Print version ISSN 0001-3714
Rev. Microbiol. vol. 29 n. 3 São Paulo Sept. 1998
Eleni Gomes1 *; Roberto da Silva1; Alcides Serzedello2
1Instituto de Biociências, Letras e Ciências Exatas, Laboratório de Bioquímica dos Processos e Microbiologia Aplicada, Universidade Estadual Paulista, São José do Rio Preto, SP, Brasil. 2Instituto de Biociências, Departamento de Bioquímica e Microbiologia, Universidade Estadual Paulista, Rio Claro, SP, Brasil.
Ribonuclease production by Aspergillus flavipes, A. sulphureus and A. fischeri in semi-synthetic medium, after 24-144 hours at 30ºC under shaking, was studied. After cultivation, the medium was separated from micelia by filtration and the resultant solution was used as enzymatic extract. The highest amount of biomass and RNase was obtained after 96 hours of cultivation. The enzymes produced by three species presented similar characteristics, with optimum temperature at 55ºC and two peaks of activity at pH 4.5 and 7.0. A. flavipes RNases were more sensitive to temperature: 50% of the initial activity was lost after 1 hour at 70ºC. After this heat treatment, RNase of A. sulphureus lost 30% of this activity and that of A. fischeri only 16%. The nucleotides released by enzimatic hydrolysis of RNA were separated by ion exchange chromatography in a AG-1X8-formiate column and identified by paper chromatography. This procedure indicated that the raw enzymatic extract of Aspergillus flavipes is able to hydrolyze RNA, releasing 3'-nucleotides monophosphate at pH 4.5 and 3' and 5'-nucleotides monophosphate at pH 7.0 and 8.5. This result suggests that this strain produces two different types of RNase, one acidic and other alcaline, with different specificities.
Key words: Ribonucleases, Aspergillus, nucleotides
INTRODUCTION Intra and extracellular ribonucleases of a wide variety of microorganisms have been described. In the last three decades, a big progress occurred in the catalogue of the RNases produced by fungi, like Aspergillus oryzae (T1, T2 and S1) (18, 17, 1), A. saitoi (M and Ms) (14, 7), Neurospora crassa (N1, N2 and N3) (21), Rhizopus sp (R) (22), Penicillium citrinum (P1) (5), among others.
These enzymes have been used commercially to produce nucleotides for clinical use (3, 9) or for the food industry (6, 8), besides constituting important tools for investigations in molecular biology (11, 2), in studies on the structure of nucleic acids structure (16) and serving as model to study of proteins structures (15, 10).
This study reports the extracellular RNase production by three species of Aspergillus and describes some properties of these enzymes.
MATERIALS AND METHODS Organism and cultivation: Aspergillus species, supplied by the Departamento de Tecnologia Agroindustrial, ESALQ, USP, Piracicaba-SP, were used in this study. The conidia formed on agar-Sabouraud were washed with a sterilized 0,01 % Tween 80 solution. This suspension was filtered, and the volume containing 107 conidia was inoculated in 50 ml of liquid medium with pH 6.2 containing (in g/l): glucose 50.0; beef extract 5.0; yeast extract 2.0; MgSO4 0.5; CaCl2 0.1; KNO3 2.0; soy bean meal 5.0 (12). After 48 hr incubation at 30ºC in an orbital shaker, 5 ml of this culture was transfered to 100 ml of the same media and mantained in identical conditions for a further 96 hr. Mycelia were separated from the culture medium by filtration and the resultant solution was used as the crude extract.
Assay of RNase: The enzyme activity was determined by incubating 6 mg of yeast RNA (Companhia Agroindustrial Ometto, Iracemápolis, SP, Brazil.) in 0.25 M acetate buffer, pH 4.5 or tris-HCl pH 7.0, with an a known volume of the enzyme solution in a total volume of 2 ml. After incubation for 60 min at 50ºC, the reaction was stopped by rapid chilling in an ice bath. The non-hydrolysed RNA was precipited with 5 ml of ice cold ethanol and mantained at -10ºC for 20 min. After centrifugation at 2000g for 10 min, the supernatant was diluted with water and the absorbance at 260 nm determined. One unit of RNase activity was defined as that amount of enzyme necessary to produce one mmol of 260 nm absorbing substance. The extinction coefficient of 10,600 (6) for hydrolysed RNA was used in the activity assays.
Analysis of the digestion products: The monophosphate nucleotides formed from yeast RNA by digestion with the RNase preparation were identified by the methods of Nakao and Ogata (12) with the following modifications. RNA was hydrolyzed at 37ºC for 8 hours and the digest, after precipitation of non-hydrolysed RNA, was concentrated under vaccum in a rotary evaporator. The pH was adjusted to 9.0 with NH4OH and the sample applied to an AG 1X8 column (1 x 6 cm, Bio Rad, 200-400 mesh - formate type), equilibrated with 0.005 M formic acid. The adsorbed material was then stepwise eluted with formic acid and sodium formiate (0.5 ml/min flow). Each 260 nm-absorbing peak was characterized by its position in the cromatogram and its ultraviolet absorption spectra using the four pure 3' and 5'- monophosphate nucleotides (Sigma) as controls for comparison. Furthermore, the identity of the monophosphate nucleotides was confirmed by paper chromatography using a Whatman nº 1 base and two different solvents: S1=i-propanol 65% and chloridric acid 2N (23), and; S2= methanol, chloridric acid and water 7:2:1 (4).
Determination of protein concentration: Protein concentration was determined by method of Seedmak and Grossberg (19).
Determination of glucose concentration: The glucose concentration was determinated by an enzymatic Kit (glucose oxidase- peroxidase) from Bio-Diagnostica.
RESULTS Activity of RNase in culture filtrates of Aspergillus species: Data on Fig. 1 show that Aspergillus flavipes (1 a), A. sulphureus (1b) and A. fischeri (1c) produce and excrete enzymes that hydrolyze RNA during the growth phase, since the increase in media ribonucleolitic activity was proportional to the biomass production. All three species reached maximum growth between 96 and 120 hours of cultivation, with very similar quantities of produced biomass. Within this period, glucose concentration reached minimum values, being entirely consumed after 120 hours.
Fig. 1: Quantification of glucose consumption and biomass and RNAse production during the cultivation of Aspergillus flavipes (a), A, sulphureus (b) and A. fischeri (c).
Effect of the pH on RNases activity: Results on Fig. 2 show two peaks of RNases activities, one at pH 4.5 and other one at pH 7.0, for all three species, suggesting the existence of, at least, two different kinds of RNases.
Fig. 2: Effect of pH on the activity of the RNases excreted by the studied species, using acetate (pH 3.0 to 5.5), succinate (pH 6.0 and 6.5) and Tris-HCl (pH 7.0 to 9.5) buffers.
Effect of temperature on RNases activity: After 96 hour of cultivation of all three species, raw enzymatic solutions were measured in various temperatures from 40 to 70ºC. As shown in Fig. 3, the optimum temperature for activity of RNases excreted by all three species was 50°C.
Fig. 3: Effect of temperature on the activity of RNases excreted by the studied species.
Effect of thermal treatment on RNases stability: The thermal stability of the RNases was determined after keeping the raw enzymatic extract at 50, 60 and 70°C for one hour. A. flavipes RNases showed to be the most sensitive to temperature, with 50% loss of the initial activity at 70°C, followed by A. sulphureus (30%) and A. fischeri (16%) (Fig.4).
Fig. 4: Thermal treatment effect on RNases with substract absence. Residual ribonucleolitic activity was determined at pH 4.5 and pH 7.0, following procedure described in the text. The full symbols corresponds to pH 4.5.
The effect of pH on nucleotides formation: For the identification of the RNA hydrolysis products, A. flavipes 96 hour cultivation medium was used with hydrolysis done at pH 4.5, 7.0 and 8.5. Results are shown in the chromatograms of Fig. 5.
Fig. 5: Dissociation of nucleotides obtained by RNA hydrolysis, using raw enzymatic extract from Aspergillus flavipes. Chromatography of ionic change in AG 1X8 column. Fractions were eluted with formic acid and formiate, as indicated, and there were colected 10 ml fractions, in a 0.5 ml/min flow. Absorbed material consisted of :
5A: Authentic nucleotides mixture ( a= 5'- CMP, b= 5"-AMP, c= 3'-AMP, d= 5'- UMP, e= 4'-UMP, f= 3'-GMP and g= 3GMP)
5B: RNA enzymatially hidrolysed at pH 4.5 ( a= 3'-CMP, b=3'-AMP, c=3'-UMP and d= 3'-GMP)
5C: RNA enzynatially hidrolysed at pH 7.0 (b= 3'-CMP, c=5'-AMP, d=3'-AMP, e=5'-UMP, f= 3'-UMP, h=3'-GMP ; a and g was not confirmed by paper chromato-graphy).
5D: RNA enzynatially hidrolysed at pH 8.5 ( a= 5'=CMP, b= 3'-CMP, c=5'- d=3'-AMP, e=5'-UMP, f= 3'-UMP, g=3'-GMP
Peaks on Fig. 5B, correspondent to enzymatic hydrolysis at pH 4.5, were identified as a=3'-CMP, b=3'-AMP, c=3'-UMP and d=3'-GMP. On Fig. 5C, correspondent to hydrolysis at pH 7.0, peaks b, c, d, e, f and h were identified as 3'-CMP, 5'-AMP, 3AMP, 5'-UMP, 3'-UMP and 3'-GMP, respectively. Peaks a and g could not be confirmed by paper chromatography because their concentration was too low. However, their absorption spectra and their positions on chromatograms indicate that they are 5'-CMP and 5'-GMP, respectively. Peaks in Fig. 5D (a, b, c, d, e, f and g) which correspond to hydrolysis at pH 8,5, were identified as 5', 3'-CMP, 5',3'-AMP, 5,3'-UMP and 5'-GMP.
DISCUSSION The three Aspergillus species excreted RNases during the growth period. The amount of enzyme produced by each species seem to be a characteristic of the microorganism and is not influenced by cultivation conditions since the biomass production by the different species was very similar.
The RNases from A. flavipes, A. sulphureus and A. fischeri presented similar properties to RNases described in a wide variety of fungi. The observed optimum pH of 4.5 and 7.0 are consistent with those described in other RNases in Aspergillus, which presented optimum pH varying from acidic, like RNases Ms in A. saitoi (7) to neutral, like RNase T1 in A. oryzae (20). The optimum temperature of 55ºC is also the same observed previously in RNases in various filamentous fungi (14, 18, 21,22,13).
The A. fischeri and A. sulphureus RNases were stable at 70°C. The thermostability is a characteristic of ribonucleases from fungi which allows the heat-treatment during the purification procedure (20). However, the RNase from A. flavipes was more sensitive to temperature, loosing 50% of activity at 70°C.
The analysis of the enzimatically hydrolysed RNA products from A. flavipes indicated that RNase releases nucleotides 3'-monophosphate in acid pH and nucleotides 5'-monophosphate in neutral and alcaline pH, suggesting the presence of two types of RNases, one active on acid pH and other active in neutral or alcaline pH.
A. oryzae produces three kinds of well defined and highly purified nucleolitic enzymes: RNase T1, which hydrolyses RNA at pH 7.5, releasing nucleotides 3'-monophosphate; RNase T2, with optimum pH at 4.5, also releasing nucleotides 3'-monophosphate; and nuclease S1, specific for single-strand nucleic acids, which is active at pH 4.6 and releasing nucleotides 5'-monophosphate (20, 1).
Enzymes similar to T1 and T2 RNases were already isolated from other Aspergillus species (13, 14). However, an alcaline RNase which releases nucleotides 5'-monophosphate was not described before in this genus, although Nakao and Ogata, (12), suggested the presence of this kind of RNase in A. quercinus.
However, the presence of any RNase with such trait in A. flavipes can only be confirmed with the purification of the excreted enzymes.
Produção de ribonucleases por Aspergillus sp.
A produção de ribonucleases extracelulares pelos fungos Aspergillus flavipes, A. sulphureus e A. fischeri foi estudada em meio semi-sintético por períodos de 24 a 144 horas, em "shaker" a 30ºC. Após o cultivo, o meio foi separado da massa micelial por filtração, sendo o filtrado utilizado como solução enzimática bruta. As três espécies produziram maior quantidade de biomassa e ribonuclease após 96 horas de cultivo. O estudo das RNases como extrato enzimático bruto demonstrou que existe grande similaridade entre as enzimas das três espécies, com temperaturas ótimas de 55ºC e dois picos de atividade a pH 4,5 e a pH 7,0. A RNAse produzida pelo fungo A. flavipes demonstrou ser mais sensível à temperatura, com perda de 50% de sua atividade inicial após 1 hora a 70ºC, enquanto que RNase de A. sulphureus perdeu 30% e a RNase de A. fischeri perdeu somente 16%. A separação dos nucleotideos liberados por hidrólise enzimática do RNA, por cromatografia de troca iônica em coluna de AG-1X8-formiato e a identificação por cromatografia de papel, indicaram que o extrato enzimático bruto de A. flavipes hidrolisou RNA liberando 3'-nucleotídeos monofosfato a pH 4,5 e 3' e 5'-nucleotídeos monofosfato a pH 7,0 e 8,5, sugerindo a existência de pelo menos dois tipos diferentes de RNases, uma acídica e outra alcalina, com diferentes especificidades.
Palavras-chave: Ribonuclease, Aspergillus, nucleotídeos
REFERENCES 1- Ando, T. A nuclease specific for heat-denatured DNA isolated from a product of Aspergillus oryzae. Biochem. Biophys. Acta,114:158-168, 1966. [ Links ]
2- Bartok, K.; Fraser, M.J.; Fareed, G.C. Detection of sequence heterology by use of the Neurospora crassa nuclease. Biochem. Res. Cooun., 60:507-511, 1974. [ Links ]
3- Benaiges, M.D.; Lopez- Santin, J.; Solà, C. Proceso de producción enzimática de 5'-ribonucleótidos a partir de RNA microbiano. Rev. Agroquim. Tecnol. Aliment., 29:285-296, 1989. [ Links ]
4- Carvalho, A.; Molinari, A. Contribuição à identificação de compostos purínicos e pirimídicos de interesse biológico. Ecl. Quim., 4:65-69, 1979. [ Links ]
5- Fujimoto, M.; Kuninaka, A.; Yoshino, H. Specificity of nuclease from Penicillium citrinum. Agric. Biol. Chem., 33:1517-1518, 1969. [ Links ]
6- Fujimoto, M., Kuninaka, A.; Yoshino, H. Purification of nuclease from Penicillium citrinum. Agric. Biol. Chem. 38: 777-783, 1974. [ Links ]
7- Irie, M.; Watanabe, H.; Ohgi, K. Site of a major ribonuclease from Aspergillus saitoi with iodoacetate. J. Biochem., 99:627-633, 1986. [ Links ]
8- Kamekura, M.; Onishi, H. Properties of the halophilic nuclease of a moderate halophilic Micrococcus varians subsp. Halophilus. J. Bacterol., 133: 59-65, 1978. [ Links ]
9- Kuninaka, A. Studies on taste of ribonucleic acids derivatives. J. Agric. Chem. Soc., 34:489-501, 1969. [ Links ]
10- Lenz, A.; Choe, H.W.; Granzin, J.; Heinemann, U.; Sanger, W. Three dimensional structures of the ternary complex between ribonuclease T1, guanosine 3'-5'-biphosphate and inorganic phosphate at 0.19 nm resolution. Eur. J. Biochem., 211:311-316, 1993. [ Links ]
11- Marka, A.; Spencer, J.H. Isolation of Escherichia coli transfer RNA-gene hybrids. J. Mol. Biol., 51:115-120, 1970. [ Links ]
12- Nakao, Y.; Ogata, K. Degradation of nucleic acids and their related compounds by microbial enzymes. V. Degradation of ribonucleic acids by Aspergillus quercinus. Agric. Biol. Chem., 27:291-301, 1963. [ Links ]
13- Nomachi, Y.; Komano, T. Purification and some properties of two acid ribonucleases from the mycelia of Aspergillus niger. J. Gen. Appl. Microbiol.,26:375-385, 1980. [ Links ]
14- Ohgi, K.; Irie, M. Purification and properties of a new ribonuclease from Aspergillus saitoi.. J. Biochem., 77:1085-1090, 1975. [ Links ]
15- Pace, C.N. Ribonuclease T1: Structure, function and stability. Angew. Chemie, 30:343:359, 1991. [ Links ]
16- Rushizky, G.W. ; Sober, H.A. Characterization of the major compounds in ribonuclease T1 digests of ribonucleic acids. J. Biol. Chem., 237:834-840, 1962. [ Links ]
17- Rushizky, G.W.; Sober, H.A. Studies on the specificity of ribonuclease T2. J. Biochem., 238: 371-376, 1963. [ Links ]
18- Sato,K.; Egami, F. Studies on ribonucleases in Takadiastase. I. J. Biochem., 44 : 753-767, 1957. [ Links ]
19- Seedmak, J.J. ; Grossberg, S.R.. A rapid, sensitive and versatile assay for protein using Coomassie Brilhante Blue G 250. Anal. Chem., 70:544-552, 1977. [ Links ]
20- Takahashi, K. The structure and function of ribonuclease T1. J. Biochem., 49 : 1-8 1961. [ Links ]
21- Takai, N.; Uchida, T.; Egami, F. Purification and properties of ribonuclease N1, an extracelular ribonuclease of Neurospora crassa. Bichem. Biophys. Acta, 128 : 218-220, 1966. [ Links ] 22- Tomoyeda, M.; Eto, Y.; Yoshino, T. Studies on ribonuclease produced by Rhizopus sp. I. Crystalization and some properties of the ribonuclease. Arch. Biochem. Biophys.,131:191-202, 1969. [ Links ] 23- Wyatt, G.R. The purine and pirimidine composition of desoxypentose nucleic acids. Biochem., 48:584-590, 1951. [ Links ]
22- Tomoyeda, M.; Eto, Y.; Yoshino, T. Studies on ribonuclease produced by Rhizopus sp. I. Crystalization and some properties of the ribonuclease. Arch. Biochem. Biophys.,131:191-202, 1969. [ Links ]
23- Wyatt, G.R. The purine and pirimidine composition of desoxypentose nucleic acids. Biochem., 48:584-590, 1951. [ Links ]