- Email: [email protected]
Preparation and properties of pectic enzymes produced by Trichoderma lignorum
Zbl. Bakt . II. Ab t . 134 (1979) , 282- 286
[Laboratory of Microbial Che mistry, National R esearch Centre , Do kk i, a nd Aca de my of Scientifi c R esearch an d T echnology, Cairo, E gypt]
Preparation and Properties of Pectic Enzymes Produced by Trichoderma ligrwrum SOUHAIR S . MABROUK, AHMED F. ABDEL-FATTAH,
and
ABDEL-MoHSEN S. I SMAIL
Summary Polygalacturonase and pe ct in-methylesterase were isolated from shaken culture of Trichoderm a lignorum. Isolation was carried ou t with various agents. Methanol was the most suitable precipitant for isolating polygalacturonase, y ieldin g en zym e preparations 6.6 times more active than that of cul ture filtrate. Likewise, tannin afforded a ctive fracti ons at p H 4 and 0.05 % conc entration . Similarly, 50 % ammonium sul phate satu ra t ion gave a ctive fraction s. The lea st polygalacturona se activity was ob taine d fr om ethanol. I n any of t he organic solvents used, high est en zy mic a ct ivity was ob taine d whe n us in g only one volume. As regards poct in -methyl eaterase, n o corre lation exi sted between it s a ctivity a nd concentration of the precipi tant used. A subs trate concentration ab ove 0.8 % was a limiting fa ct or for polygala cturonase act ivi ty , wh ile optimum en zyme conce ntra tion was 40 flg prot einjml at 40 °C an d p H 4.4 5.
Zusammenfassung P olygal akturonase un d P ek t inm ethyl est erase wurde n aus Sch iittelkulturen von Trichoderma lignoru m isoli erb, AlsFallungsmitt elliefer t eM ethanol b efri edigende E rgebnisse. Di e EO gewonnenen E n zyrnpraparate ware n 6,6mal a ktiver al s di e Kulturfiltrat e. Auch 0,05 % Gerbsaure (p H 4) so wie eine 50 % Ammon sulfa tl osu n g en thielt en aktive Frakt ionen. B ei den organisch en L osungs mitteln wurde die hochst e E nzym akti vi tiit im me r dann er halte n , wenn nur ein Volum en d es L osungsmittels verwe n de t wurd e. B ei d el' P ektinmeth ylest era se best eh t k eine K orrela t ion zwischen Enzymakt ivi tiit u nd Konzentration des F allungsm itt els. E in e Substratkon zentration vo n 0, 8 % erwies sich als limitier ender Faktor fur die P olygala kt u ronaseak t.ivit a t . Di e optimale E nz ymkonz en t ration bet rug 40 flg Proteinjml bei 40 °C und p H 4,45.
P ectinases are known t o be imp ortant in the food and t extile industries. Numerou s organisms have been r eported t o produce these enzy mes (8). Extracellular and intracellular pectinases have been also reported to be ext racte d from moulds (1,4). Th e pre sent work deals with isolat ion of polygalacturonase and pectin-methylest erase from shaken cultures of T richoderma lignorum . In ad ditio n some properties of the par ti ally purified enzymes were investi gated. Thi s mould pr oved in a pr evious work (3) t o produce t he highest extrace llular polygalacturonase and pect in-m ethyleste rase using shaken cultures. The mould was grown on a medium t hat afford ed best enzy mic acti vit ies among ot her media investi gated.
Materials and Methods Microorgani sm T ri choderm a lig norum, a local isola t e, wa s ki n dly provided by Dr. D. A. ZARr, Botany Depa rt. m en t , Faculty of Science , Cairo Univer sity, Giza, E gypt .
Preparation and Properties of Pectic Enzymes
283
Pectin This was apple pectin, a preparation of Fluka AG. Buchs SG, Switzerland.
Culture medium The following culture medium was used (gil): pectin, 15; KH2P 0 4 , 0.5; MgS0 4 KN03 , 2.5.
•
7 H 2 0 , 0.5;
Cultivation Transfers were made from the subcultures to Dox's agar plates, which were then incubated at 30°C for 7 days. Liquid cultures were grown in 250 ml flat-bottomed flasks, each containing 50 ml sterile medium. Two discs, each of 1 em diameter, were cut from the 7 day-old culture plates and used for inoculating each flask. After incubation for different periods at 30°C (shaken cultures) the culture medium from each flask was filtered off and made up to 50 ml with distilled water.
Protein determination This was done by the method of LOWRY, ROSEBROUGH, FARR and RANDALL (1951).
Assay for polygalacturonase activity This was done by measuring the decrease in the viscosity of buffered pectin solution. 1.5 ml of the enzyme solution portions were added to 6.0 ml of 0.5 % pectin solution (0.02 M acetate buffer of pH 4.05) and the reaction mixtures were incubated at 40°C for 20 min. Thereafter, 5 ml portions of the reaction mixture were pipetted into an Ostwald viscosimeter in a water bath at 40°C and the time of run was recordea. In control tests heated enzyme solutions were employed.
Detection of pectin-methylesterase activity This was achieved using the hydroxamate method of MCCOMB and MCCREADY (1958). The appearance of a clear zone on the buffered pectin-agar plate, where the enzyme sample was applied, indicated a positive reaction for pectin-methylesterase.
Results and Discussion With all the precipitants used, a parallel relationship existed between the protein content and the concentration of the precipitant. This relation, however, was not the same with polygalacturonase activity except in the case of using ammonium SUlphate. This may be due to precipitation of some proteins in the culture medium other than the enzymes involved. It was of interest to notice that with any of the organic solvents used, the highest polygalacturonase activities were obtained when using only one volume. On the other hand, a sharp decrease in enzymic activity occurred when the culture filtrate was treated with two volumes. The same results were obtained with tannin. Methanol was the most suitable precipitant for isolating polygalacturonase (Table 1). Thus the fraction precipitated out by one volume yielded polygalacturonase preparations 6.6 times more active than that of culture filtrate. Likewise, tannin afforded active fractions at pH 4 and 0.05 %concentration (Tables 2 and 3). The enzymic activity of this fraction was 5.5 times that of the culture filtrate. Tannin was recommended by BERGKVIST (1963) for isolation of proteolytic enzymes from Aspergillus oryzae and it was also used for isolation of fungal milk- clotting and proteolytic enzymes (2, 11). As far as we are aware, this compound had not been used for pectolytic enzyme isolation. Isolation of polygalacturonase with acetone, similarly gave active fractions (Table 4) amounting to about 4 times that of the unprecipitated fractions.
284
S. S. MABROUK et al.
Table 1. Polygalacturonase activity and pectin-methylesterase reaction of methanol precipitates of Trichoderma lignorum enzyme preparations CF + methanol (v/v) Protein content of ppt Ipg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
1+ 1 16.75
1+ 2 49.5
1+ 4 61.75
1+ 6 76.00
1+ 8 80.50
1 + 10 114.75
26.56
45.87
43.86
44.83
46.27
46.67
1.58 6.60 +1
0.92 3.84 +4
0.66 2.74 +3
0.58 2.46 +1
0.58 2.38 +2
0.40 1.70 +2
In this and the following Tables: CF = Culture filtrate, ppt = Precipitate, PG = Polygalacturonase, P /CF = Precipitate/culture filtrate polygalacturonase activity ratio, PME = Pectin-mothylosterase, + 1 = Weak reaction, + 2 = Moderate reaction, + 3 and + 4 = Strong reaction.
Table 2. The effect of tannin concentration on the polygalacturonase activity and pectin-methylesterase reaction of the precipitates of Trichoderma lignorum enzyme preparations from culture filtrate of pH 4.0 Tannin(%) Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
0.05 23.2
0.10 87.8
0.30 132.0
0.50 219.0
0.70 216.0
0.90 259.5
1.0 258.0
30.31
40.41
47.78
57.87
56.71
57.46
57.11
1.30
0.48
0.36
0.28
0.28
0.24
0.24
5.50 +1
1.96 +1
1.52 +1
1.12 +2
1.12 +2
0.92 +1
0.92 +1
Table 3. The effect of the pH value of the culture filtrate on the polygalacturonase activity and pectin-methylesterase reaction of precipitates by 0.05 % tannin pH ofCF
Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
4.0 23.2
4.5 63.0
5.0 76.5
5.5 63.0
6.0 200.0
6.5 100.0
7.0 266.0
30.31
18.86
15.0
6.2
4.80
6.1
2.43
1.30
0.30
0.20
0.10
0.02
0.06
0.01
5.50 +1
1.26 +1
0.80 +1
0.42 - ve
0.08 - ve
0.24 - ve
0.04 - ve
- ve: No reaction. Table 4. Polygalacturonase activity and pectin-methylesterase reaction of acetone precipitates of Trichoderma lignorum enzyme preparations CF + acetone (v/v) Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
1+ 1 50.0
1+ 2 121.0
1+ 4 124.0
1+ 6 132.0
1+ 8 134.2
1 + 10 125.5
49.97
48.13
51.58
49.69
50.25
51.70
1.00 4.2 +1
0.40 1.66 +3
0.42 1.72 +4
0.36 1.56 +3
0.36 1.56 +2
0.40 1.70 +2
Preparation and Properties of Pectic Enzymes
285
Table 5. Polygalacturonase activity and pectin-methylesterase reaction of ethanol precipitates of Trichoderma lignorum enzyme preparations CF + ethanol (v/v) Protein content of ppt (ltg/ml) PG activity (% reduction in viscosity) PG activity/ltg protein (P) P/CF PME reaction
1+ 1 93.0
1+ 2 131.0
1+ 4 178.0
1+ 6 178.0
1+ 8 185.0
1 + 10 194.0
55.24
54.72
54.45
55.66
49.14
55.72
0.58 2.84 +3
0.42 1.98 +3
0.30 1.46 +3
0.32 1.48 +3
0.26 1.26 +4
0.28 1.38 +4
Table 6. Polygalacturonase activity and pectin-methylesterase reaction of ammonium sulphate precipitates of Trichoderma lignorum enzyme preparations Ammonium sulphate saturation (%) 25.0 7.5 Protein content of ppt (ltg/ml) 14.1 PG activity (% reduction in viscosity) 1.88 PG activitY/ltg protein (P) 4.36 P/CF PME reaction - ve
40.0 9.0
50.0 12.0
66.0 22.0
80.0 26.0
100.0 24.5
18.6
25.5
24.4
52.0
43.5
2.06 4.80 +1
2.12 4.94 +2
1.10 2.58 +2
2.00 2.64 +4
1.78 4.12 +3
The least polygalacturonase activity was obtained from ethanol (Table 5). However higher results were obtained by EFREMOVA, KOSTIK and OSIPOVA (1971) for the isolation of exopolygalacturonase from Rhieopus arrhieus. As regards ammonium sulphate, the data recorded in Table 6 indicated that the fractions precipitated were characterized by lower values of both protein content and polygalacturonase activity. However, 50 % ammonium sulphate saturation afforded active fractions about 5 times that of culture filtrate which was more or less double that obtained by BLIEVA (1971). Generally, no loss of polygalacturonase activity occurred with all the precipitants investigated. Concerning pectin-methylesterase, the results did not show any correlation between its activity and concentration of the precipitant used. Similarly no relation existed either between the enzyme activity and protein content, or between enzyme activity and polygalacturonase activity. It is worthwhile to mention that the highest peetin.methylesterase activity was obtained by ethanol which exhibited the least polygalacturonase activity. The results demonstrated that substrate concentration above 0.8 % was a limiting factor for polygalacturonase activity between 0.5-0.8 % substrate concentration. However, 0.2 % concentration showed about 50 % loss in enzymic activity when compared to the optimum obtained at 0.8 %. This loss may be due to an insufficient amount of substrate to be acted upon by the enzyme. An inverse relationship occurred between the enzyme concentration and polygalacturonase activity up to a concentration of 40 fig proteinjml, On the other hand, higher enzyme concentration showed no influence on the enzymic activity even at concentrations 3 and 4 times that of the optimum activity. The rate of increase of polygalacturonase activity was slow at low temperatures and reached a maximum at 40°C. Further rise of temperature from 45 °C to 60°C did not cause marked loss of activity. Thus the enzyme retained 90 % of its activity at 55 °0 and 60 °0. Similarly, at 65 °C the loss in enzymic activity amounted only
286
S. S. MABROUK et al. , P reparation a n d Properties of P ectic Enzymes
Table 7. Effect of p H value on t ho act iv it y of polygalacturonase pH value
P G act ivi ty
3.45 3.72 4.05 4.45 4.99 5.2 7 5.89
51.45 53.44 55.53 60.72 55.8 3 39.23 39.93
(% re duct ion in viscos ity)
to about 24 %. However, the enzyme activity dropped to more than half its act ivity by further r ise of temperature from 65 °0 to 70 °0. This may show that t he enzyme was st ill active at 70 °0, and that the polygalacturonase preparation involved may be heat stable. The data recorded in Table 7 indicate that the enzym e showed a maximal activity at pH 4.45. This result is consist ent with the results obta ined by SHIGETAKA and TAMoTsu (1972). Generally, the polygalacturonase activity did not show a marked change with increasing pH of th e reaction mixture from 3.45- 4.99 in the acetate buffer. However, the enzy me lost about 35 % and 40 % of its activity at pH 5.27 and 5.89, r espectively. These p H values were evidently unfavour abl e for polygalact uronase act ivity and indica ted that t he enzyme involved was acid ic in nature.
References I. ABDEL-FATTAH, A . F . : Polyga la cturonase fr om citric a ci d producing mycelia. J . Che rn. U .A. R. 12 (1969), 559 -568. 2. - :MABROUK, S. S. , a n d EL.HAWWARY, N. J\L: Producti on a nd so me properties of r en n in like m ilk -olotbing enzyme fr om P enicillium citrinum . J. Gen . Microbiol. 70 (1972), 151 -1 55. 3. - - a n d I S~IAIL , A . S . : P rod uction of pol yg al acturon a se a nd p ect.in -me thylesterase by fungi . Chemie, Mik r obi ol. , T echnologi e der L eb en smit t el (in pre ss). 4. B ARTFEY, J. , a nd GAv ALYA, S .: P reparation of p ect oly t ic enzymes from m y celi a of Aspergillus niger. Elelmez esi Ipar 15 (1961),371 -3 75. 5. BERGKVIST, R . : The proteolytic e nzym es of Aeperqillus oryzae. I . Me th ods for t he estimation and iso lation of t he proteolytic e nzymes. Acta Chern . Scand. 17 (1963), 1521-1 540. 6. B LIEVA,R . K . : I sola t ion of a p cctolytic en zym e com plex from a su bm erged culture of Aspergillus nige r a nd m ethod s of purifying the preparati on. Tr . I n st. Mik r obiol. Viru sol. Akad , K a z. SS R No. 17 (1971) ,1 23-1 29. 7. E FREMOVA, L. L., KOSTIK, F . D ., a n d OSIPOVA, R. A.: E x op olygal a cturonase of Rh.izop us arrhizus. Izv. Akad. Nauk Mold. SS R , Ser. Biol. Khim. N auk 4 (1971),36-40. 8. KERTESZ, Z. 1.: The P ect ic Sub stances . New York 1951. 9. L OWRY, O. H., ROSEBROUGH, N . J ., FARR, A. L., and RANDALL, R . J.: Protein measurem en t with t he folin phenol r eage n t . J. B iol, Che rn. 193 (1951) , 265 - 275. 10. MCCOMB , E. A. , and MCCREADY, R . M.: Use of t he h y droxa m ic acid r ea ct ion for d etermin ing pe ctinesterase a ctiv it y . Stain T echn ol. 33 (1958) , 129-1 31. II. OSMAN, H . G., ABDEL-FATTAII, A. F ., ABDEL-SAMIE, 1\1., and MABROUK, S. S .: Product ion of a m ilk -clot t ing enzym e prepa ra t ion by Aspergillu 8 niger and t he effe ct of vario us fa ct ors on its activ ity. J . Gen. Microbiol. 59 (1969) ,1 25-1 29. 12. SWGETAKA, 1. , a nd TA~IOTsu , Y . F . : Purifica ti on and proper ti es of en d o-polygalac t uron ase fr om A sp erqillus [ap onicus . Ag ri c. Biol, Chern. 36 (1972). 1885 -1 893. Author's addre ss : As soc. P ro f. Dr. SOUHAIR S. MABROUK, L a bora t ory of Microbi al Che mi stry, Nat ional R esearch Centre of E gypt , Dokki - Cai ro, E gy pt.
[Laboratory of Microbial Che mistry, National R esearch Centre , Do kk i, a nd Aca de my of Scientifi c R esearch an d T echnology, Cairo, E gypt]
Preparation and Properties of Pectic Enzymes Produced by Trichoderma ligrwrum SOUHAIR S . MABROUK, AHMED F. ABDEL-FATTAH,
and
ABDEL-MoHSEN S. I SMAIL
Summary Polygalacturonase and pe ct in-methylesterase were isolated from shaken culture of Trichoderm a lignorum. Isolation was carried ou t with various agents. Methanol was the most suitable precipitant for isolating polygalacturonase, y ieldin g en zym e preparations 6.6 times more active than that of cul ture filtrate. Likewise, tannin afforded a ctive fracti ons at p H 4 and 0.05 % conc entration . Similarly, 50 % ammonium sul phate satu ra t ion gave a ctive fraction s. The lea st polygalacturona se activity was ob taine d fr om ethanol. I n any of t he organic solvents used, high est en zy mic a ct ivity was ob taine d whe n us in g only one volume. As regards poct in -methyl eaterase, n o corre lation exi sted between it s a ctivity a nd concentration of the precipi tant used. A subs trate concentration ab ove 0.8 % was a limiting fa ct or for polygala cturonase act ivi ty , wh ile optimum en zyme conce ntra tion was 40 flg prot einjml at 40 °C an d p H 4.4 5.
Zusammenfassung P olygal akturonase un d P ek t inm ethyl est erase wurde n aus Sch iittelkulturen von Trichoderma lignoru m isoli erb, AlsFallungsmitt elliefer t eM ethanol b efri edigende E rgebnisse. Di e EO gewonnenen E n zyrnpraparate ware n 6,6mal a ktiver al s di e Kulturfiltrat e. Auch 0,05 % Gerbsaure (p H 4) so wie eine 50 % Ammon sulfa tl osu n g en thielt en aktive Frakt ionen. B ei den organisch en L osungs mitteln wurde die hochst e E nzym akti vi tiit im me r dann er halte n , wenn nur ein Volum en d es L osungsmittels verwe n de t wurd e. B ei d el' P ektinmeth ylest era se best eh t k eine K orrela t ion zwischen Enzymakt ivi tiit u nd Konzentration des F allungsm itt els. E in e Substratkon zentration vo n 0, 8 % erwies sich als limitier ender Faktor fur die P olygala kt u ronaseak t.ivit a t . Di e optimale E nz ymkonz en t ration bet rug 40 flg Proteinjml bei 40 °C und p H 4,45.
P ectinases are known t o be imp ortant in the food and t extile industries. Numerou s organisms have been r eported t o produce these enzy mes (8). Extracellular and intracellular pectinases have been also reported to be ext racte d from moulds (1,4). Th e pre sent work deals with isolat ion of polygalacturonase and pectin-methylest erase from shaken cultures of T richoderma lignorum . In ad ditio n some properties of the par ti ally purified enzymes were investi gated. Thi s mould pr oved in a pr evious work (3) t o produce t he highest extrace llular polygalacturonase and pect in-m ethyleste rase using shaken cultures. The mould was grown on a medium t hat afford ed best enzy mic acti vit ies among ot her media investi gated.
Materials and Methods Microorgani sm T ri choderm a lig norum, a local isola t e, wa s ki n dly provided by Dr. D. A. ZARr, Botany Depa rt. m en t , Faculty of Science , Cairo Univer sity, Giza, E gypt .
Preparation and Properties of Pectic Enzymes
283
Pectin This was apple pectin, a preparation of Fluka AG. Buchs SG, Switzerland.
Culture medium The following culture medium was used (gil): pectin, 15; KH2P 0 4 , 0.5; MgS0 4 KN03 , 2.5.
•
7 H 2 0 , 0.5;
Cultivation Transfers were made from the subcultures to Dox's agar plates, which were then incubated at 30°C for 7 days. Liquid cultures were grown in 250 ml flat-bottomed flasks, each containing 50 ml sterile medium. Two discs, each of 1 em diameter, were cut from the 7 day-old culture plates and used for inoculating each flask. After incubation for different periods at 30°C (shaken cultures) the culture medium from each flask was filtered off and made up to 50 ml with distilled water.
Protein determination This was done by the method of LOWRY, ROSEBROUGH, FARR and RANDALL (1951).
Assay for polygalacturonase activity This was done by measuring the decrease in the viscosity of buffered pectin solution. 1.5 ml of the enzyme solution portions were added to 6.0 ml of 0.5 % pectin solution (0.02 M acetate buffer of pH 4.05) and the reaction mixtures were incubated at 40°C for 20 min. Thereafter, 5 ml portions of the reaction mixture were pipetted into an Ostwald viscosimeter in a water bath at 40°C and the time of run was recordea. In control tests heated enzyme solutions were employed.
Detection of pectin-methylesterase activity This was achieved using the hydroxamate method of MCCOMB and MCCREADY (1958). The appearance of a clear zone on the buffered pectin-agar plate, where the enzyme sample was applied, indicated a positive reaction for pectin-methylesterase.
Results and Discussion With all the precipitants used, a parallel relationship existed between the protein content and the concentration of the precipitant. This relation, however, was not the same with polygalacturonase activity except in the case of using ammonium SUlphate. This may be due to precipitation of some proteins in the culture medium other than the enzymes involved. It was of interest to notice that with any of the organic solvents used, the highest polygalacturonase activities were obtained when using only one volume. On the other hand, a sharp decrease in enzymic activity occurred when the culture filtrate was treated with two volumes. The same results were obtained with tannin. Methanol was the most suitable precipitant for isolating polygalacturonase (Table 1). Thus the fraction precipitated out by one volume yielded polygalacturonase preparations 6.6 times more active than that of culture filtrate. Likewise, tannin afforded active fractions at pH 4 and 0.05 %concentration (Tables 2 and 3). The enzymic activity of this fraction was 5.5 times that of the culture filtrate. Tannin was recommended by BERGKVIST (1963) for isolation of proteolytic enzymes from Aspergillus oryzae and it was also used for isolation of fungal milk- clotting and proteolytic enzymes (2, 11). As far as we are aware, this compound had not been used for pectolytic enzyme isolation. Isolation of polygalacturonase with acetone, similarly gave active fractions (Table 4) amounting to about 4 times that of the unprecipitated fractions.
284
S. S. MABROUK et al.
Table 1. Polygalacturonase activity and pectin-methylesterase reaction of methanol precipitates of Trichoderma lignorum enzyme preparations CF + methanol (v/v) Protein content of ppt Ipg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
1+ 1 16.75
1+ 2 49.5
1+ 4 61.75
1+ 6 76.00
1+ 8 80.50
1 + 10 114.75
26.56
45.87
43.86
44.83
46.27
46.67
1.58 6.60 +1
0.92 3.84 +4
0.66 2.74 +3
0.58 2.46 +1
0.58 2.38 +2
0.40 1.70 +2
In this and the following Tables: CF = Culture filtrate, ppt = Precipitate, PG = Polygalacturonase, P /CF = Precipitate/culture filtrate polygalacturonase activity ratio, PME = Pectin-mothylosterase, + 1 = Weak reaction, + 2 = Moderate reaction, + 3 and + 4 = Strong reaction.
Table 2. The effect of tannin concentration on the polygalacturonase activity and pectin-methylesterase reaction of the precipitates of Trichoderma lignorum enzyme preparations from culture filtrate of pH 4.0 Tannin(%) Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
0.05 23.2
0.10 87.8
0.30 132.0
0.50 219.0
0.70 216.0
0.90 259.5
1.0 258.0
30.31
40.41
47.78
57.87
56.71
57.46
57.11
1.30
0.48
0.36
0.28
0.28
0.24
0.24
5.50 +1
1.96 +1
1.52 +1
1.12 +2
1.12 +2
0.92 +1
0.92 +1
Table 3. The effect of the pH value of the culture filtrate on the polygalacturonase activity and pectin-methylesterase reaction of precipitates by 0.05 % tannin pH ofCF
Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
4.0 23.2
4.5 63.0
5.0 76.5
5.5 63.0
6.0 200.0
6.5 100.0
7.0 266.0
30.31
18.86
15.0
6.2
4.80
6.1
2.43
1.30
0.30
0.20
0.10
0.02
0.06
0.01
5.50 +1
1.26 +1
0.80 +1
0.42 - ve
0.08 - ve
0.24 - ve
0.04 - ve
- ve: No reaction. Table 4. Polygalacturonase activity and pectin-methylesterase reaction of acetone precipitates of Trichoderma lignorum enzyme preparations CF + acetone (v/v) Protein content of ppt (pg/ml) PG activity (% reduction in viscosity) PG activity/pg protein (P) P/CF PME reaction
1+ 1 50.0
1+ 2 121.0
1+ 4 124.0
1+ 6 132.0
1+ 8 134.2
1 + 10 125.5
49.97
48.13
51.58
49.69
50.25
51.70
1.00 4.2 +1
0.40 1.66 +3
0.42 1.72 +4
0.36 1.56 +3
0.36 1.56 +2
0.40 1.70 +2
Preparation and Properties of Pectic Enzymes
285
Table 5. Polygalacturonase activity and pectin-methylesterase reaction of ethanol precipitates of Trichoderma lignorum enzyme preparations CF + ethanol (v/v) Protein content of ppt (ltg/ml) PG activity (% reduction in viscosity) PG activity/ltg protein (P) P/CF PME reaction
1+ 1 93.0
1+ 2 131.0
1+ 4 178.0
1+ 6 178.0
1+ 8 185.0
1 + 10 194.0
55.24
54.72
54.45
55.66
49.14
55.72
0.58 2.84 +3
0.42 1.98 +3
0.30 1.46 +3
0.32 1.48 +3
0.26 1.26 +4
0.28 1.38 +4
Table 6. Polygalacturonase activity and pectin-methylesterase reaction of ammonium sulphate precipitates of Trichoderma lignorum enzyme preparations Ammonium sulphate saturation (%) 25.0 7.5 Protein content of ppt (ltg/ml) 14.1 PG activity (% reduction in viscosity) 1.88 PG activitY/ltg protein (P) 4.36 P/CF PME reaction - ve
40.0 9.0
50.0 12.0
66.0 22.0
80.0 26.0
100.0 24.5
18.6
25.5
24.4
52.0
43.5
2.06 4.80 +1
2.12 4.94 +2
1.10 2.58 +2
2.00 2.64 +4
1.78 4.12 +3
The least polygalacturonase activity was obtained from ethanol (Table 5). However higher results were obtained by EFREMOVA, KOSTIK and OSIPOVA (1971) for the isolation of exopolygalacturonase from Rhieopus arrhieus. As regards ammonium sulphate, the data recorded in Table 6 indicated that the fractions precipitated were characterized by lower values of both protein content and polygalacturonase activity. However, 50 % ammonium sulphate saturation afforded active fractions about 5 times that of culture filtrate which was more or less double that obtained by BLIEVA (1971). Generally, no loss of polygalacturonase activity occurred with all the precipitants investigated. Concerning pectin-methylesterase, the results did not show any correlation between its activity and concentration of the precipitant used. Similarly no relation existed either between the enzyme activity and protein content, or between enzyme activity and polygalacturonase activity. It is worthwhile to mention that the highest peetin.methylesterase activity was obtained by ethanol which exhibited the least polygalacturonase activity. The results demonstrated that substrate concentration above 0.8 % was a limiting factor for polygalacturonase activity between 0.5-0.8 % substrate concentration. However, 0.2 % concentration showed about 50 % loss in enzymic activity when compared to the optimum obtained at 0.8 %. This loss may be due to an insufficient amount of substrate to be acted upon by the enzyme. An inverse relationship occurred between the enzyme concentration and polygalacturonase activity up to a concentration of 40 fig proteinjml, On the other hand, higher enzyme concentration showed no influence on the enzymic activity even at concentrations 3 and 4 times that of the optimum activity. The rate of increase of polygalacturonase activity was slow at low temperatures and reached a maximum at 40°C. Further rise of temperature from 45 °C to 60°C did not cause marked loss of activity. Thus the enzyme retained 90 % of its activity at 55 °0 and 60 °0. Similarly, at 65 °C the loss in enzymic activity amounted only
286
S. S. MABROUK et al. , P reparation a n d Properties of P ectic Enzymes
Table 7. Effect of p H value on t ho act iv it y of polygalacturonase pH value
P G act ivi ty
3.45 3.72 4.05 4.45 4.99 5.2 7 5.89
51.45 53.44 55.53 60.72 55.8 3 39.23 39.93
(% re duct ion in viscos ity)
to about 24 %. However, the enzyme activity dropped to more than half its act ivity by further r ise of temperature from 65 °0 to 70 °0. This may show that t he enzyme was st ill active at 70 °0, and that the polygalacturonase preparation involved may be heat stable. The data recorded in Table 7 indicate that the enzym e showed a maximal activity at pH 4.45. This result is consist ent with the results obta ined by SHIGETAKA and TAMoTsu (1972). Generally, the polygalacturonase activity did not show a marked change with increasing pH of th e reaction mixture from 3.45- 4.99 in the acetate buffer. However, the enzy me lost about 35 % and 40 % of its activity at pH 5.27 and 5.89, r espectively. These p H values were evidently unfavour abl e for polygalact uronase act ivity and indica ted that t he enzyme involved was acid ic in nature.
References I. ABDEL-FATTAH, A . F . : Polyga la cturonase fr om citric a ci d producing mycelia. J . Che rn. U .A. R. 12 (1969), 559 -568. 2. - :MABROUK, S. S. , a n d EL.HAWWARY, N. J\L: Producti on a nd so me properties of r en n in like m ilk -olotbing enzyme fr om P enicillium citrinum . J. Gen . Microbiol. 70 (1972), 151 -1 55. 3. - - a n d I S~IAIL , A . S . : P rod uction of pol yg al acturon a se a nd p ect.in -me thylesterase by fungi . Chemie, Mik r obi ol. , T echnologi e der L eb en smit t el (in pre ss). 4. B ARTFEY, J. , a nd GAv ALYA, S .: P reparation of p ect oly t ic enzymes from m y celi a of Aspergillus niger. Elelmez esi Ipar 15 (1961),371 -3 75. 5. BERGKVIST, R . : The proteolytic e nzym es of Aeperqillus oryzae. I . Me th ods for t he estimation and iso lation of t he proteolytic e nzymes. Acta Chern . Scand. 17 (1963), 1521-1 540. 6. B LIEVA,R . K . : I sola t ion of a p cctolytic en zym e com plex from a su bm erged culture of Aspergillus nige r a nd m ethod s of purifying the preparati on. Tr . I n st. Mik r obiol. Viru sol. Akad , K a z. SS R No. 17 (1971) ,1 23-1 29. 7. E FREMOVA, L. L., KOSTIK, F . D ., a n d OSIPOVA, R. A.: E x op olygal a cturonase of Rh.izop us arrhizus. Izv. Akad. Nauk Mold. SS R , Ser. Biol. Khim. N auk 4 (1971),36-40. 8. KERTESZ, Z. 1.: The P ect ic Sub stances . New York 1951. 9. L OWRY, O. H., ROSEBROUGH, N . J ., FARR, A. L., and RANDALL, R . J.: Protein measurem en t with t he folin phenol r eage n t . J. B iol, Che rn. 193 (1951) , 265 - 275. 10. MCCOMB , E. A. , and MCCREADY, R . M.: Use of t he h y droxa m ic acid r ea ct ion for d etermin ing pe ctinesterase a ctiv it y . Stain T echn ol. 33 (1958) , 129-1 31. II. OSMAN, H . G., ABDEL-FATTAII, A. F ., ABDEL-SAMIE, 1\1., and MABROUK, S. S .: Product ion of a m ilk -clot t ing enzym e prepa ra t ion by Aspergillu 8 niger and t he effe ct of vario us fa ct ors on its activ ity. J . Gen. Microbiol. 59 (1969) ,1 25-1 29. 12. SWGETAKA, 1. , a nd TA~IOTsu , Y . F . : Purifica ti on and proper ti es of en d o-polygalac t uron ase fr om A sp erqillus [ap onicus . Ag ri c. Biol, Chern. 36 (1972). 1885 -1 893. Author's addre ss : As soc. P ro f. Dr. SOUHAIR S. MABROUK, L a bora t ory of Microbi al Che mi stry, Nat ional R esearch Centre of E gypt , Dokki - Cai ro, E gy pt.