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Properties of milk-clotting enzyme from Aspergillus versicolor and isolation of rennin-like enzyme
Biological Wastes 25 (1988) 109-115
Properties of Milk-clotting Enzyme from Aspergillus versicolor and Isolation of Rennin-like Enzyme A h m e d F. Abdel-Fattah & Soad A. Saleh Laboratory of Natural and Microbial Products Chemistry, National Research Centre,
Dokki, Cairo, Egypt (Received 28 November 1987; revised version received 15 January 1988; accepted 21 January 1988)
A BS TRA C T Some properties of the crude milk-clotting enzyme produced by Aspergillus versicolor in surface culture were studied. The enzyme action was optimal at p H 6 and 45°C. Addition of increasing amounts of calcium chloride at constant level of skim milk enhanced milk-clotting. The enzyme possessed a high milk-clotting activity/proteolytic activity ratio and compared well to calf rennin. Electrophoresis in O'OIM acetate buffer at pH3.42 separated the enzyme into three protein components. Two components showed only proteolytic activity while the third component showed high milk-clotting activity and feeble proteolytic action. That third component was the renninlike enzyme fraction, which showed 2"8-fold purification. The rennin-like enzyme fraction was activated 5-fold by treatment with either maleic or iodoacetic acid. It was partially inhibited by reduced glutathione and parachloromercuribenzoate, and completely inhibited by iodine and potassium cyanide.
INTRODUCTION Due to the increasing demands on rennin for cheese-making, a great deal of work has been carried out in search of rennin substitutes. A rennin or renninlike enzyme is characterized by high milk-clotting activity and feeble proteolytic action. Attention has therefore been directed toward microbial milk-clotting enzymes possessing these properties (Veselov et al., 1965; Abdel-Fattah et aL, 1974, 1984, 1987; Abdel-Fattah & Amr, 1987). 109
Biological Wastes 0269-7483/88/$03.50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
110
Ahrned F. Abdel-Fattah, Soad A. Saleh
In a previous work (Abdel-Fattah & Saleh, 1979), we reported the production and isolation of a milk-clotting enzyme from Aspergillus versicolor. The results indicated that complex organic compounds favoured the production of the enzyme. The present work was undertaken to investigate the properties of the crude milk-clotting enzyme produced by A. versicolor and its suitability as a rennin-like enzyme compared to calf rennin. Electrophoresis studies were also carried out to separate the rennin-like enzyme fraction and investigate the effect of some activators and inhibitors on its activity.
METHODS The fungal strain used was Aspergillus versicolor, a local isolate, obtained from the fungal collection of the Centre of Cultures of the National Research Centre, Cairo, Egypt. The culture medium contained (g/litre): sucrose, 50; yeast extract, 3; peptone, 5; KH2PO4, 1; MgSO4.7H20, 0-5. The calf rennin used (Rennin N F X 1) was a preparation of Difco from the glandular layer of calf stomach.
Cultivation Transfers were made from the stock cultures to Dox's agar plates, which were then incubated at 30°C for 7 days. Liquid cultures were grown in 250ml Erlenmeyer flasks, each containing 50ml of sterile medium and inoculated with four discs, each of 0.5 cm diameter, cut from the 7-day-old culture plates. After incubation for 5 days at 30°C (surface culture), the culture medium was filtered off to obtain the culture filtrate.
Analyses Protein was determined by the method of Lowry et al. (1951). Milk-clotting activity was measured by the method of Berridge (1952). Unless otherwise specified, 2.5 ml of the enzyme sample in 0.02M acetate buffer (pH 4.0) were incubated with 10 ml of reconstituted skim milk (12 g dry skim milk/100 ml 0-01 ~t CaCI2) at 40°C and the clotting time recorded. One unit of enzyme activity was taken to be that which clotted 10 ml of milk in 10 rain. The pH of this standard activity test was raised by the presence of the milk (see Results). Proteolytic activity of the culture filtrate was determined by the method of Bergkvist (1963) from the hydrolysis of 1-5% casein in phosphate buffer (pH 7-0) containing the enzyme, after incubation for 30 min at 37°C. The
Rennin-like enzyme from A. versicolor
111
a m o u n t of tyrosine released was determined in the trichloroacetic acid filtrate by the method of Lowry et al. (1951) (using tyrosine for the standard curve) and taken as a measure of caseinase activity. Paper electrophoresis was performed, with Elphor apparatus, with 19 buffer solutions including acetate, McIlvaine citrate-phosphate and S6rensen-phosphate buffers of different concentrations and at different pH values. A potential of 300 V (0.2 mA/strip) was applied at room temperature for 4 h. Staining of the pherograms was effected with Amido Black dye. The electrophoresed protein components were separately eluted from several unstained pherograms with (R)2M acetate buffer (pH 5) for assay of milkclotting activity, or with 0"2M sodium phosphate buffer (pH 7) for assay of caseinase activity.
RESULTS A N D DISCUSSION Some properties of the crude milk-clotting enzyme produced by A. versicolor were investigated. The results indicated that 8 mg enzyme protein in 2.5 ml were sufficient to clot 1.2 g reconstituted skim milk in 10 ml within 3 rain at pH 5.98 and 40°C. Thus the enzyme quickly clotted 150 times its own weight of skim milk. Addition of increasing amounts of calcium chloride to reaction mixtures containing skim milk (0.8 g/10 ml) and enzyme protein (8 mg/2.5 ml) resulted in significant increase in clotting activity (Table 1). Increase in calcium chloride from 7.4 to 19.2 mg/10 ml milk resulted in about 7.7-fold increase in enzyme activity, whereby the enzyme could clot 100 times its own weight of milk in about 20 s. These results are in agreement with those reported for TABLE 1
Effect of the Addition of Calcium Chloride to Skim Milk on the Milk-clotting Activity of the Crude Fungal Enzyme Preparation from Aspergillus versicolor Calcium chloride concentration (mg/ l Oml milk)
Clotting time (rain)
Milk-clotting activity (units~rag of protein)
2"50 2"23
0-50 0-56
12'0
1-62
0'77
14"4 16"8 19"2
1"52 0-43 0-33
0-82 2-90 3"86
7"4 9"6
Concentration of skim milk=0.8g/lOml; enzyme protein concentration= 8 mg/2"5mi.
112
Ahmed F. Abdel-Fattah, Soad A. Saleh TABLE 2
Effect of Temperature of the Reaction on the Milk-clotting Activity of Aspergillus versicolor Crude Enzyme Preparation Temperature (°C)
Clotting time (min)
Milk-clotting activity (units/mg of protein)
40 45 50 55
2.5 1.7 2.1 41.6
0.50 0-74 0.60 0.03
Calcium chloride concentration = 7-4 mg/10 ml skim milk; skim milk concentration=0.8g/10ml; enzyme protein concentration = 8 mg/2.5 ml. other microbial milk-clotting enzymes (Tsugo et ai., 1964; Osman et al., 1969a; Abdel-Fattah & E1-Hawwary, 1972). At p H 5.98, the enzyme showed its maximal activity at 45°C (Table 2). At 55°C the enzyme lost 9 6 % o f its activity at 45°C in presence of substrate. The crude milk-clotting enzyme showed a pH optimum of 6.4 at 45°C (Table 3). It should be noted that, due to the high buffering capacity of milk, the final p H values o f the reaction mixtures varied within a very limited range on using acetate buffers of pH values from 3.42 to 5.89. Similar pH optima have been found for other fungal milk-clotting enzymes (Veselov et al., 1965; Osman et al., 1969a). The crude fungal milk-clotting enzyme was compared with calf rennin enzyme. The milk-clotting activities (units/mg o f protein) of the fungal enzyme and calf rennin were 1.54 and 2, while their proteolytic (caseinase) TABLE 3 EffectofpH of the Reaction Mixture on the Milk-clotting Activity of Aspergillus versicolor Crude Enzyme Preparation pH of enzyme solution
pH of reaction mixture
Clotting time (rain)
Milk-clotting activity (units~rag of protein)
3"42 4'00 4"45 5"00 5"57 5"89
5-94 5'98 6-32 6'40 6-42 6'44
2-00 1'70 1-31 0'8 ! 2.44 2'44
0-63 0"74 0-95 1"54 0.51 0'51
Calcium chloride concentration=7.4mg/lOml skim milk; skim milk concentration = 0-8 g/lO ml; enzyme protein concentration = 8 mg/2.5 ml.
Rennin-like enzyme flora A. versicoior
113
activities (mg tyrosine/mg of protein) were 0"12 and 0.14, respectively. Thus, the ratio of milk-clotting activity to proteolytic activity of the fungal enzyme and calfrennin were 12.8 and 14-3, indicating that the former possessed good rennetic properties. Electrophoresis of the crude fungal enzyme preparation was studied using 19 buffer solutions. Of these, only 0-01M acetate buffer of pH 3-42 was successful in separating the crude enzyme into three enzyme protein components which migrated towards the cathode. Components 1 and 2 were nearer the starting line and possessed no milk-clotting activity but showed proteolytic action on casein of 0.05 and 0.16mg tyrosine/mg of protein, respectively. Component 3 moved faster; it possessed milk-clotting activity (4.35 units/mg of protein) and proteolytic activity (0.12 mg tyrosine/mg of protein) in the ratio of 36.3, indicating a 2.8-fold purification. Due to its high ratio of milk-clotting to proteolytic activity, component 3 was therefore the rennin-like enzyme fraction in the fungal milk-clotting enzyme preparation. Determination of the protein contents of the three electrophoretic components revealed a high proportion (60%) of the rennin-like enzyme fraction (component 3) compared to components 1 (10%) and 2 (30%). The rennin-like enzyme fraction thus comprised the major portion of the crude fungal milk-clotting enzyme. TABLE 4 Effect of Some Activators and lnhibitors on the Activity of A,pergillus versicolor Rennin-like Enzyme Fraction 3
Substance added
None Maleic acid Iodoacetic acid Iodine Reduced glutathione Potassium cyanide p-Chloromercuribenzoate
Concentration of substance a (mol)
Relative activity (%)
-0-1 04)01 0-1 0-001 0-1 0"001 0-01 if001 0-1 0.1 0-01 if001
100 508 100 508 75 0 75 33 50 0 75 58 50
* In the pretreated enzyme solution; see Results and Discussion. Calcium chloride concentration = 7-4 mg/10 ml skim milk; skim milk concentration = (>8 g/ml; enzyme protein concentration = 8 rag/2"5 ml.
114
Ahmed F. AbdeI-Fattah, Soad A. Saleh
The effect of some activators and inhibitors on the activity of the renninlike enzyme fraction (3) was studied by pretreating the enzyme solution, containing the test substance, for 10 min at 45°C and thereafter determining milk-clotting activity at 45°C. As shown in Table 4, pretreatment o f the enzyme with either maleic or iodoacetic acid at 0.1M brought about significant (5 fold) enzyme activation. On the other hand, iodine and potassium cyanide (0.1M) brought about complete enzyme inhibition. Reduced glutathione and p-chloromercuribenzoate had partial inhibitory effects on the enzyme activity. These results collectively suggest the absence o f sulphhydryl groups in the active site o f the fungal rennin-like enzyme. No evidence could be provided in support for the presence o f reactive sulphhydryl groups in fungal rennin-like enzymes (Osman et al., 1969b; Abdel-Fattah & E.-Hawwary, 1973).
REFERENCES AbdeI-Fattah, A. F. & Amr, A. S. (1987). Preparation and some properties of a partially purified rennin-like enzyme from Penicillium expansum. Biol. Wastes, 20, 35. AbdeI-Fattah, A. F. & EI-Hawwary, N. M. (1972). Purification and proteolytic action of milk-clotting enzyme produced by Penicillium citrinum. J. Gen. Appl. Microbiol., 18, 341. Abdel-Fattah, A. F. & EI-Hawwary, N. M. (1973). Some characteristics and rennetic activity of the pure rennin-like enzyme from Penicillium citrinum. Acta Biol. Acad. Sci. Hung., 24, 95. Abdel-Fattah, A. F. & Saleh, S. A. (I 979). Production and isolation of milk-clotting enzyme from Aspergillus versicolor. Zbl. Bakt. II. Abt., 134, 547. Abdel-Fattah, A. F., EI-Hawwary, N. M. & Amr, A. S. (1974). Studies on the production of milk-clotting enzymes, proteolytic enzymes and mucilage by fungi. J. Gen. Microbiol., 84, 327. Abdel-Fattah, A. F., Ismaii, A. S. & EI-Aassar, S. A. (1984). Production of renninlike enzyme by Absidia cylindrospora. Agric. Wastes, 11, 125. Abdel-Fattah, A. F., Ismail, A. S. & El-Aassar, S. A. (1987). Purification and properties of rennin-like enzyme from Absidia cylindrospora. Zbl. Microbiol., 142, 37. Bergkvist, R. (1963). The proteolytic enzymes of Aspergillus oryzae. I. Methods for the estimation and isolation of the proteolytic enzymes. Acta Chem. Scand., 17, 1521. Berridge, N. J. (1952). Some observations on the determination of the activity of rennet. Analyst Lond., 77, 57. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265. Osman, H. G., Abdel-Fattah, A. F., Abdel-Samie, M. & Mabrouk, S. S. (1969a). Production of a milk-clotting enzyme preparation by Aspergillus niger and the effect of various factors on its activity. J. Gen. Microbiol., 59, 125.
Rennin-like enzyme from A. versicolor
115
Osman, H. G., Abdel-Fattah, A. F. & Mabrouk, S. S. (1969b). Purification and some properties of milk-clotting enzyme from Aspergillus niger. J. Gen. Microbioi., 59, 131. Tsugo, T., Yoshino, U., Taniguchi, K., Ozawa, A., Miki, Y., Iwasaki, S. & Arima, K. (1964). Cheese-making by using the milk-clotting enzyme of Mucor pusillus. I. Rennetic properties of the enzyme. Jap. J. Zootech. Sci., 35, 221. Veselov, I. Y., Tipograph, D. Y. & Petina, T. A. (1965). Aspergillus candidus as a producer of rennin. PrikL Biokhim. Microbiol., 1, 52.
Properties of Milk-clotting Enzyme from Aspergillus versicolor and Isolation of Rennin-like Enzyme A h m e d F. Abdel-Fattah & Soad A. Saleh Laboratory of Natural and Microbial Products Chemistry, National Research Centre,
Dokki, Cairo, Egypt (Received 28 November 1987; revised version received 15 January 1988; accepted 21 January 1988)
A BS TRA C T Some properties of the crude milk-clotting enzyme produced by Aspergillus versicolor in surface culture were studied. The enzyme action was optimal at p H 6 and 45°C. Addition of increasing amounts of calcium chloride at constant level of skim milk enhanced milk-clotting. The enzyme possessed a high milk-clotting activity/proteolytic activity ratio and compared well to calf rennin. Electrophoresis in O'OIM acetate buffer at pH3.42 separated the enzyme into three protein components. Two components showed only proteolytic activity while the third component showed high milk-clotting activity and feeble proteolytic action. That third component was the renninlike enzyme fraction, which showed 2"8-fold purification. The rennin-like enzyme fraction was activated 5-fold by treatment with either maleic or iodoacetic acid. It was partially inhibited by reduced glutathione and parachloromercuribenzoate, and completely inhibited by iodine and potassium cyanide.
INTRODUCTION Due to the increasing demands on rennin for cheese-making, a great deal of work has been carried out in search of rennin substitutes. A rennin or renninlike enzyme is characterized by high milk-clotting activity and feeble proteolytic action. Attention has therefore been directed toward microbial milk-clotting enzymes possessing these properties (Veselov et al., 1965; Abdel-Fattah et aL, 1974, 1984, 1987; Abdel-Fattah & Amr, 1987). 109
Biological Wastes 0269-7483/88/$03.50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
110
Ahrned F. Abdel-Fattah, Soad A. Saleh
In a previous work (Abdel-Fattah & Saleh, 1979), we reported the production and isolation of a milk-clotting enzyme from Aspergillus versicolor. The results indicated that complex organic compounds favoured the production of the enzyme. The present work was undertaken to investigate the properties of the crude milk-clotting enzyme produced by A. versicolor and its suitability as a rennin-like enzyme compared to calf rennin. Electrophoresis studies were also carried out to separate the rennin-like enzyme fraction and investigate the effect of some activators and inhibitors on its activity.
METHODS The fungal strain used was Aspergillus versicolor, a local isolate, obtained from the fungal collection of the Centre of Cultures of the National Research Centre, Cairo, Egypt. The culture medium contained (g/litre): sucrose, 50; yeast extract, 3; peptone, 5; KH2PO4, 1; MgSO4.7H20, 0-5. The calf rennin used (Rennin N F X 1) was a preparation of Difco from the glandular layer of calf stomach.
Cultivation Transfers were made from the stock cultures to Dox's agar plates, which were then incubated at 30°C for 7 days. Liquid cultures were grown in 250ml Erlenmeyer flasks, each containing 50ml of sterile medium and inoculated with four discs, each of 0.5 cm diameter, cut from the 7-day-old culture plates. After incubation for 5 days at 30°C (surface culture), the culture medium was filtered off to obtain the culture filtrate.
Analyses Protein was determined by the method of Lowry et al. (1951). Milk-clotting activity was measured by the method of Berridge (1952). Unless otherwise specified, 2.5 ml of the enzyme sample in 0.02M acetate buffer (pH 4.0) were incubated with 10 ml of reconstituted skim milk (12 g dry skim milk/100 ml 0-01 ~t CaCI2) at 40°C and the clotting time recorded. One unit of enzyme activity was taken to be that which clotted 10 ml of milk in 10 rain. The pH of this standard activity test was raised by the presence of the milk (see Results). Proteolytic activity of the culture filtrate was determined by the method of Bergkvist (1963) from the hydrolysis of 1-5% casein in phosphate buffer (pH 7-0) containing the enzyme, after incubation for 30 min at 37°C. The
Rennin-like enzyme from A. versicolor
111
a m o u n t of tyrosine released was determined in the trichloroacetic acid filtrate by the method of Lowry et al. (1951) (using tyrosine for the standard curve) and taken as a measure of caseinase activity. Paper electrophoresis was performed, with Elphor apparatus, with 19 buffer solutions including acetate, McIlvaine citrate-phosphate and S6rensen-phosphate buffers of different concentrations and at different pH values. A potential of 300 V (0.2 mA/strip) was applied at room temperature for 4 h. Staining of the pherograms was effected with Amido Black dye. The electrophoresed protein components were separately eluted from several unstained pherograms with (R)2M acetate buffer (pH 5) for assay of milkclotting activity, or with 0"2M sodium phosphate buffer (pH 7) for assay of caseinase activity.
RESULTS A N D DISCUSSION Some properties of the crude milk-clotting enzyme produced by A. versicolor were investigated. The results indicated that 8 mg enzyme protein in 2.5 ml were sufficient to clot 1.2 g reconstituted skim milk in 10 ml within 3 rain at pH 5.98 and 40°C. Thus the enzyme quickly clotted 150 times its own weight of skim milk. Addition of increasing amounts of calcium chloride to reaction mixtures containing skim milk (0.8 g/10 ml) and enzyme protein (8 mg/2.5 ml) resulted in significant increase in clotting activity (Table 1). Increase in calcium chloride from 7.4 to 19.2 mg/10 ml milk resulted in about 7.7-fold increase in enzyme activity, whereby the enzyme could clot 100 times its own weight of milk in about 20 s. These results are in agreement with those reported for TABLE 1
Effect of the Addition of Calcium Chloride to Skim Milk on the Milk-clotting Activity of the Crude Fungal Enzyme Preparation from Aspergillus versicolor Calcium chloride concentration (mg/ l Oml milk)
Clotting time (rain)
Milk-clotting activity (units~rag of protein)
2"50 2"23
0-50 0-56
12'0
1-62
0'77
14"4 16"8 19"2
1"52 0-43 0-33
0-82 2-90 3"86
7"4 9"6
Concentration of skim milk=0.8g/lOml; enzyme protein concentration= 8 mg/2"5mi.
112
Ahmed F. Abdel-Fattah, Soad A. Saleh TABLE 2
Effect of Temperature of the Reaction on the Milk-clotting Activity of Aspergillus versicolor Crude Enzyme Preparation Temperature (°C)
Clotting time (min)
Milk-clotting activity (units/mg of protein)
40 45 50 55
2.5 1.7 2.1 41.6
0.50 0-74 0.60 0.03
Calcium chloride concentration = 7-4 mg/10 ml skim milk; skim milk concentration=0.8g/10ml; enzyme protein concentration = 8 mg/2.5 ml. other microbial milk-clotting enzymes (Tsugo et ai., 1964; Osman et al., 1969a; Abdel-Fattah & E1-Hawwary, 1972). At p H 5.98, the enzyme showed its maximal activity at 45°C (Table 2). At 55°C the enzyme lost 9 6 % o f its activity at 45°C in presence of substrate. The crude milk-clotting enzyme showed a pH optimum of 6.4 at 45°C (Table 3). It should be noted that, due to the high buffering capacity of milk, the final p H values o f the reaction mixtures varied within a very limited range on using acetate buffers of pH values from 3.42 to 5.89. Similar pH optima have been found for other fungal milk-clotting enzymes (Veselov et al., 1965; Osman et al., 1969a). The crude fungal milk-clotting enzyme was compared with calf rennin enzyme. The milk-clotting activities (units/mg o f protein) of the fungal enzyme and calf rennin were 1.54 and 2, while their proteolytic (caseinase) TABLE 3 EffectofpH of the Reaction Mixture on the Milk-clotting Activity of Aspergillus versicolor Crude Enzyme Preparation pH of enzyme solution
pH of reaction mixture
Clotting time (rain)
Milk-clotting activity (units~rag of protein)
3"42 4'00 4"45 5"00 5"57 5"89
5-94 5'98 6-32 6'40 6-42 6'44
2-00 1'70 1-31 0'8 ! 2.44 2'44
0-63 0"74 0-95 1"54 0.51 0'51
Calcium chloride concentration=7.4mg/lOml skim milk; skim milk concentration = 0-8 g/lO ml; enzyme protein concentration = 8 mg/2.5 ml.
Rennin-like enzyme flora A. versicoior
113
activities (mg tyrosine/mg of protein) were 0"12 and 0.14, respectively. Thus, the ratio of milk-clotting activity to proteolytic activity of the fungal enzyme and calfrennin were 12.8 and 14-3, indicating that the former possessed good rennetic properties. Electrophoresis of the crude fungal enzyme preparation was studied using 19 buffer solutions. Of these, only 0-01M acetate buffer of pH 3-42 was successful in separating the crude enzyme into three enzyme protein components which migrated towards the cathode. Components 1 and 2 were nearer the starting line and possessed no milk-clotting activity but showed proteolytic action on casein of 0.05 and 0.16mg tyrosine/mg of protein, respectively. Component 3 moved faster; it possessed milk-clotting activity (4.35 units/mg of protein) and proteolytic activity (0.12 mg tyrosine/mg of protein) in the ratio of 36.3, indicating a 2.8-fold purification. Due to its high ratio of milk-clotting to proteolytic activity, component 3 was therefore the rennin-like enzyme fraction in the fungal milk-clotting enzyme preparation. Determination of the protein contents of the three electrophoretic components revealed a high proportion (60%) of the rennin-like enzyme fraction (component 3) compared to components 1 (10%) and 2 (30%). The rennin-like enzyme fraction thus comprised the major portion of the crude fungal milk-clotting enzyme. TABLE 4 Effect of Some Activators and lnhibitors on the Activity of A,pergillus versicolor Rennin-like Enzyme Fraction 3
Substance added
None Maleic acid Iodoacetic acid Iodine Reduced glutathione Potassium cyanide p-Chloromercuribenzoate
Concentration of substance a (mol)
Relative activity (%)
-0-1 04)01 0-1 0-001 0-1 0"001 0-01 if001 0-1 0.1 0-01 if001
100 508 100 508 75 0 75 33 50 0 75 58 50
* In the pretreated enzyme solution; see Results and Discussion. Calcium chloride concentration = 7-4 mg/10 ml skim milk; skim milk concentration = (>8 g/ml; enzyme protein concentration = 8 rag/2"5 ml.
114
Ahmed F. AbdeI-Fattah, Soad A. Saleh
The effect of some activators and inhibitors on the activity of the renninlike enzyme fraction (3) was studied by pretreating the enzyme solution, containing the test substance, for 10 min at 45°C and thereafter determining milk-clotting activity at 45°C. As shown in Table 4, pretreatment o f the enzyme with either maleic or iodoacetic acid at 0.1M brought about significant (5 fold) enzyme activation. On the other hand, iodine and potassium cyanide (0.1M) brought about complete enzyme inhibition. Reduced glutathione and p-chloromercuribenzoate had partial inhibitory effects on the enzyme activity. These results collectively suggest the absence o f sulphhydryl groups in the active site o f the fungal rennin-like enzyme. No evidence could be provided in support for the presence o f reactive sulphhydryl groups in fungal rennin-like enzymes (Osman et al., 1969b; Abdel-Fattah & E.-Hawwary, 1973).
REFERENCES AbdeI-Fattah, A. F. & Amr, A. S. (1987). Preparation and some properties of a partially purified rennin-like enzyme from Penicillium expansum. Biol. Wastes, 20, 35. AbdeI-Fattah, A. F. & EI-Hawwary, N. M. (1972). Purification and proteolytic action of milk-clotting enzyme produced by Penicillium citrinum. J. Gen. Appl. Microbiol., 18, 341. Abdel-Fattah, A. F. & EI-Hawwary, N. M. (1973). Some characteristics and rennetic activity of the pure rennin-like enzyme from Penicillium citrinum. Acta Biol. Acad. Sci. Hung., 24, 95. Abdel-Fattah, A. F. & Saleh, S. A. (I 979). Production and isolation of milk-clotting enzyme from Aspergillus versicolor. Zbl. Bakt. II. Abt., 134, 547. Abdel-Fattah, A. F., EI-Hawwary, N. M. & Amr, A. S. (1974). Studies on the production of milk-clotting enzymes, proteolytic enzymes and mucilage by fungi. J. Gen. Microbiol., 84, 327. Abdel-Fattah, A. F., Ismaii, A. S. & EI-Aassar, S. A. (1984). Production of renninlike enzyme by Absidia cylindrospora. Agric. Wastes, 11, 125. Abdel-Fattah, A. F., Ismail, A. S. & El-Aassar, S. A. (1987). Purification and properties of rennin-like enzyme from Absidia cylindrospora. Zbl. Microbiol., 142, 37. Bergkvist, R. (1963). The proteolytic enzymes of Aspergillus oryzae. I. Methods for the estimation and isolation of the proteolytic enzymes. Acta Chem. Scand., 17, 1521. Berridge, N. J. (1952). Some observations on the determination of the activity of rennet. Analyst Lond., 77, 57. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265. Osman, H. G., Abdel-Fattah, A. F., Abdel-Samie, M. & Mabrouk, S. S. (1969a). Production of a milk-clotting enzyme preparation by Aspergillus niger and the effect of various factors on its activity. J. Gen. Microbiol., 59, 125.
Rennin-like enzyme from A. versicolor
115
Osman, H. G., Abdel-Fattah, A. F. & Mabrouk, S. S. (1969b). Purification and some properties of milk-clotting enzyme from Aspergillus niger. J. Gen. Microbioi., 59, 131. Tsugo, T., Yoshino, U., Taniguchi, K., Ozawa, A., Miki, Y., Iwasaki, S. & Arima, K. (1964). Cheese-making by using the milk-clotting enzyme of Mucor pusillus. I. Rennetic properties of the enzyme. Jap. J. Zootech. Sci., 35, 221. Veselov, I. Y., Tipograph, D. Y. & Petina, T. A. (1965). Aspergillus candidus as a producer of rennin. PrikL Biokhim. Microbiol., 1, 52.