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Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase
Enzyme and Microbial Technology 32 (2003) 231–235
Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase S. Gargova, M. Sariyska∗ Department of Biotechnology, Higher Institute of Food and Flavour Industries, 26 Maritza Blvd., Plovdiv, Bulgaria Received 4 June 2002; received in revised form 20 June 2002; accepted 11 September 2002
Abstract The reduction of phytate content in plant feeds is advisable for increasing of their nutritional values. The dephosphorylation of phytates is believed to be mainly effected by phytase. A strain of Aspergillus niger shows a high potential for phytase production. In this study the possibilities to increase the enzyme production by alteration of the growth conditions of Aspergillus niger 307 are investigated. The effect of the type, age and quantity of the inoculum was established. The strain showed maximal productivity when about 50-day-old spores were used as inoculum and the augmentation of phytase and acid phosphatase activities was 3.29-fold and 2.17-fold, respectively. Two-fold increase of the phytase activity was observed when the inorganic phosphorus concentration in the media was 10 mg%. The highest level of acid phosphatase activity was produced at 17.5 mg% Pi . The unpurified phytase from Aspergillus niger 307 shows two pH optima—at 2.5 and 5.0 and temperature optimum (Topt ) is 56 and 58 ◦ C, respectively. The pH optimum for acid phosphatase action is 2.1 and Topt is 73 ◦ C. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Aspergillus niger; Phytase; Acid phosphatase; Growth conditions
1. Introduction Phytase (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) belongs to the family of histidine acid phosphatases [1]. It catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) to inositol and ortho-phosphoric acid. The salts of phytic acid, i.e. the phytates, are permanent ingredients of plant tissues and are the major storage form of phosphorus in them [2]. Monogastric animals are not able to utilize phytic acid phosphorus, since they have no or very low levels of phytase activity in their digestive tracts. This necessitates additional supplementation with inorganic phosphate (Pi ) of their diets or preliminarly treatment of plant raw materials for phosphorus liberation. On the other hand, the phytate excreted by animals causes environmental problems (eutrophication), especially in areas with intensive pig and poultry farming. Simultaneously, the interaction of phytic acid with essential dietary minerals (Fe, Zn, Mg, Ca, etc.), protein or vitamins is considered to be one of the primary factors limiting the nutritional values of cereals and legumes in man and animals, which makes it an antinutritional factor. Therefore, ∗
Corresponding author. Tel.: +359-32-603-646. E-mail address: [email protected] (M. Sariyska).
decreasing the phytate content in plant materials to increase their nutritional values is advisable. This helps explain the increased interest in phytate-degrading enzymes, which is evident from the published reviews [3–7]. Phytase has been reported in cereals, legumes, oilseeds [8,9] and in animal tissues [3], but it is also produced by some microorganisms such as bacteria, yeast and fungi. Several phytase-producing bacteria have been examined— Klebsiella aerogenes [10], Bacillus subtilis [11,12], Escherichia coli [13] and some ruminal bacteria [14,15]. Some studies on phytase from Saccharomyces cerevisiae [16,17] and Schwanniomyces castellii [18–20] have been reported. The most active producers of extracellular phytase are fungi belonging to the genus Aspergillus. Well studied is the enzyme from Aspergillus ficuum [21–25]. Phytases have also been characterized from other aerobic fungi— Aspergillus carbonarius [26,27], Aspergillus oryzae [28] and Aspergillus niger [29]. A wild type strain of A. niger was isolated earlier in our investigations [30]. In the previous studies the maximal phytase activity at pH 5.0 was 52.1 nkat/cm3 and at pH 2.5–62.5 nkat/cm3 . We directed our efforts to enhance the enzyme production of A. niger 307 by more extensive investigation of some growth conditions and biosynthesis of the dephosphorylating enzymes phytase and acid phosphatase.
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 4 7 - 8
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2. Materials and methods 2.1. Chemicals Phytic acid dodecasodium salt was purchased from Sigma Chemical Co. (St. Louis, MO, USA); p-nitrophenol (p-NP), p-nitrophenylphosphate (p-NPP), H2 SO4 and glycine were obtained from Merck (Darmstadt, F.R., Germany). All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Strain and media Stock cultures of A. niger 307 were maintained at 30 ◦ C on malt agar slants. The cultivation was performed at 30 ◦ C on a rotary shaker (220 rpm) in 300 cm3 Erlenmeyer flasks containing 30 cm3 liquid fermentation media. The fermentation media used was the corn starch media described by Shieh and Ware [31] in which 40 g/l starch was added instead of 80 g/l (our unpublished data). 2.2.2. Analytical methods The enzyme activities were assayed on fermentation broths obtained after removing of the biomass at the end of the process. Phytase activity (PhA) was determined according to Ullah and Gibson with substrate sodium phytate at concentration 500 nmol in final volume of 1 cm3 [32]. The reaction was carried out at two pH values—5.0 and 2.5 at 58 ◦ C for 10 min. The liberated inorganic ortho-phosphate was measured according to Heinonen and Lahti [33] at 355 nm in a Secomam spectrophotometer (Secomam ANTHELIE Light V 1.0, Domont Cedex, France). The phytase activity (nkat) was expressed as nmol inorganic phosphate released per second. Acid phosphatase (APhA) was detected by measuring the release of p-NP from p-NPP at pH 2.5 and 30 ◦ C (Ullah and Gummins, [34]). The enzyme activity (nkat) was defined as nmol p-NP released per second. The biomass (BM) was measured after drying at 105 ◦ C. The phosphorus content in the starch was determined after mineralization and the sample was assayed for Pi according to Heinonen and Lahti [33]. Protein concentrations of fermentation broths were determined according to Lowry et al. [35]. Bovine serum albumin was used as standard.
When a conidial inoculum was used for spore propagation A. niger 307 was grown on malt agar (Difco, Detroit, MI, USA) for 7–56 days. The conidial suspensions were obtained by washing the stock tube cultures with sterile salt solution (0.7%, w/v) and the suspensions were filtered through a cotton swab in a glass tube. The liquid medium was inoculated with 2–3 × 107 spores/cm3 of media. The increase of the age of the spore inoculum correlates with increase in both studied enzyme activities (Fig. 1A). The strain shows maximal productivity when about 50-day-old spores were used as inoculum and the augmentation of the activities compared to the control (7-day-old spores) was 3.29-fold for PhA5.0, 3.78-fold for PhA2.5 and 2.17-fold for APhA. This is the first step in our investigations leading to a considerable increase of the phytase production. The time course of the cultivation was also influenced by the age of the spores (Fig. 1B). When 7-day-old conidia were used as inoculum maximal PhA5.0 was detected at 11th day of cultivation, but, with the increase in spore age the highest levels of phytase were achieved for 7 days. To obtain primary cultures of A. niger 307, the liquid starch medium was inoculated with the spore suspension described above and 1, 5 or 10% (v/v) of it was then added to the secondary culture media. Our assumption, that using a vegetative inoculum would shorten the time for maximal phytase production was not confirmed. The results of the analysis on the 3rd and 5th
3. Results and discussion 3.1. Type, age and quantity of inoculum It is known that the degree of influence of this factor depends on the specific physiological features of the microorganisms and it must be determined on a strictly individual basis.
Fig. 1. Influence of the age of spore inoculum on: (A) biosynthesis of dephosphorylating enzymes and (B) duration of the cultivation (phytase activity at pH 5.0 is shown).
S. Gargova, M. Sariyska / Enzyme and Microbial Technology 32 (2003) 231–235
Fig. 2. Phytase activity at pH 5.0 depending on the age and quantity of the primary culture.
days secondary culture development were lower than those determined on the 7th day. It is worth noting that PhA was higher when 1% (v/v) of primary culture was used as compared with inoculation with higher volumes of inoculum (Fig. 2). This is due to the faster exhausting of the nutrition substances in the media in case of higher amount of biomass imported with the inoculum. Most appropriate was the inoculation with 1% (v/v) of 24-h old primary culture, but the activity was not higher than that obtained by using of conidial inoculum. 3.2. Phosphorus in the nutritient media Several investigators established that phytase production is limited by the amount of Pi in the nutrition media— 20 mg/100 ml for A. ficuum [36], 0.006% (w/v) for A. niger [37]. High levels of Pi are known to repress the biosynthesis of the enzyme. In our studies, the Pi was supplied as K2 HPO4 in concentrations from 0 to 40 mg%. When a spore inoculum was used and the medium was not supplied with Pi , good growth and enzyme synthesis were observed (Fig. 3). Obviously, the strain satisfied its physiological needs of phosphorus from the starch, where the Pi concentration determined by us was 2.3 mg/g starch. Concentrations of Pi higher than 20 mg% did not effect the cell growth but strongly suppressed the biosynthesis of phytase. At a phosphorus concentration of 10 mg% the PhA at both pH values of determination was almost two times higher compared to the control (20 mg% Pi )—the PhA found at pH 5.0 and 2.5 was 315 and 559 nkat/cm3 , respectively, and APhA was 175 nkat/cm3 . This was the second step in our research resulting in considerable increase of the productivity of the strain. A maximal value of APhA was found at 17.5 mg% Pi . Those values were close to activities reported for genetically transformed strain Aspergillus terreus—22 mol/min, and higher than those of Myceliophtora thermophila (also a recombinant strain)—8 mol/min [1], A. carbonarius (2.75 U/g wet solid state culture) [26], A. oryzae (0.4 U/cm3 ) [28], A.
233
Fig. 3. Effect of Pi on development of the strain, and, phytase and acid phosphatase activities.
niger—132 nkat/cm3 [29], A. ficuum—22 nkat/cm3 [38], Aspergillus fumigatus—21.6 nkat/cm3 [39]. A two-fold increase of the PhA was also observed when a vegetative inoculum was used and the media contained 10 mg% Pi (data not shown). 3.3. Initial pH of the nutritient medium Under the modified conditions of use of old inoculum (50-day) and decreased amount of Pi (10 mg%) in the medium it was of interest to investigate the effect of the initial pH value on the studied activities, which was ranged from 3 to 8. The optimal value of the initial pH of the media for phytase production from A. niger 307 was 5.0 (Fig. 4). 3.4. Dynamics of the process of cultivation The strain developed most intensively up to the 72-h, as about 98% of the maximal amount of biomass was accumulated (Fig. 5). For 3 days the quantity of mycelium remained at an almost constant level, whereupon, it began to decrease. Maximal PhA was observed at the end of the
Fig. 4. Effect of the initial pH value on phytase and acid phosphatase activities.
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Fig. 5. Dynamics of growth, phytase and acid phosphatase production of Aspergillus niger 307.
stationary phase of the development of the strain and at the beginning of the phase of lysis, which was due to the intensified secretion of the enzyme from the cells. At the end of the process the PhA sharply diminished in contrast to APhA which continues to increase. 3.5. Some properties of the unpurified phytase and acid phosphatase
that at pH 2.5, which can be explained with its combined action with the acid phosphatase at the lower pH values. The optimal temperature (Topt ) for the action of the enzyme was 58 ◦ C at pH 5.0 and 56 ◦ C at pH 2.5 (Fig. 7). Optimal pH value for acid phosphatase action was 2.1 and Topt was 73 ◦ C. In conclusion, A. niger 307 produced high levels of dephosphorylating enzymes phytase and acid phosphatase. Their catalytic and physico-chemical properties will be
The unpurified phytase showed two pH optima—at pH 2.5 and 5.0 (Fig. 6). The activity at pH 5.0 is about 70% of
Fig. 6. pH profiles of the action of phytase and acid phosphatase.
Fig. 7. Temperature profiles of the action of phytase and acid phosphatase of Aspergillus niger 307.
S. Gargova, M. Sariyska / Enzyme and Microbial Technology 32 (2003) 231–235
further investigated in order to obtain an enzyme suitable for application.
References [1] Mitchell DB, Vogel K, Weimann BJ, Pasamontes L, van Loon A. The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophtora thermophila. Microbiology 1997;143:245–52. [2] Lott JNA, Ockenden I, Raboy V, Batten GD. Phytic acid and phosphorus in crop seeds and fruits: a global estimate. Seed Sci Res 2000;10:11–33. [3] Zyla K. Mould phytases and their application in the food industry. World J Microbiol Biotechnol 1992;8:467–72. [4] Liu BL, Rafiq A, Tzeng YM, Rob A. The induction and characterization of phytase and beyond. Enzyme Microb Technol 1998;22: 415–24. [5] Wyss M, Pasamontes L, Friedlein A, Remy R, Tessier M, Kronenberger A, et al. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of protoelitic resitance. Appl Environ Microbiol 1999;65:359–66. [6] Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, et al. Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 1999;65:367–73. [7] Frossard E, Bucher M, Machler F, Mozafar A, Hurrell R. Potential for increasing the content and bioavailability of Fe, Zn and Ca in plants for human nutrition. J Sci Food Agric 2000;80:861–79. [8] Greiner R, Alminger ML. Purification and characterization of a phytate-degrading enzyme from germinated oat (Avena sativa). J Sci Food Agric 1999;79:1453–60. [9] Nakano T, Joh T, Narita K, Hayakawa T. The pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases from wheat bran of Triticum aestivum L. cv. Nourin # 61A. Biosci Biotechnol Biochem 2000;64(5):995–1003. [10] Tambe SM, Kaklij GS, Kelkar SM, Parekh LJ. Two distinct molecular forms of phytase from Klebsiella aerogenes: evidence for unusually small active enzyme peptide. J Ferment Bioeng 1994;77(1):23–7. [11] Shimizu M. Purification and characterization of phytase from Bacillus subtilis (natto) N-77. Biosci Biotech Biochem 1992;56(8):1266–9. [12] Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J. Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 1998;64(6):2079–85. [13] Greiner R, Konietzny U, Jany KD. Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys 1993;303:107–13. [14] Yanke LJ, Bae HD, Selinger LB, Cheng KJ. Phytase activity of anaerobic ruminal bacteria. Microbiology 1998;144:1565–73. [15] D’Silva CG, Bae HD, Yanke LJ, Cheng KJ, Selinger LB. Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy. Can J Microbiol 2000;46: 391–5. [16] Zyla K. Phytate dephosphorylation by free and immobilized cells of Saccharomyces cerevisiae. J Ind Microbiol 1994;13:30–4. [17] Nayini NR, Markakis P. The Phytase of Yeast. Lebensm Wiss Technol 1984;17:24–6.
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[18] Lambrechts C, Boze H, Moulin G, Galzy P. Utilization of phytate by some yeasts. Biotechnol Lett 1992;14(1):61–6. [19] Lambrechts C, Boze H, Segueilha L, Moulin G, Galzy P. Influence of culture conditions on the biosynthesis of Schwanniomyces castellii phytase. Biotechnol Lett 1993;15(4):399–404. [20] Segueilha L, Moulin G, Galzy P. Reduction of phytate content in wheat bran and glandless cotton flour by Schwanniomyces castellii. J Agric Food Chem 1993;41:2451–4. [21] Gibson DM. Production of extracellular phytase from Aspergillus ficuum on starch media. Biotechnol Lett 1987;9(5):305–10. [22] Han YW, Gallagher DJ, Wilfred AG. Phytase production by Aspergillus ficuum on semisolid substrate. J Ind Microbiol 1987;2: 195–200. [23] Ullah A. Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization. Prep Biochem 1998;18(4):459–71. [24] Ullah A, Phillippy BQ. Substrate selectivity in Aspergillus ficuum phytase and acid phosphatase using myo-Inositol phosphates. Agric Food Chem 1994;42:423–5. [25] Liu BL, Jong CH, Tzeng YM. Effect of immobilization on pH and thermal stability of Aspergillus ficuum phytase. Enzyme Microb Technol 1999;25:517–21. [26] Al-Asheh S, Duvnjak Z. The effect of surfactants on the phytase production and the reduction of the phytic acid content in canola meal by Aspergillus carbonarius during a solid state fermentation process. Biotechnol Lett 1994;16(2):183–8. [27] Al-Asheh S, Duvnjak Z. Characteristicts of phytase produced by Aspergillus carbonarius NRC 401121 in canola meal. Acta Biotechnol 1994;14(3):223–33. [28] Shimizu M. Purification and characterization of phytase and acid phosphatase produced by Aspergillus oryzae K1. Biosci Biotech Biochem 1993;57(8):1364–5. [29] Volfova O, Dvorakova J, Hanzlikova A, Jandera A. Phytase from Aspergillus niger. Folia Microbiol 1994;39(6):481–4. [30] Gargova S, Roshkova Z, Vancheva G. Screening of fungi for phytase production. Biotechnol Tech 1997;11(4):221–4. [31] Shieh TR, Ware JH. Survey of microorganisms for the production of extracellular phytase. Appl Microbiol 1968;16(9):1348–51. [32] Ullah A, Gibson DM. Extracellular phytase (EC 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. Prep Biochem 1987;17(1):63–91. [33] Heinonen JK, Lahti RJ. A new and convinient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 1981;113:313–7. [34] Ullah A, Gummins BJ. Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (EC 3.1.3.2) from Aspergillus ficuum. Prep Biochem 1987;17(4):397–422. [35] Lowry OH, Rosenbrough HJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. [36] Shieh TR, Wodzinski RJ, Ware JH. Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum. J Bacteriol 1969;100(3):1161–5. [37] Chelius MK, Wodzinski RJ. Strain improvement of Aspergillus niger for phytase production. Appl Microbiol Biotechnol 1994;41:79–83. [38] Ullah A. Production, rapid purification and catalytic characterization of extracellular phytase from Aspergillus ficuum. Prep Biochem 1988;18(4):443–58. [39] Mullaney EJ, Daly CB, Sethumadhavan K, Rodriquez E, Lei XG, Ullah A. Phytase activity in Aspergillus fumigatus isolates. Biochem Biophys Res Commun 2000;275:759–63.
Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase S. Gargova, M. Sariyska∗ Department of Biotechnology, Higher Institute of Food and Flavour Industries, 26 Maritza Blvd., Plovdiv, Bulgaria Received 4 June 2002; received in revised form 20 June 2002; accepted 11 September 2002
Abstract The reduction of phytate content in plant feeds is advisable for increasing of their nutritional values. The dephosphorylation of phytates is believed to be mainly effected by phytase. A strain of Aspergillus niger shows a high potential for phytase production. In this study the possibilities to increase the enzyme production by alteration of the growth conditions of Aspergillus niger 307 are investigated. The effect of the type, age and quantity of the inoculum was established. The strain showed maximal productivity when about 50-day-old spores were used as inoculum and the augmentation of phytase and acid phosphatase activities was 3.29-fold and 2.17-fold, respectively. Two-fold increase of the phytase activity was observed when the inorganic phosphorus concentration in the media was 10 mg%. The highest level of acid phosphatase activity was produced at 17.5 mg% Pi . The unpurified phytase from Aspergillus niger 307 shows two pH optima—at 2.5 and 5.0 and temperature optimum (Topt ) is 56 and 58 ◦ C, respectively. The pH optimum for acid phosphatase action is 2.1 and Topt is 73 ◦ C. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Aspergillus niger; Phytase; Acid phosphatase; Growth conditions
1. Introduction Phytase (myo-inositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) belongs to the family of histidine acid phosphatases [1]. It catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) to inositol and ortho-phosphoric acid. The salts of phytic acid, i.e. the phytates, are permanent ingredients of plant tissues and are the major storage form of phosphorus in them [2]. Monogastric animals are not able to utilize phytic acid phosphorus, since they have no or very low levels of phytase activity in their digestive tracts. This necessitates additional supplementation with inorganic phosphate (Pi ) of their diets or preliminarly treatment of plant raw materials for phosphorus liberation. On the other hand, the phytate excreted by animals causes environmental problems (eutrophication), especially in areas with intensive pig and poultry farming. Simultaneously, the interaction of phytic acid with essential dietary minerals (Fe, Zn, Mg, Ca, etc.), protein or vitamins is considered to be one of the primary factors limiting the nutritional values of cereals and legumes in man and animals, which makes it an antinutritional factor. Therefore, ∗
Corresponding author. Tel.: +359-32-603-646. E-mail address: [email protected] (M. Sariyska).
decreasing the phytate content in plant materials to increase their nutritional values is advisable. This helps explain the increased interest in phytate-degrading enzymes, which is evident from the published reviews [3–7]. Phytase has been reported in cereals, legumes, oilseeds [8,9] and in animal tissues [3], but it is also produced by some microorganisms such as bacteria, yeast and fungi. Several phytase-producing bacteria have been examined— Klebsiella aerogenes [10], Bacillus subtilis [11,12], Escherichia coli [13] and some ruminal bacteria [14,15]. Some studies on phytase from Saccharomyces cerevisiae [16,17] and Schwanniomyces castellii [18–20] have been reported. The most active producers of extracellular phytase are fungi belonging to the genus Aspergillus. Well studied is the enzyme from Aspergillus ficuum [21–25]. Phytases have also been characterized from other aerobic fungi— Aspergillus carbonarius [26,27], Aspergillus oryzae [28] and Aspergillus niger [29]. A wild type strain of A. niger was isolated earlier in our investigations [30]. In the previous studies the maximal phytase activity at pH 5.0 was 52.1 nkat/cm3 and at pH 2.5–62.5 nkat/cm3 . We directed our efforts to enhance the enzyme production of A. niger 307 by more extensive investigation of some growth conditions and biosynthesis of the dephosphorylating enzymes phytase and acid phosphatase.
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 2 4 7 - 8
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S. Gargova, M. Sariyska / Enzyme and Microbial Technology 32 (2003) 231–235
2. Materials and methods 2.1. Chemicals Phytic acid dodecasodium salt was purchased from Sigma Chemical Co. (St. Louis, MO, USA); p-nitrophenol (p-NP), p-nitrophenylphosphate (p-NPP), H2 SO4 and glycine were obtained from Merck (Darmstadt, F.R., Germany). All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Strain and media Stock cultures of A. niger 307 were maintained at 30 ◦ C on malt agar slants. The cultivation was performed at 30 ◦ C on a rotary shaker (220 rpm) in 300 cm3 Erlenmeyer flasks containing 30 cm3 liquid fermentation media. The fermentation media used was the corn starch media described by Shieh and Ware [31] in which 40 g/l starch was added instead of 80 g/l (our unpublished data). 2.2.2. Analytical methods The enzyme activities were assayed on fermentation broths obtained after removing of the biomass at the end of the process. Phytase activity (PhA) was determined according to Ullah and Gibson with substrate sodium phytate at concentration 500 nmol in final volume of 1 cm3 [32]. The reaction was carried out at two pH values—5.0 and 2.5 at 58 ◦ C for 10 min. The liberated inorganic ortho-phosphate was measured according to Heinonen and Lahti [33] at 355 nm in a Secomam spectrophotometer (Secomam ANTHELIE Light V 1.0, Domont Cedex, France). The phytase activity (nkat) was expressed as nmol inorganic phosphate released per second. Acid phosphatase (APhA) was detected by measuring the release of p-NP from p-NPP at pH 2.5 and 30 ◦ C (Ullah and Gummins, [34]). The enzyme activity (nkat) was defined as nmol p-NP released per second. The biomass (BM) was measured after drying at 105 ◦ C. The phosphorus content in the starch was determined after mineralization and the sample was assayed for Pi according to Heinonen and Lahti [33]. Protein concentrations of fermentation broths were determined according to Lowry et al. [35]. Bovine serum albumin was used as standard.
When a conidial inoculum was used for spore propagation A. niger 307 was grown on malt agar (Difco, Detroit, MI, USA) for 7–56 days. The conidial suspensions were obtained by washing the stock tube cultures with sterile salt solution (0.7%, w/v) and the suspensions were filtered through a cotton swab in a glass tube. The liquid medium was inoculated with 2–3 × 107 spores/cm3 of media. The increase of the age of the spore inoculum correlates with increase in both studied enzyme activities (Fig. 1A). The strain shows maximal productivity when about 50-day-old spores were used as inoculum and the augmentation of the activities compared to the control (7-day-old spores) was 3.29-fold for PhA5.0, 3.78-fold for PhA2.5 and 2.17-fold for APhA. This is the first step in our investigations leading to a considerable increase of the phytase production. The time course of the cultivation was also influenced by the age of the spores (Fig. 1B). When 7-day-old conidia were used as inoculum maximal PhA5.0 was detected at 11th day of cultivation, but, with the increase in spore age the highest levels of phytase were achieved for 7 days. To obtain primary cultures of A. niger 307, the liquid starch medium was inoculated with the spore suspension described above and 1, 5 or 10% (v/v) of it was then added to the secondary culture media. Our assumption, that using a vegetative inoculum would shorten the time for maximal phytase production was not confirmed. The results of the analysis on the 3rd and 5th
3. Results and discussion 3.1. Type, age and quantity of inoculum It is known that the degree of influence of this factor depends on the specific physiological features of the microorganisms and it must be determined on a strictly individual basis.
Fig. 1. Influence of the age of spore inoculum on: (A) biosynthesis of dephosphorylating enzymes and (B) duration of the cultivation (phytase activity at pH 5.0 is shown).
S. Gargova, M. Sariyska / Enzyme and Microbial Technology 32 (2003) 231–235
Fig. 2. Phytase activity at pH 5.0 depending on the age and quantity of the primary culture.
days secondary culture development were lower than those determined on the 7th day. It is worth noting that PhA was higher when 1% (v/v) of primary culture was used as compared with inoculation with higher volumes of inoculum (Fig. 2). This is due to the faster exhausting of the nutrition substances in the media in case of higher amount of biomass imported with the inoculum. Most appropriate was the inoculation with 1% (v/v) of 24-h old primary culture, but the activity was not higher than that obtained by using of conidial inoculum. 3.2. Phosphorus in the nutritient media Several investigators established that phytase production is limited by the amount of Pi in the nutrition media— 20 mg/100 ml for A. ficuum [36], 0.006% (w/v) for A. niger [37]. High levels of Pi are known to repress the biosynthesis of the enzyme. In our studies, the Pi was supplied as K2 HPO4 in concentrations from 0 to 40 mg%. When a spore inoculum was used and the medium was not supplied with Pi , good growth and enzyme synthesis were observed (Fig. 3). Obviously, the strain satisfied its physiological needs of phosphorus from the starch, where the Pi concentration determined by us was 2.3 mg/g starch. Concentrations of Pi higher than 20 mg% did not effect the cell growth but strongly suppressed the biosynthesis of phytase. At a phosphorus concentration of 10 mg% the PhA at both pH values of determination was almost two times higher compared to the control (20 mg% Pi )—the PhA found at pH 5.0 and 2.5 was 315 and 559 nkat/cm3 , respectively, and APhA was 175 nkat/cm3 . This was the second step in our research resulting in considerable increase of the productivity of the strain. A maximal value of APhA was found at 17.5 mg% Pi . Those values were close to activities reported for genetically transformed strain Aspergillus terreus—22 mol/min, and higher than those of Myceliophtora thermophila (also a recombinant strain)—8 mol/min [1], A. carbonarius (2.75 U/g wet solid state culture) [26], A. oryzae (0.4 U/cm3 ) [28], A.
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Fig. 3. Effect of Pi on development of the strain, and, phytase and acid phosphatase activities.
niger—132 nkat/cm3 [29], A. ficuum—22 nkat/cm3 [38], Aspergillus fumigatus—21.6 nkat/cm3 [39]. A two-fold increase of the PhA was also observed when a vegetative inoculum was used and the media contained 10 mg% Pi (data not shown). 3.3. Initial pH of the nutritient medium Under the modified conditions of use of old inoculum (50-day) and decreased amount of Pi (10 mg%) in the medium it was of interest to investigate the effect of the initial pH value on the studied activities, which was ranged from 3 to 8. The optimal value of the initial pH of the media for phytase production from A. niger 307 was 5.0 (Fig. 4). 3.4. Dynamics of the process of cultivation The strain developed most intensively up to the 72-h, as about 98% of the maximal amount of biomass was accumulated (Fig. 5). For 3 days the quantity of mycelium remained at an almost constant level, whereupon, it began to decrease. Maximal PhA was observed at the end of the
Fig. 4. Effect of the initial pH value on phytase and acid phosphatase activities.
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Fig. 5. Dynamics of growth, phytase and acid phosphatase production of Aspergillus niger 307.
stationary phase of the development of the strain and at the beginning of the phase of lysis, which was due to the intensified secretion of the enzyme from the cells. At the end of the process the PhA sharply diminished in contrast to APhA which continues to increase. 3.5. Some properties of the unpurified phytase and acid phosphatase
that at pH 2.5, which can be explained with its combined action with the acid phosphatase at the lower pH values. The optimal temperature (Topt ) for the action of the enzyme was 58 ◦ C at pH 5.0 and 56 ◦ C at pH 2.5 (Fig. 7). Optimal pH value for acid phosphatase action was 2.1 and Topt was 73 ◦ C. In conclusion, A. niger 307 produced high levels of dephosphorylating enzymes phytase and acid phosphatase. Their catalytic and physico-chemical properties will be
The unpurified phytase showed two pH optima—at pH 2.5 and 5.0 (Fig. 6). The activity at pH 5.0 is about 70% of
Fig. 6. pH profiles of the action of phytase and acid phosphatase.
Fig. 7. Temperature profiles of the action of phytase and acid phosphatase of Aspergillus niger 307.
S. Gargova, M. Sariyska / Enzyme and Microbial Technology 32 (2003) 231–235
further investigated in order to obtain an enzyme suitable for application.
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