Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

ABSTRACT

This work studied the acid phosphatase (APase) activity from culture medium (extracellular, eAPase) and mycelial extract (intracellular, iAPase) of Aspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km) for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with V_max = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V _max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH ₂PO₄ and 5 mM (NH₄)₂MoO₄; eAPase was also inhibited by 1 mM CoCl_2.

Key words:
Enzyme inhibitors; molybdenum; overexpression; phosphate; soil chemical characteristics

INTRODUCTION

Phosphorus is one of the major macronutrients needed by the plants after nitrogen, and is indispensable to many biological processes. The most visible effect of its omission or reduction is the substantial decrease in plant growth, as has been reported in potato crops (Balemi et al. 2009). Approximately 95-99% of phosphorus is present in the insoluble form, either organic or inorganic, which is unavailable for plant growth. The amount of phosphorus available is very low in many soils and requires the application of fertilisers (Shen et al. 2011). However, soil microorganisms perform the mineralisation of organic phosphorus compounds and the solubilisation of insoluble mineral compounds, making them available to the plants. This mechanism depends on the environmental conditions and biological processes taking place in the soil (Malboobi et al. 2012).

Organic phosphorus content ranges from 30 to 50% of total soil P (Mullen 2005). Several hydrolytic enzymes play a major role in the mineralization processes of organic P substrates (Annaheim et al. 2010). Organic compounds, which appear as phosphate esters, are hydrolysed by phosphatases to produce phosphate, which can then be assimilated by the plants (Hidayat et al. 2006; Nannipieri et al. 2011). These enzymes are secreted by the plants and microorganisms when the content of soluble phosphate in the soil is low (Samuel et al. 2010). When the phosphorus content is high, these enzymes are repressed. Derepression occurs when the levels of phosphate become limiting. It can be inferred that in the soil, which is a complex system, the activity of phosphatases can also be controlled by other factors such as the concentration of substrates of the enzymes, the presence or absence of metals and others. Many elements are important for plant growth (Kevresan et al. 2001) and biological nitrogen fixation (Chagas et al. 2010) but amounts in excess can inhibit phosphatase acid activity (Huang and Shindo 2000; Tsekova et al. 2002, Quiquampoix and Mousain 2005). For example, acid phosphatase from Ustilago sp. was inhibited by Ca and Al (Onthong et al. 2007). Addition of Ni II ions to the culture medium caused a substantial decrease in Rhizopus delemar growth and acid phosphatase activity (Açikel and Ersan 2010).

Studies of phosphatases characteristics and control have been examined in A. niger. Four fungal strains of this fungus produced both acid phosphatase and alkaline phosphatase activities (Nopparat et al. 2007). Both intracellular and extracellular phosphatase activity was detected in A. niger, but the extracellular phosphatase activity of other species of fungi was repressed by the insoluble phosphorus (Rinu and Pandey 2010). Activity of phosphatase enzymes is affected by the chemical and physical characteristics of soil (Nannipieri et al. 2010; Fitriatin et al. 2011). Although there has been considerable research on the acid phosphatases in fungi, there is relatively little information about the effect of soil chemical characteristics on these enzymes. Further characterization of phosphatases is required to understand the mechanisms underlying the fungus response to soil Pi starvation. Also, the soil characteristics that affect the process of hydrolysis in P-organic compounds need to be known.

Aspergillus niger, a soil isolate showed a high solubilisation activity of insoluble phosphates, such as Ca, Fe and Al phosphate (Barroso and Nahas 2005). A preliminary study showed that this fungus possessed high phosphatase activity, which was important for the release of soluble phosphate from organic phosphate in the soil. Until now, no other study reported the overexpression of acid phosphatase in fungi. The purpose of this work was to describe the biochemical characteristics of both the extracellular and intracellular acid phosphatases from A. niger under the influence by chemical soil characteristics.

MATERIAL AND METHODS

Growth studies

The A. niger F111 strain was isolated from the soil in São Paulo, Brazil (Barroso and Nahas 2005). The fungus was grown on Sabouraud agar slants for seven days and was subsequently maintained at 4^oC. It was grown in Czapek-Dox broth that contained (g/L): 2.0 NaNO₃, 0.5 KCl, 0.5 MgSO₄.7H₂O, 0.01 FeSO₄, 0.01 and 30.0 sucrose. The pH was adjusted to 6.0. K₂HPO₄ was added at a concentration of 50 µM. When the effect of phosphate was studied, K₂HPO₄ was added at 50, 100, 200, 500, 1000 and 5000 µM. Spores were collected from the agar slants and counted in a Neubauer chamber. Then 0.5 mL of a spore suspension containing 22.3 x 10⁶spores mL^-1 was inoculated into 50 mL of the culture medium contained in Petri dishes. Plates were incubated at 30ºC for up to 96 h. Cultures were harvested by filtration at 24 h intervals in the time-course assay up to 96 h. Mycelial growth was determined after the mycelium was washed with 50 mL deionised water and dried in the oven at 105^oC for 24 h.

Enzyme activity determination

The cell-free filtrates and mycelial extract were used as enzyme source. Mycelia extract was obtained from the washed mycelia with 50 mL deionised water after being ground in a pre-cooled mortar with washed sand and 50 mM-sodium acetate buffer (pH 5.4). Measurement of enzyme activity was done using 4 mM p-nitrophenyl phosphate (p-NPP) in 0.1 M sodium acetate buffer, pH 5.4 (acid phosphatase), or in 0.3 M glycine buffer, pH 9.0 (alkaline phosphatase) as the enzyme substrate. The reaction mixture contained 1.8 and 0.2 mL of cell-free filtrate, or 1.9 mL of substrate and 0.1 mL of mycelium extract. The mixture was incubated at 37^oC in water bath for 5 (mycelia extract) or 30 min (cell-free filtrates). After incubation, 1.0 mL 1.0 M NaOH was added to the test tubes. The yellow colour complex of p-nitrophenol (pNP) was measured using a spectrophotometer at 405 nm. The amount of p-nitrophenol (pNP) released was calculated by referring to a standard curve. A unit of activity (U) was defined as the amount of enzyme required to liberate 1.0 μmol of pNP per minute under the assay conditions. Specific activities (U mg^-1 mycelium) were expressed as units mL^-1 hour^-1 mg^-1 mycelium dry weight.

Acid phosphatases properties

A set of assays was performed to examine the properties of acid phosphatase from cell-free filtrates and mycelia extract on the reaction mixture.

Effect of pH: The effect of pH was determined using 0.5 M buffer: citrate (pH 2.5-3.0), citrate-acetate (pH 3.5), acetate (pH 4.0-5.0), citrate (pH 5.5-6.0) and Tris-maleate (pH 6.5-7.0).

Effect of substrate concentration: The influence of substrate concentration was determined using p-NPP at 0.5, 1, 2, 4, 8 and 16 mM. The Michaelis constant (K_m) and maximum velocity (V_max) values were determined from a Lineweaver-Burk plot.

Effect of inorganic and organic compounds: Various compounds were added to the reaction mixture. The relation of these substances and the concentration are shown in Table 1.

Effect of molybdate: Enhancing concentrations of (NH₄)₂MoO₄ ranging from 0 to 250 mM were added in the reaction mixture.

Table 1
Effect of organic and inorganic compounds on the activity of extracellular (eAPase) and intracellular (iAPase) acid phosphatases fromAspergillus niger.

Compound	Concentration	eAPase	iAPase	(mM)	Residual activity (%)
Control	-	100.00	100.00
(NH4)2MoO4	5	43.84	3.88
CaCl2	1	86.49	72.32
Na2SO4	5	94.28	98.57
MgCl2	1	99.12	101.96
ZnSO4	1	86.96	106.56
KH2PO4	0.05	92.05	102.93
KH2PO4	0.1	92.41	103.49
KH2PO4	1	83.56	94.76
KH2PO4	10	53.54	51.26
(NH4)2SO4	1	90.02	107.85
CoCl2	1	28.04	100.14
Na and K tartarate	10	93.45	101.26
Na citrate	1	106.06	105.18
EDTA	0.1	97.85	110.26
EDTA	1	89.90	108.53
EDTA	10	98.15	108.68
Glucose	1	74.36	93.41
Tris*	1	69.73	92.72
Fenol	1	89.93	91.95
Fenol	10	98.05	90.57
Na acetate	0.1	79.18	103.07
Na acetate	1	91.94	118.82
Na acetate	10	102.03	91.03
Acetic acid	0.1	100.75	93.78
Acetic acid	1	110.83	97.85
Acetic acid	10	97.06	108.40

• *Tris (hydroxymethyl) aminomethane

Statistical analysis

All the results are sum means of three replicates. Data were subjected to regression analysis using the SAS statistical package for ANOVA Maximum and minimum points were calculated using Maple 8 software.

RESULTS AND DISCUSSION

Effect of inorganic phosphate (Pi)

According to Bünemann (2008), up to 60% of the total organic P can be hydrolyzed by phosphatases. The present results showed that the activity of phosphatases also depended on the content of the available soil P. In general, the P content of Brazilian soils is very low, ranging from 23 to 58 µg P mL^-1 soil (Marcelo et al. 2009) and from 15 to 79 µg P mL^-1 soil (Garcia and Nahas 2012). According to results (Fig. 1), the concentration of P of these soils was still sufficient to inhibit the activity of acid phosphatases from A. niger. However, depending on the concentrations of C and N, these amounts of P are consumed by microorganisms in the soil; the scavenge for new sources of P stimulates soil microorganisms in the production and secretion of phosphatases for P organic hydrolysis.

Figure 1
Effect of phosphate concentration on Aspergillus niger growth and acid phosphatases activities. (-●-) intracellular acid phosphatase; mycelium. (-○-) extracellular acid phosphatase.

Figure 1 shows the active growth of A. niger on the medium with increasing concentrations of Pi ranging from 50 to 5000 µM and decreasing activity of acid phosphatase (APase) from mycelial extract (intracellular,iAPase) and culture medium (extracellular,eAPase). A. niger grew up to Pi 200 µM and growth stopped at higher concentrations. The results clearly demonstrated that A. niger contained an intra and extracellular acid phosphatase, but not an alkaline phosphatase (data not included). The effect of the Pi concentration on the growth and phosphatase activity have been shown on A. niger (Gargova and Sariyska 2003; Hidayat et al. 2006). Various fungi exhibit both intracellular and extracellular APases; for example, A.fumigatus (Bernard et al. 2002) and A. niger, A. awamori, Emmericella nidulans, E. rugulosa, Penicillium simplicissimum and P. rubrum(Yadav and Tarafdar 2003). The present results suggested that both APase from A. niger were repressed by high concentrations of Pi. eAPase activity decreased by 86% and iAPase by 99% when Pi concentration was enhanced from 50 µM to 5000 µM. The activity of iAPase decreased more rapidly than that ofeAPase, suggesting that the iAPase activity was more repressed than the secreted enzyme. Repression of PhoAp activity by the presence of Pi in the culture medium was found in A. fumigatus (Bernard et al. 2002), A. niger 307 (Gargova and Sariyska 2003) and A. niger N402A (Hydayat et al. 2005). The results obtained in this study indicated that the APases seemed to be different molecular species; however, this needed to be confirmed.

iAPase activity was 12,128.17 and eAPase 263.09 U mg^-1 mycelium at a Pi concentration of 50 µM, which was 46.1 times higher. Higher activity of intracellular acid phosphatase in relation to extracellular was also found in A. niger, A. terreus and A. rugulosus, but in the ratio 10.1-10.9 (Tarafdar et al. 2001).A. niger is a fungus that is recognised by the high solubilising activity of insoluble phosphates, using a mechanism that involves the production of organic acids (Richardson and Simpson 2011; Braz and Nahas 2012). Thus, overexpression of iAPase seems to contribute in a major way to the metabolic pathway involved in the synthesis of organic acids from C sources. However, the overexpression ofiAPase by A. niger seemed to be an intriguing result and needed to be clarified. Yadav and Tarafdar (2003) reported that the extracellular/intracellular acid phosphatase ratio from seven species of fungi, including A. niger, ranged from 0.39 to 0.86. The ratio found in this study was very low, ranging from 0.02 (Pi 50 µM) to 0.15 (Pi 5000 µM), due to the low eAPase activity in relation to iAPase (Fig. 1). In contrast to these findings, various authors found a higher activity of extracellular APase in relation to the intracellular form from N. crassa (Han and Rossi 1989) and from Humicola lutea ( Aleksieva et al. 2002).

Time-course of the cultivation

At a Pi concentration of 50 µM, A. niger grew according to a 2^nd degree polynomial curve (y = -0.0199x²+ 0.3162x - 0.0005; R² = 0.98) and the pH of culture media (Fig. 2) decreased rapidly up to 3.0 according to a 3^rd degree polynomial curve (y = -0.035x³ + 0.4912x² - 2.1784x + 5.8974; R² = 0.98). The maximal theoretical values were obtained after 7.95 days of incubation for mycelium growth and after 5.74 days for pH decreases. Maximal activity of iAPase was found after 96h incubation, but eAPase activity decreased after 24 h (Fig. 2). In contrast, Tsekova et al. (2002) reported that the maximal activity of intra and intracellular enzyme was found after 12h ofA. niger growth. In addition, the extracellular phosphatase activity continued to increase even when the growth of A. niger307 had stabilised after three days of culture incubation in the stationary phase (Gargova and Sariyska 2003).

Figure 2
Time-course of Aspergillus niger growth and acid phosphatase activities. (-●-) intracellular acid phosphatase, mycelium; (-○-) extracellular acid phosphatase, final pH.

The decrease of eAPase activity during later fungus growth could be due to the repression of Pi production in the environment Phosphate is necessary for fungal growth and the APase secretion in Pi-limited concentration is essential for scavenging organic phosphorus for Pi uptake by the fungus. Intracellular acid phosphatases have multiple functions involved in the utilisation of Pi (Pawar and Thaker 2009) in the metabolic process of fungus growth. The synthesis of these enzymes is controlled by the concentration of Pi and is regulated by the interactions of regulatory genes in N. crassa(Davis 2002; Nozawa et al. 2002). It was concluded that the variation of Pi concentration in the soil could affect both the fungus growth and, in consequence, the production and secretion of phosphatases.

Effect of pH

Both the intracellular and extracellular APases had pH optimum 4.5 (Fig. 3). The eAPase activity decreased by 92% at pH 7.0 and the iAPase by 98% in relation to optimum pH 4.5. However, these results disagreed with those reported by Gargova and Sariyska (2003), who found the optimum pH to be 2.1 for acid phosphatase from A. niger 307. At 0-10 cm soil depth, the pH ranged from 5.4 to 6.5 in four crop sequences (Marcelo et al. 2009) and from 4.4 to 6.2 in agricultural and agricultural-pastoral systems (Garcia and Nahas 2012). It was concluded that secreted acid phosphatase activity from A. niger could be severely inhibited in these soils.

Figure 3
Effect of pH in the reaction mixture on the acid phosphatases activities. (-●-) intracellular acid phosphatase; (-○-) extracellular acid phosphatase.

Effect of substrate concentration

The activity of both APases increased when p-nitrophenyl phosphate was used at enhancing concentrations from 0.5 to 16.0 mM (Fig. 4). The optimum concentration corresponded to 8.0 mM p-nitrophenyl phosphate. Both phosphatases had Michaelis-Menten type behaviour. The Michaelis-Menten constant (Km) was 0.31 mM with V_max= 147.06 U mg^-1 mycelium for the eAPase, showing a relatively high specificity to the substrate when compared witheAPase 0.57 mM with V_max = 14,285.71 U mg^-1 mycelium. Similar values of Km for the extracellular acid phosphatases as 0.38-0.53 mM were found for A. nidulans (Nozawa et al. 1998). However, higher values of Km as 0.95 and 1.3mM were calculated for the acid phosphatases of A. niger (Gargova et al. 2006) and Humicola lutea (Micheva-Viteva et al.2000), respectively. Also, Km values of 0.029 and 0.325mM were reported for the intracellular enzymes of A. caespitosus(Guimarães et al. 2003) and R. delemar (Tsekova and Galabova 2003), which were low in comparison with the value found in the present work, suggesting that these enzymes had more affinity to the substrate than iAPase. Orthophosphate monoesters was the dominated fraction of organic P compounds found in the soil (Turner et al. 2003) and could be used as a source of P for soil organisms after phosphate release by phosphatases enzymes. The higher specificity for substrate presented by eAPase fromA. niger would allow using the P compounds more efficiently than iAPase.

Figure 4
Effect of substrate concentration in the reaction mixture on the acid phosphatases activities. A) (-○-) extracellular acid phosphatase; B) (-●-) intracellular acid phosphatase.

Effect of inorganic and organic compounds

Organic and inorganic compounds were added to the reaction mixture (see Table 1). Organic substances had little effect on APases when compared to the influence of the salts. The activity of eAPase ranged from 70 to 111% in relation to the control. Tris 1 mM (70%), glucose (74%) and 1 mM Na acetate (79%) were the organic substances that decreased eAPase activity. The activity of iAPase ranged from 91 to 119% in relation to the control and the largest activity was found following the addition of 1 mM Na acetate to the reaction mixture. From the inorganic compounds, the residual activity of eAPase and iAPase ranged from 28 to 99% and 4 to 108% in relation to the control, respectively. The activity of eAPase and iAPase decreased (54 and 51%) following the addition of 10 mM KH₂PO₄ and CaCl₂ (86 and 72%, respectively). The residual activity of extra- and intracellular acid phosphatases from A. caespitosus were inhibited by 500 mM phosphate, resulting in residual activity of 20 and 31%, respectively (Guimarães et al. 2004). Thus, these phosphatases were less inhibited by phosphate concentrations than found in this study. Salts such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate were reported to be inhibitors of acid phosphatase from Cryptococcus neoformans(Collopy-Junior et al. 2006) and Candida parapsilosis (Kiffer-Moreira et al. 2007). Cobalt is known to induce the activity of phosphatases in bacteria (Gong et al. 2005; Palacios et al. 2005; Wang et al. 2005) and fungi (Venkateswerlu and Sivarama Sastry 1982). The present results showed that 1 mM CoCl₂ severely inhibited the activity of eAPase but not iAPase. This could be a negative response because cobalt influenced the absorption of nitrogen by symbiosis (Mengel and Kirkby 2001).

Effect of Mo

Molybdenum from soil is used by several enzymes from eukaryotes and prokaryotes in reduction and oxidation reactions (Tomatsu et al. 2007). However, various authors have reported that acid phosphatase activity decreased due to the presence of molybdate. This effect was reported in A. niger, where the activity was inhibited 44% by 8 mM Na molybdate (Shimada et al. 1977) and corn roots that were also inhibited by 0.01-1.0 mM molybdate (Gallagher and Leonard 1982). Acid phosphatases from A. fumigatus were inhibited by NaF, molybdate and vanadate (Bernard et al. 2002). The activity of both APases was severely decreased by 5 mM ammonium molybdate (Table 1). Based on these results, ammonium molybdate was added to the reaction mixture in increasing concentrations of 0-250 mM. Strong inhibition of the iAPase activity was still observed, even at concentration up to 1 mM, with the residual activity of 18%, and decreased up to 5% at 250 mM (Fig. 5).eAPase was less inhibited by molybdate, with residual activities of 81 and 19%, respectively Fig. 6). The importance of these results was related to the content of Mo in the soil (0.1-7 mg kg^-1) and that required by the plants (0.1-2.4 mg kg^-1) (Kabata-Pendias and Mukherjee 2007). This suggested that the activity of APase from A. niger and other microorganisms could be inhibited by the Mo from the environment.

Figure 5
Effect of molybdate concentration in the reaction mixture on the acid phosphatase activities. (-●-) intracellular acid phosphatase; (-○-) extracellular acid phosphatase.

CONCLUSIONS

A. niger plays a very significant role in the mineralisation of P-organic compounds in the soil, after which free phosphates are released by the action of acid phosphatases (APases). This mechanism occurred in aPi-limited manner, which was shown by the fact that both extracellular (eAPase) and intracellular (iAPase) APase were repressed at increasing concentrations of phosphate. From the results, it could be concluded that the necessity of Pi by A. niger was not severely limited to up 200 µM, with growth only stopping at higher concentrations. The time-course of fungus growth in 50 µMPi showed that the pH of the media decreased up to 3.0 according to 3^rd degree equations and the maximal activity ofiAPase and eAPase was found after 96h and 24h, respectively. The decrease of APase activity in the environment could be interpreted as a mechanism of self-repression by the Pi available from the organic phosphate sources. The optimum pH and substrate concentration (pNP) for both APases activities were 4.5 and 8.0 mM. The other biochemical properties showed that these enzymes had different characteristics, suggesting that they were not similar proteins. iAPase activity was overexpressed and had less affinity to the substrate pNP when compared to eAPase. Both the enzymes were inhibited by Pi and ammonium molybdate; however, onlyeAPase was severely inhibited by CoCl₂ and Tris (hydroxymethyl) aminomethane. Finally, these results suggested that A. niger growth in the soil was dependent on various factors that influenced the synthesis and secretion of acid phosphatases.

REFERENCES

• Açikel Ü, Ersan M. Acid phosphatase production by Rhizopus delemar: a role played in the Ni II bioaccumulation process. J. Hazard Mater 2010; 184: 632-639.
• Aleksieva P, Spasova D, Radoevska S. Acid phosphatase distribution and localisation in the fungus Humicola lutea Z Naturforsch2002; 58: 239-243.
• Annaheim E, Frossar E, Bünemann EK. Characterization of organic phosphorus compounds in soil by phosphatase hydrolysis. 19th World Congress of Soil Science, Soil Solutions for a Changing World, 2010. Brisbane, Australia; p. 9-11.
• Balemi T. Effect of phosphorus nutrition on growth of potato genotypes with contrasting phosphorus efficiency. African Crop Sci J2009; 17: 199-212.
• Barroso CB, Nahas E. The status of soil phosphate fractions and the ability of fungi to dissolve hardly soluble phosphates. Appl Soil Ecol. 2005; 29: 73-83.
• Bernard M, Mouyna I, Dubreucq G, Debeaupuis JP, Fontaine T, Vorgias C, et al. Characterisation of a cell-wall acid phosphatase (PhoAp) inAspergillus fumigatus Microbiol2002; 148: 2819-2829.
• Braz RR, Nahas E. Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilisation in growth medium. FEMS Microbiol Lett 2012; 332: 84-90.
• Bünemann EK. Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients. Soil Biol Biochem2008; 40: 2116-2129.
• Chagas E, Araujo AP, Alves BJR, Teixeira MG. Seeds enriched with phosphorus and molybdenum improve the contribution of biological nitrogen fixation to common bean as estimated by ¹⁵N isotope dilution.Rev Bras Ci Solo. 2010; 34: 1093-1101.
• Collopy-Junior I, Esteves FF, Nimrichter L, Rodrigues ML, Alviano CS, Meyer-Fernandes JR. An ectophosphatase activity in Cryptococcus neoformansFEMS Yeast Res 2007; 7: 621-628.
• Davis RH. Neurospora: Contributions of a model organism. New York: Oxford University, 2000.
• Fitriatin BN, Arief DH, Simarmata T, Santosa DA, Joy B. Phosphatase- producing bacteria isolated from Sanggabuana forest and their capability to hydrolyze organic phosphate. J Soil Sci Environ Manage2011; 2: 299-303.
• Gallagher RS, Leonard RT. Effect of vanadate, molybdate, and azide on membrane-Associated ATPase and soluble phosphatase activities of corn roots. Plant Physiol 1982; 70: 1335-1340.
• Garcia MRL, Nahas E. Microbial populations and the activity of the soil under agricultural and agricultural-pastoral systems. Arch Agron Soil Sci 2012; 58: 511-525.
• Gargova S, Sariyska M. Effect of culture conditions on the biosynthesis of Aspergillus niger phytase and acid phosphatase. Enzyme Microb Technol 2003; 32: 231-235.
• Gargova S, Sariyska M, Angelov A, Stoilova I. Aspergillus niger pH 2.1 optimum acid phosphatase with high affinity for phytate. Folia Microbiol 2006; 51: 541-545.
• Gong N, Chen C, Xie L, Chen H, Lin X, Zhang R. Characterisation of a thermostable alkaline phosphatase from a novel species Thermus yunnanensis sp. nov. and investigation of its cobalt activation at high temperature. Biochim Biophys Acta. 2005; 1750: 103-111.
• Guimarães LHS, Jorge JA, Terenzi HF, Jamur MC, Oliver C, Polizeli MLTM. Effect of carbon source on alkaline phosphatase production and excretion in Aspergillus caespitosus J Basic Microbiol 2003; 43: 210-217.
• Han SW, Rossi A. Acid Phosphatase (EC 3.1.3.2) synthesis by phosphorus regulatory mutant strains of Neurospora crassa Braz J Med Biol Res 1989; 22: 423-431.
• Hidayat BJ, Eriksen NT, Wiebe MG. Acid phosphatase production byAspergillus niger N402A in continuous flow culture. FEMS Microbiol Lett 2006; 254: 324-331.
• Huang Q, Shindo H. Effects of copper on the activity and kinetics of free and immobilized acid phosphatase. Soil Biol Biochem 2000; 32: 1885-1892.
• Kabata-Pendias A, Mukherjee AB. Trace elements from soils to human. Berlin: Springer-Verlag, 2007, 550p.
• Kevresan S, Petrovic N, Popovic M, Kandrac J. Nitrogen and protein metabolism in young pea plants as affected by different concentrations of nickel, cadmium, lead, and molybdenum. J Plant Nutr 2001; 24: 1633-1644.
• Kiffer-Moreira T, Pinheiro AAS, Alviano WS, Barbosa FM, Souto-Padrón T, Nimrichter L, et al. An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells. FEMS Yeast Res 2007; 7: 621-628.
• Malboobi MA, Owlia P, Behbahani M, Sarokhani E, Moradi S, Yakhchali B, et al. Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates. World J Microbiol Biotechnol2012; 25: 1471-1477.
• Marcelo AV, Corá JE, Fernandes C, Martins MR, Jorge RF. Crop sequences in no-tillage system: effects on soil fertility and soybean, maize and rice yield.. Rev Bras Ci Solo 2009; 33: 417-428.
• Mengel K, Kirkby EA. Principles of plant nutrition. Dordrecht: Kluwer, 2001, 859p.
• Micheva-Viteva S, Tchorbanov B, Aleksieva P, Lazarova V. Acid phosphatases excreted by Humicola lutea 120-5 in casein-containing medium. World J Microbiol Biotechnol 2000; 16: 859-863.
• Mullen MD. Phosphorus and other elements. In: Sylvia DM, Hartel PG, Fuhrmann JJ, Zuberer DA, editors. Principles and applications of soil microbiology. New Jersey: Pearson Prentice Hall; 2005. p. 463-488.
• Nannipieri P, Giagnoni L, Landi L, Renella G. Role of phosphatase enzymes in soil phosphorus in action, biological processes in soil phosphorus cycling. In: Bunemann EK, editor. Phosphorus in action, soil biology. Berlin: Springer; 2010. p. 215-243.
• Nopparat C, Jatupornpipat M, Rittiboon A. Isolation of phosphate solubilizing fungi in soil from Kanchanaburi, Thailand. KMITL Sci Tech J. 2007; 7: 137-146.
• Nozawa SR, ThedeiJr G, Luciana SP, Crott LSP, Barbosa JE, Rossi A. The synthesis of phosphate-repressible alkaline phosphatase do not appear to be regulated by ambient pH in the filamentous mould Neurospora crassaBraz J Microbiol 2002; 33: 92-95.
• Nozawa SR, Maccheroni Jr W, Stábeli RG, Thedei Jr G, Rossi A. Purification and properties of Pi-repressible acid phosphatases fromAspergillus nidulans Phytochemistry 1998; 49: 1517-1523.
• Onthong J, Gimsanguan S, Pengnoo A, Nilnond C, Osaki M. Effecf of pH and some cations on activity of acid phosphatase secreted from Ustilagosp. isolated from acid sulphate soil. Songklanakarin J Sci Technol 2007; 29: 275-286.
• Palacios MC, Haros M, Rosell CM, Sanz Y. Characterisation of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties. J Appl Microbiol 2005; 98: 229-237.
• Pawar VC, Thaker VS. Acid phosphatase and invertase activities ofAspergillus niger Mycoscience. 2009; 50: 323-330.
• Quiquampoix H, Mousain D. Enzymatic hydrolysis of organic phosphorus. In: Turner BL, Frossard E, Baldwin D, editors. Organic Phosphorus in the Environment. UK, Wallingford: CABI; 2005. p. 89-112.
• Richardson AE, Simpson RJ. Soil microorganisms mediating phosphorus availability. Plant Physiol 2011; 156: 989-996.
• Rinu K, Pandey A. Temperature-dependent phosphate solubilisation by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil.. Mycoscience 2010; 51: 263-271.
• Samuel AD, Domuţa C, Şandor M, Vuşcan A, Domuţa C. The estimation of phosphatase activity in soil. Res J Agricult Sci 2010; 42: 311-314.
• Shen J, Yuan L, Zhang J, Li H, Bai Z, Chen X, Zhang W, Zhang F. Phosphorus dynamics from soil to plant. Plant Physiol. 2011; 156: 997-1005.
• Shimada Y, Shinmyo A, Enatsu T. Purification and properties of one component of acid phosphatase produced by Aspergillus niger. Biochim Biophys Acta 1977; 480: 417-427.
• Tarafdar JC, Yadav RS, Meena SC. Comparative efficiency of acid phosphatase originated from plant and fungal sources. J Plant Nutr Soil Sci 2001; 164: 279-282.
• Tomatsu H, Takano J, Takahashi H, Watanabe-Takahashi A, Shibagaki N, Fujiwara T. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil. Proc Natl Acad Sci USA. 2007; 104: 18807-18812.
• Tsekova K, Galabova D. Phosphatase production and activity in copper (II) accumulating Rhizopus delemar Enzyme Microbial Tech 2003; 33: 926-931.
• Tsekova K, Galabova D, Todorova K, Ilieva S. Phosphatase activity and copper uptake during growth of Aspergillus niger Process Biochem 2002; 37: 753-758.
• Turner BL, Mahieu N, Condron LM. The phosphorus composition of temperate pasture soils determined by NaOH-EDTA extraction and solution P-31 NMR spectroscopy. Org Geochem 2003; 34: 1199-1210.
• Venkateswerlu G, SivaramaSastry K. A new repressible alkaline phosphatase in Neurospora crassa EM 5297a. J Biosci 1982; 4: 275-279.
• Wang J, Stieglitz KA, Kantrowitz ER. Metal specificity is correlated with two crucial active site residues in Escherichia colialkaline phosphatase. Biochemistry2005; 44: 8378-8386.
• Yadav RS, Tarafdar JC. Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds. Soil Biol Biochem 2003; 35: 1-7.

Publication Dates

• Publication in this collection
  Sep-Oct 2015

History

• Received
  27 Apr 2015
• Accepted
  16 June 2015