC 3.2.1.15, 3.2.1.67 and 3.2.1.82), cellulases (EC 3.2.1.4., 3.2.1.91, 3.2.1.74 and 3.2.1.21) and xylanases (EC 3.2.1.37 and 3.2.1.8). Some optimum conditions for enzyme activities are also investigated.
Peterson of the US Department of Agriculture and Development Division, Peoria, IL, USA (NRRL). Orange peels A number of similar orange fruits (Citrus sinensis var. Balady) were obtained from a local market in summer 1992. Pigmented peels were separated from some fruits while the thin pigmented skins from others were removed with a suitable grater and the unpigmented peels separated. Peels were cut into small pieces, washed in tap water several times, dried at 50°C and finally milled.
MATERIALS AND METHODS Pectin Pure citrus pectin was obtained from the EINasr Company for Preserved Foods (Kaha), Kalyubia, Egypt.
Microorganisms The fungi used in this work were obtained from the culture collection of the Centre of Cultures 645
646
A.-M. S. Ismail
Media
Czapek Dox's agar medium was used for culture maintenance and stock cultures. Growth enhancement medium was used for preparation of activated fungal inoculum and had the following composition (g/litre): glucose, 10; peptone, 5.0; yeast extract, 1; MgSO4, 0"5; KH2P04, l'0. The following culture media (g/litre) were used in the present investigation. Medium 1 (Abdel-Fattah et al. 1) contained (g/litre): citrus pectin, 15; KH2PO4 , 0"5; MgSO4, 0"5; KNO3, 2.5. Media 2 and 3 had the same composition as medium 1, but pectin, in equal basis, was substituted with dry grated peels (72-2, g/litre) and dry pigmented peels (61.04 g/litre), respectively. Moisture and ash content
A known weight of the sample (dry peels) was heated to 105°C to constant weight; the values were calculated on dry basis. Ashing was done in muffle furnace at 600°C. Preparation of corn cobs hemicellulose
This was carried out according to the method of Chen & Anderson. 2 Cultivation
Stock cultures were subcultured on Czapek Dox's agar slopes, incubated at 30°C for 7 days and then stored on a refrigerator at 4°C. Cultivation was carried out in 250 ml Erlenmeyer flasks, each containing 50 ml of sterile medium. 2.5 ml of mycelial suspension was used as inoculum. In some cases the culture flasks were incubated at 30°C on a rotary shaker at 200 rpm for different periods (3 and 5 days). In other cases, surface cultures were performed at 30°C for 4 and 7 days. Isolation of the crude multienzyme preparation
At the end of the incubation period, the culture broth was filtered off to separate mycelium from filtrate, this filtrate was centrifuged in a refrigerated centrifuge (1500g). The transparent culture filtrate was taken, as the multienzyme preparation, for different investigations. In shaken cultures with media 2 or 3 the mycelium and peel residue are associated together. Protein estimation and enzyme assays
Estimation of protein was carried out either by the Micro-Kjeldahl3 or Lowry method. 4
Pectinase activity was expressed as percentage reduction in viscosity of 0.5% buffered citrus pectin solution upon the enzyme action.1 Cellulase activity was carried out as filter paper-ase (FP-ase) activity according to the method of Mandels and Sternberg. 5 To 50 mg (1 × 6 cm) of Whatman No. 1 filter paper 1.0 ml of 0-05 M citrate-phosphate buffer (pH 4.8) and 1.0 ml diluted culture filtrate were added (the paper strip was coiled by touching the tube on to a vibratory mixer). The reaction mixture was incubated at 50°C for 60min. The released reducing sugars were determined by the method reported by Neish 6 and based on Somogyi7 and Nelson 8 methods. One enzyme activity unit was defined as the amount of enzyme that released 1/~mol of glucose per min under the assay conditions. Xylanase activity was determined according to the method described by Ismail et al. 9 One enzyme unit was defined as the amount of enzyme that released 1.0#mol xylose from xylan per min under assay conditions. a-Amylase activity was assayed by measuring the reducing sugars released during action on soluble starch according to the method of Bergmann et alJ ° T h e released reducing sugars (as glucose) were estimated with a Stanbio enzymatic glucose kit (GOD/POD) procedure No. 1075 and based on the method described by Trinder. t~ One enzyme unit was defined as the amount of enzyme which liberates 1.0 pmol of reducing sugars as glucose per min reaction under assay conditions. Lipase activity was assayed to the method of Parry et al. 12
RESULTS AND DISCUSSION
Chemical analysis of dry orange peels (grated and pigmented) revealed that the two samples were rich in pectin (21.5 and 25.6%), a-cellulose (16.98 and 21.71%) and hemicellulose (38.98 and 29.20%), while soluble materials comprised 20-7 and 22.2%, respectively. Low ash content was recorded (1-84 and 1.6%) in the same order. These values therefore justify the study of the utilization of peels by some fungal strains for the production of multienzyme complex containing pectinases, cellulases and xylanases. Some cellulose-rich plant residues including sugar cane bagasse (Abdel-Fattach et al. 13) and
Production of multienzyme complexes by fungi
wheat straw (Ismail et al. 9) were reported to be utilized by microorganisms for the production of cellulases and hemicellulases. The aforementioned authors also pointed out that these residues were devoid of pectin. The use of grated peels (medium 2) extensively favoured fungal growth, which reached maximal values in both static and shaken cultures. Pigmented peels (medium 2) also enhanced growth but only to a small extent. The synthesis of pectinases, cellulases and xylanases by all the fungal isolates tested depended on the presence of their substrates in the culture medium adopted. Using medium 1, none of the fungal isolates yielded cellulases, xylanases or amylases, indicating the inducible nature of these enzymes. The final pH of the culture depended on the fungal species and type of the culture. Culture filtrates of Aspergillus niger 2 and A. niger A-20, were acidic in all cases, while those of A. oryzae 1911 changed from acidic (pH 3.65) to alkaline (pH 8.1). The data also confirmed that the protein contents of the culture filtrates essentially depended upon the culture medium used. Media 2 and 3 afforded the highest protein level with all fungal strains tested. This may be attributed to biosynthesis of many enzymes (pectinases, cellulases, xylanases and sometimes amylases). It seemed that no consistent relationship existed between either the mycelial growth or the protein content of the culture filtrate and any of the investigated enzyme activities. The results recorded in Tables 1-4 indicated that the sequence of production of enzyme activities during incubation depended on the fungal strain, type and age of the culture and, above all, the culture medium used. Some
647
strains produced highly active pectinase in shaken cultures while static cultures were more suitable for the other strains. The alkalinity of the culture filtrate had the most adverse effect on pectinase activity and all alkaline filtrates possessed moderate or feeble pectinase activity. This is in accord with other reports on pectinase producing fungi (Abdel-Fattah et al.~). Of all the strains tested A. niger 2 and A. niger A-20 produced the most potent pectinases in shaken cultures with all media used. Pectinase activity amounted to 57 and 56% by the two strains, respectively. These values are similar to that of the potent pectinase producer Trichoderma lignorum (Abdel-Fattah et al.~). Table 2 shows the productivity of cellulases by the six fungal isolates. AspergiIlus niger A-20 in 5-day shaken cultures using medium 2 produced the highest cellulase activity (4.39 U/ml enzyme) and yielded the highest saccharifying activity (94.8%) after 60 min reaction at 50°C. On the other hand Memmoniella sp. 6 and Penicillium oxalicum 7 also yielded active cellulases in 4-day static culture using medium 2 (3-39 and 2.39 U/ml enzyme) and 73 and 51.6% saccharification under the assay conditions, respectively. The data also show that medium 3 was unsuitable and had an adverse effect on cellulase productivity by all the fungal strains used. This provides conclusive evidence of the necessity for the removal of pigments which are barriers for cellulase attack on cellulose. Similar findings have been reported by many authors with other agricultural wastes containing lignin which inhibits cellulase activity on cellulose thus needs to be removed (Ghose & Ghose; ~4 Ryu & Mandels; ~5 Chang et al.16). Table 3 shows xylanase activities in different
Table 1. Pectinase activities (% reduction in viscosity of 0"5% buffered pectin solution) in different static and shaken fungal cultures following different incubation periods a
Microorganism
Medium 1 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P chrysogenum 3486 P oxalicum 7
Shaken
Medium 2 Static
Medium 3 Shaken
Static
Shaken
4-day
7-day 3-day 5-day 4-day 7-day 3-day 5-day 4-day 7-day 3-day 5-day
4.33 34"90 13.90 5.30 51.10 44.40
38.70 40-10 36.40 8.37 28.70 44"60
5 7 " 0 5 55.29 21-07 43.00 5 2 " 5 4 53.54 36"70 44"60 3.85 24.78 2 9 " 0 0 31.50 1 2 " 7 1 11-00 4.76 10.80 5-25 1 4 . 4 5 54.70 26-70 1"08 3.76 14.90 48"20
~For media composition see Materials and Methods section.
47.80 39.58 5"00 36"99 28.02 20-93
55"90 56"00 6-49 42.34 23"75 21.45
53.92 35-99 52"56 6"48 41"27 11.15
53.72 51.05 53-26 18.58 24-99 47.19
47.59 46.69 4 0 " 3 6 51"51 4.97 3"05 2.26 7.23 0-00 0.00 4.52 3.01
A.-M. S. Ismail
648
Table 2. Cellulase activities (a) (U/ml enzyme) and saccharification (b) (%) in different static and shaken fungal cultures following different incubation periods a
Microorganism
Medium 2
Medium 3
Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P chrysogenum 3486 P. oxalicum 7
a b a b a b a b a b a b
Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
1.03 22.20 1.28 27.60 1-05 22-70 3.39 73.20 0.92 19.90 2.39 51.60
0.26 5.62 0.64 13.80 0"58 12.53 1-29 27.78 1.24 26.80 0.73 15.77
0.380 8.260 0.810 17-500 0.044 0.960 2.760 59.500 0.098 2-110 1.090 26.060
0.12 2.59 4.39 94-80 0.17 3.72 1.02 22.00 1.84 39.84 0.08 1.75
0.000 0.000 0.000 0.000 0.000 0.000 0.026 0-550 0.000 0.000 0.167 3.600
0.000 0.000 0.017 0-360 0"028 0.600 0.039 0.840 0.290 6.400 0.000 0.000
0.128 2.760 0.160 3.460 0.002 0.480 0.017 0.360 0.011 0.238 0.034 0.730
0.133 2.880 0.330 7.200 0.240 5.280 0"078 1"690 0.000 0.000 0.039 0.840
"No cellulases detected with culture medium 1.
funiculosum 258 (Ismail et al. TM) for utilization of sugar cane bagasse. Screening of a-amylase activities in the fungal cultures (Table 4) revealed moderate activities in static cultures only using media 2 or 3, by A. oryzae 1911. It is well known that the aforementioned fungus is a good producer of diastase. Other cultures showed feeble or no amylase activities and this may be attributed to the diminished content of starch in orange peels and to the inducible nature of a-amylases. The data recorded in Table 5 indicated that the optimum conditions for enzymic activities are pH 4-5 and reaction temperature 45-50°C.
culture media. Of all the tested strains, A. niger A-20 in 3- and 5-day shaken culture using medium 2 yielded the highest xylanase activity (3.27 and 3.33 U/ml enzyme) and 78.6 and 80% saccharification under the assay conditions, respectively. On the other hand, A. niger 2 and P oxalicum 7 in 3- and 5-day shaken cultures, respectively, yielded good xylanase activity using culture medium 2 or 3. Medium 3 with other fungal isolates afforded weak xylanase activities. In this respect, the xylanase of A. niger A-20 is superior to those yielded by Trichoderma viride 253-M16 (Abdel-Fattah et al.~3), AspergiUus terreus 603 (Abdel-Fattah et all 7) and Penicillium
Table 3. Xylanase activities (a) (U/ml enzyme) and saccharification (b) (%) in different static and shaken fungal cultures following different incubation periods"
Microorganism
Medium 2 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P. chrysogenum 3486 P. oxalicum 7
a b a b a b a b a b a b
Medium 3 Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
0.087 2.090 0.000 0-000 0.000 0.000 0.000 0.000 0.000 0.000 0.029 0.700
0-420 10.090 0.840 25"100 0"000 0.000 0"186 5.590 1.480 44.500 0.093 2.780
2"45 58"81 3"27 78-56 0"54 12.88 0-42 10"09 0.00 0.00 1.33 32"03
0"58 13.92 3.33 80-00 0.71 17.05 1-33 32.02 0.46 11.05 2-05 49.24
0.000 0'000 0.000 0"000 0.493 11.830 0"000 0.000 0.186 6"590 0"000 0"000
0.000 0.000 0-000 0.000 0-000 0.000 0.000 0.000 0-217 5"220 0-000 0"000
1"040 25.040 1.130 27.140 0"435 10"450 1.043 25"060 0.000 0.000 0.695 16-700
2"40 59"00 0"00 2"00 1.10 27"80 0"40 11"80 0"50 12"50 1"80 44'50
"No xylanases detected with culture medium 1.
Production of multienzyme complexes by fungi
649
Table 4. a-Amylase activities (U/ml enzyme) in different static and shaken fungal cultures following different incubation periods"
,rvIicroorganism
Medium 2 Static
A. niger 2 A. niger A-20 A. oryzae 1911 Memmoniella sp. 6 P. chrysogenum 3486 P. oxalicum 7
Medium 3 Shaken
Static
Shaken
4-day
7-day
3-day
5-day
4-day
7-day
3-day
5-day
0"220 0.890 0"070 0.050 0.190 0.005
0.460 0.460 0.380 0.070 0.340 0-006
0"02 0.04 0.00 0.10 0"00 0' 15
0-030 0.010 0-000 0.004 0"000 0.000
0'000 0.097 1"300 0.000 0.250 0.000
0"36 0.29 1.24 0"00 0.00 0.00
0-000 0.396 0.430 0.340 0.210 0.000
0"160 0.000 0.190 0.290 0.030 0.000
"No a-amylase activities detected with culture medium 1.
Mabrouk et al. 19 reported the optimum pH and temperature for pectinase produced by Trichoderma lignorum to be pH 4.45 and 40°C, respectively. On the other hand, Abdel-Fattah et al. 13 found that the optimum pH and temperature were 5.5 and 45°C for both cellulase and hemicellulase produced by Trichoderma viride 253 M-16. The data collectively indicated that the local fungal strain A. niger A-20 was the most potent and produced a highly active multienzyme preparation containing pectinase, cellulase and xylanase, besides feeble amylase activity, in a 5-day shaken culture using grated orange peels instead of pure pectin. In unrecorded data, very weak lipase activity was also found in this enzyme preparation.
Table 5. Effect of temperature and pH value of the reaction on the different activities of A. niger A-20 enzyme preparation
Pectinase Cellulase Xylanase (% reduction (U/ml (U/ml in viscosity) enzyme) enzyme) Reaction temperature (°C) 35 40 45 50 55
45 56 58 59 58
3.00 4"20 4.80 4.39 4"00
2"5 3-5 4"0 4.2 4.0
42 58 54 50 40
3"00 4-40 4.80 5"00 2.00
3"2 4.0 4-5 4.0 3'8
Reaction pH" 3.40 4.05 4.45 4-99 5.89
"Enzymatic reactions were conducted at optimum temperatures.
The crude lyophilized multienzyme of A. niger A-20 was a deep yellow colour, readily soluble in water and stable following storage over 2 years at room temperature. Accordingly, this enzyme preparation is, to a considerable extent, superior to Novo Nordisk multienzyme complex product, Viscozyme L, which is produced from selected strains of the Aspergilli. The optimum conditions for this enzyme complex are pH 3.5-5.5 and a temperature of 40-50°C and a loss in its activity of 1-2% per month may occur in storage periods of more than 3 months (Novo Nordisk2°). REFERENCES 1. Abdel-Fattah, A. F., Mabrouk, S. S. & Ismail, A. S., Production of polygalacturonase and pectin methylesterase by fungi. Chem. Mikrobiol. Technol. Lebensm, 5 (1977) 38-41. 2. Chen, W. P. & Anderson, A. W., Extraction of hemicellulose from ryegrass straw for the production of glucose isomerase and use of the resulting straw residue for animal feed. Biotechnol. Bioeng., 20 (1980) 519-31. 3. A.O.A.C., Official Methods of Analysis of Association of Official Agricultural Chemists. Washington D.C., 1970. 4. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265-75. 5. Mandels, M. & Sternberg, D., Recent advances in cellulase technology. J. Ferment. Technol., 54 (1976) 267-86. 6. Neish, A. C., Analytical Methods for Bacterial Fermentations. Report No. 46-2-3, National Research Council of Canada, Second Revision 34, 1952. 7. Somogyi, M. J., Determination of blood sugar. J. Biol. Chem., 160 (1945) 61-8. 8. Nelson, N. A., A photometric adaptation of the Somogyi method for the determination of glucose. J. BioL Chem., 153 (1944) 375-80. 9. Ismail, A. S., Abdel-Naby, M. A. & Abdel-Fattah, A.
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10.
11. 12. 13.
14. 15.
A.-M. S. Ismail F., Production of hemicellulytic enzymes by fungi. Agricultural Wastes, 18 (1986) 283-8. Bergmann, F. W., Abe, J. & Hizukurt, S., Selection of microorganisms which produce raw starch degrading amylases. Appl. Microbiol. Biotechnol., 27 (1988) 443-6. Trinder, P., Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J. Clin. Pathol., 22 (2) (1989) 158-61. Parry, R. M., Chandan, R. C. & Shahani, K. M., Rapid assay for milk-lipase. Dairy Science, 49 (4) (1966) 356-60. Abdel-Fattah, A. F. et al., Utilization of Cane-Sugar Bagasses. Final Technical Report, National Research Centre, Cairo/US Department of Agriculture, Grant No. FG-EG-180, 1981, pp. 388-415. Ghose, T. K. & Ghose, P., Bioconversion of cellulosic substances. J. Appl. Chem. Biotechnol., 28 (1978) 309-20. Ryu, D. Y. & Mandels, M., Cellulases: biosynthesis
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and application. Enz. Microb. Technol., 2 (1980) 91-102. Chang, M. M., Chou, T. Y. & Tsao, G. T., Structure, pretreatment and hydrolysis of cellulose. Adv. Biochem. Eng., 20 (1981) 15-42. Abdel-Fattah, A. F., Abdel-Naby, M. A. & Ismail, A. S., Purification and properties of xylan-degrading enzyme from Aspergillus terreus 603. Biological Wastes, 20 (1987) 143-51. Ismail, A. S., Sary, H. H. & Abdel-Fattah, A. F., Production of xylanase by Penicillium funiculosum 258. Bull. Fac. Pharm. Cairo Univ., 32 (2) (1994) 299-302. Mabrouk, S. S., Abdel-Fattah, A. F. & Ismail, A. S., Preparation and properties of pectic enzymes produced by Trichoderma lignorum. Zbl. Bakt. II. Abt., 134 (1979) 282-6. Novo-Nordisk Ferment Ltd, Viscozyme L. Novo Nordisk A/5 Enzyme process Division, Product Sheet, 4 pp. Novo Alle, 2880, Bagsvaerd, Denmark, 1994.