Print version ISSN 0001-3714
Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998
Vinícius DArcadia Cruz, Juliana Gisele Belote, Márcia Zilioli Belline, Rubens Cruz*
Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, Universidade Estadual Paulista, Assis, SP, Brasil.
Submitted: October 17, 1997; Returned to authors for corrections: February 17, 1998;
Approved: September 17, 1998
A strain of Aspergillus niger isolated from soil samples showed great capacity to produce extracellular inulinase. Although the enzyme has been synthesized in presence of monosaccharides, sucrose and sugar cane molasse, the productivity was significantly higher (p<0.05) when the microorganism was inoculated in media formulated with dahlia extract and pure inulin, as carbon sources. With regard to the nitrogen source, the best results were obtained with casein and other sources of proteic nitrogen, comparatively to the mineral nitrogen. However, statistic significance (p<0.01) only was found between the productivity obtained in the medium prepared with casein and ammonium sulphate. The optimum pH of the purified enzyme for inulin hydrolysis was found between 4.0 and 4.5 and the optimun temperature at 60oC. When treated by 30 minutes in this temperature no loss of activity was observed. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. Acting on inulins from several sources, the enzyme showed larger hydrolysis speed on the polissaccharide from chicory (Cichorium intibus), comparatively, to the inulins from dahlia (Dahlia pinnata) and Jerusalem artichoke (Helianthus tuberosus) roots.
Key words: inulinase, Aspergillus niger, fructose syrup, inulin
a-D-Fructose is a monosaccharide widely distributed in nature that shows sweetening power 70% higher than sucrose. It consistis in a suitable table sugar, ingredient for some food formulation and in substrate for fermentative processes (13,17,18). It is the principal component of the fructans as the inulin found in the roots of artichoke (Heliantus tuberosus), dahlia (Dahlia pinnata) and chicory (Cichorium intibus) corresponding to 12.5% of its wet weight (1,10,14). According to Barta (1), at this moment, these are the more suitable inulin sources for utilization in industrial scale, whose productivity is estimated, respectively, in 4.5 ton/ha, 2.5 ton/ha and 0.9 ton/ha. Besides, in the last years several authors have showed that other plants of the Liliaceae and Asteraceae families found in Brazilian savannahs can store amounts up to 85% of fructans in their roots, exhibiting potentiality for economical application as fructose source (7,11).
Enzymatic hydrolysis by inulinases (1-ß-D-Fructan-Fructanohydrolase - EC 220.127.116.11) has been proposed as the most promising technique to obtain the fructose syrups from inulin (10,13,18). So, some fungi, yeasts and bacteria have been described as producers of potentially adequated ß-fructosidases, but without a definite valuation for industrial employment. In this work, approximatelly 350 Aspergillus niger strains were evaluated by its capacity to synthesize inulinase with potentiality for industrial employment. A strain codified as Aspergillus niger-245 showed the best productivity and inulinase thermostability and was selected for the work seqüence. The enzyme was applied on the fructans from several origins and its action, was evaluated.
MATERIALS AND METHODS
Microorganisms and culture media. It was utilized a collection culture of Aspergillus niger isolated from soil that has been mantained in slants with Sabouraud-dextrose-agar (Difco) medium in our laboratories. For the screening procedure, 0.1 ml of a spores suspension (4 x 107 spores/ml) was inoculated in Erlenmeyers flasks containing 50 ml of dahlia extract (2.0% of total sugars) enriched with 1.5% (NH4)2SO4, 0.5% yeasts extrat, 0.5% K2HPO4, 0.2% NaNO3, 0.05% KCl, 0.05% MgSO4.7H2O, and final pH, 5.5. The flasks were agitated in a rotary shaker, for 60 h, at 200 rpm and 28ºC. After the mycelia separation by filtration, the filtrate was analysed for enzyme activity. Dahlia extract was replaced by inulin as carbon source, for the studies of the screened strain growth. The fermentation condition as pH, remaining carbohidrates, intra and extracellular enzyme and mycelia dry weight were analysed at each 12 h.
Enzyme assay. Amounts of 0.5 ml of enzyme solution were incubated with 1.0 ml of 2.0% inulin solution in 50 mM acetate buffer, pH 5.0 for 30 min and the reaction stopped by maintenance in boiling water for 5 min.The content of liberated fructose was estimated by the Somogyi and Nelson method (15) and expressed as reducing sugar. The invertase activity was measured by replacing the inulin for a sucrose soluction in the same buffer and concentration and the liberated glucose was estimated by the glucose oxidase method (2). One enzyme unit (EU) was defined as the enzyme amount necessary to liberate 1 µMol of fructose/glucose.min-1, in assay condition .
Inulinase production. For the experiments on the effect of the carbon sources, the media were formulated with 2.0% of glucose, fructose, sucrose, inulin, sugar cane molasse, yam extract and dahlia extract (2.0% of total sugar) and 1.5% of peptones, as nitrogen source. When the effect of the nitrogen source in the enzyme production was investigated, the spores were innoculated in media containing 1.5% of urea, (NH4)2SO4, peptones, casein, soy flour and yeast flour, as nitrogen source, and 2.0% inulin (Sigma) was utilized as carbon source. For optimization of the enzyme production, a medium composed with dahlia extract and casein was utilized. From each experimental design, 5 flasks were inoculated and the results, submitted to variance analysis (Tukey test) for verification of statistical significance.
Dahlia and yam extracts. The dahlia and yam extracts were prepared according to the procedure described by Houly et al. (10) with some modifications. Small cubes of the peeled tubercules were grounded in a homely miller and the obtained paste was suspended in destiled water in the 1:4 ratio (w:v). This suspension was agitated in magnetic stirring, for 20 min, sieved in a 120 mesh sieve, centrifuged at 3,000 rpm and the total sugar, adjusted to 2.0% in accordance with Dubois et al. method (4).
Enzyme characterization. The effect of pH on purified inulinase (not published yet) activity was investigated by measuring the enzyme activity at 50ºC, in 50 mM acetate buffer in the pH range from 2.5 to 7.0. For the pH stability determination, aliquots of 0.5 ml of enzyme plus 0.5 ml of the same buffer in mentioned pH range were maintained at 4ºC, by 96 h and the residual activity was estimated. The optimum temperature was obtained by measuring the enzymatic activity in 50 mM acetate buffer, pH 5.0 in the temperature range from 35ºC to 70ºC. For the thermal stability determination of the inulinase, a reaction medium composed with 0.5 ml of enzyme solution and 0.5 ml of 100 mM acetate buffer, pH 5.0, was mantained for 30 min in the same temperature range and the residual activity, measured as described in enzyme assay.
Inulinase action pattern. Aliquots of 0.5 ml of the purified enzyme were incubated, separately in 2.0 ml of raffinose solution (Vetec), sucrose (Reagen), and inulin (Sigma), in a final concentration of 2.0% in 50 mM acetate buffer, pH 5.0, at 50ºC. The profile of the enzymatic reaction was followed by thin layer chromatography (TLC) with a solvent system composed by ethyl acetate, acetic acid and distilled water (3:1:1) and the stains revealed as described by Walkley and Tillman (19). The liberation of glucose from sucrose and fructose from raffinose and inulin was also determined, respectively by the methods of the glucose oxidase (2 ) and Somogiy and Nelson (15). The values obtained in the raffinose reaction were divided by 2, because to each molecule of liberated fructose, corresponds 1 reducing unit of melibiose.
Hydrolysis of inulin from different sources. For the hydrolysis studies of inulin from different origins, the reaction system was composed by 1.0 ml (14 EU) of purified enzyme, 10 ml of dahlia, chicory or artichoke 5.0% inulin solutions in 50 mM acetate buffer, pH 5.0. The enzymatic action was followed by High Efficiency Liquid Chromatography (HPLC) in a Shimadzu LC-10A chromatography, equipped with a refraction index detector, RID 6-A model, and Supercosyl LC-NH2 column with 250 x 4.6 mm maintained at 20ºC. The solvent system was composed by acetonitrile-water (80:20) and the speed of flow of 2.0 ml/min.
RESULTS AND DISCUSSION
Microorganism growth and enzyme production. Fig. 1 shows the growth curve of Aspergillus niger-245. It is possible to verify that the biosynthesis of the inulinase happens simultaneously to the log phase of the mycelia growth, starting at the 24 h of incubation and that the largest enzyme liberation happened between 48 and 60 h of fermentation. Such behavior is quite uncommon for this enzyme once, generally, in other microorganisms it reaches the maximum activity in the extra-cellular medium after 72 h of fermentation (3).
Figure 1. Growth profile and inulinase production by Aspergillus niger-245
|l Extracel. inulinase||¡ Intracel. inulinase|
|n Sugar consumption||o Mycelia dry weight|
Ohta et al. (17) obtained the maximum activity of inulinase of a mutant strain of Aspergillus niger, described as high enzyme-producing, after 5 days of inoculation in a medium very similar to the one used in this work, once that medium contained inulin and ammoniun sulfate, respectively, as sources of carbon and nitrogen. Various strains of Aspergillus sp, studied by Gupta et al. (8), also exhibited maximum inulinase activity in the 9o day of submerged fermentation. The fast enzyme production for the now selected microorganism is a suitable propriety in industrial processes and represents an advantage of this one on the other microorganisms already proposed for such purpose.
Effect of carbon source. As shown in Table 1, the inulinase production in the media formulated with dahlia extract, (that contain inulin) and with the own inulin, its natural inductor, was significantly (p<0.05) higher than the one observed in other carbon sources. In spite of the apparent larger productivity in the presence of the extract, it was not registered statistical significance between both. The same table shows, also, that such media favored the biosynthesis of the inulinase activity in detriment of the invertase activity, registering the largest ratios I/S (0.42 to 0.44). That observation was already described by Ferreira et al. (6), working with a strain of Cladosporium cladosporioides. The smallest I/S ratio obtained in the medium formulated with sucrose and molasse (0.16), can suggest that other invertase molecular forms has been synthesized in those media. Kaur et al. (12) isolated four differents invertases from a growth medium of Fusarium oxysporum enriched with fructans and sucrose. The production of both activities in the media formulated with monossacarides also suggests that such enzyme activities are synthesized constitutively.
Effect of nitrogen source. The microorganism was more efficient in the inulinase production when inoculated in media containing proteic nitrogen in relation to those formulated with mineral nitrogen as it can be verified in Table 2. The urea inhibited the synthesis of the enzyme completely, and in relation to the ammonium sulphate, the averages were considerably inferior to all the media formulated with proteic nitrogen. However, statistical significance (p<0.01) was only registered between the enzyme production obtained in casein and ammonium sulphate media. In the casein medium was observed the best performance of the microorganism, although, there is no registration of statistical significance in relation to the others formulated with proteic nitrogen. The same table shows that when casein was associated with dahlia extract as carbon source the inulinase production reached 9.9 EU/ml, in a synergistic effect. Synthesis of invertase was not considered in this table because the I/S ratio was not affected by the nitrogen sources. The largest production of inulinase in media formulated with organic nitrogen was, also, described by Ha and Kim (9). Those authors verified that a strain of Streptomyces sp presented the largest enzyme productivity in a medium formulated with soy flour compared to others organic and mineral nitrogen sources.
Influence of the pH on the enzymatic activity. In acordance with the Fig. 2, the inulinase from A. niger 245 showed whole stability in the pH range from 3.5 to 7.0 and optimum pH between 4.0 and 4.5, coinciding with the results described by Vandamme and Derycke (18) for the enzyme sytnthetized by other strain of A. niger. However, Oengen et al. (16) obtained the maximum activity for the inulinase from a microorganism of the same group in a range slightly superior, that is 5.0 and 6.0.
Figure 2: Effect of the pH on the activity and stability of the inulinase from Aspergillus niger-245.
|n Activity||o Remaining activity.|
For industrial application in obtaining concentrated fructose syrups, enzymes with larger activity in pH range inferior to 5.0, as the one here described, are suitable since they make difficult the bacterial contamination of the process.
Enzyme behaviour in relation to the temperature. As it can be seen in Fig. 3, the enzyme showed the larger activity at 60ºC. In this temperature the crude enzyme stayed stable, after 30 min of treatment. It is possible to suppose, however, that the enzyme can support longer that treatment once when submitted for the same time at 65ºC, it lost only 8% of its initial activity. This supposition also is supported by the studies of Gupta et al. (8). Searching the termostability of the inulinases produced by Aspergillus sp, including several A. niger, the authors had concluded that the inulinases from this genus, in general, present thermostability higher than several ones from microbial origin.
Figure 3: Effect of temperature on the activity and stability of the inulinase from Aspergillus niger-245.
n Activity o Remaining activity.
Action pattern of the enzyme. Fig. 4 shows the reducing sugars (fructose) or glucose liberation from several substrates as function of the reaction time. With regard to the sucrose and its homologous raffinose, practically there was no progression in the reaction after 30 minutes that perhaps can be explained, by the speed with that the enzyme attacks such substrates leading to their prompt exhaustion. From the inulin, the stabilization of the process was only obtained after 180 min and no glucose was found in the reaction medium. Such results are very similar to those produced by the inulinase from Arthrobacter sp which showed ability to hydrolyze sucrose, raffinose and inulin, however with activity 5 times higher on the fructooligosacharides, when compared to the inulin (5).
Figure 4: Action of the inulinase from Aspergillus niger-245 on several substrates.
|l Inulin||¡ Raffinose|
|n Sucrose||o Mycelia dry weight|
The obtaining of fructose as the only hydrolysis product from inulin and its liberation from the sucrose and raffinose suggest an exo-action mechanism of the enzyme. This action pattern was better evidenced in the chromatogram of the Fig. 5. To the progress of the reaction it corresponds a intensity increasing of the stains produced by fructose, proportionally, to the decrease in the intensity of the stains related to the inulin, confirming, therefore, the expressed results in Fig. 4.
1- Fructose, 2- Glucose, 3- Sucrose, 4- Inulin 0, 5- Inulin 30, 6- Inulin 60, 7- Inulin 120, 8- Inulin 180, 9- Inulin 240.
Figure 5: Hydrolysis of inulin by inuliase from A. niger-245. Effect of reaction time.
Inulin hydrolysis of different origins. Fig. 6 summarizes the inulinase action on the inulin from several sources. It is possible to verify that the fructose liberation curves of the fructans from artichoke and dahlia are, extremely, similar. However, the inulinase showed larger activity on inulin from chicory, once 72% of the reaction medium sugars were identified as fructose at the 30 min of reaction, against only 27% of the dahlia and artichoke inulins. After 1 hour of reaction, these values arose, respectively, to 88% and 80% and after 3 h, total hydrolysis was obtained for all the fructans when the fructose reached to about 90%, characterizing the remainder one as glucose.
Figure 6: Action of the inulinase from Aspergillus niger-245 on inulin from several sources.
l Dahlia ¡ Chicory n Artichoke.
The largest hydrolysis speed on the inulin from chicory leads to the supposition that this fructan presents a structure more susceptible to the enzymatic attack (perhaps less condensed or with different polimerization degree) than the other ones. Unfortunately, similar studies were not found in the literature to compare with them.
The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support
Produção e mecanismo de ação de inulinase de Aspergillus niger-245: Hidrólise de inulinas de diferentes origens
Uma linhagem de Aspergillus niger isolada de amostras de solo mostrou grande capacidade de produzir inulinase extracelular. Embora a enzima tenha sido sintetizada na presença de monossacarídeos, sacarose e melaço de cana, a produtividade foi significativamente maior (p<0.01) quando o microrganismo foi inoculado em meios formulados com extrato de dália e inulina pura, como fontes de carbono. Em relação à fonte de nitrogênio, os melhores resultados foram obtidos com caseína e outras fontes de nitrogênio proteico, comparativamente ao nitrogênio mineral. Entretanto, somente foi encontrada significância (p<0.01) entre a produtividade obtida nos meios preparados com caseína e sulfato de amônia. O pH ótimo da enzima purificada foi localizado entre 4.0 e 4.5 e a temperatura ótima a 60ºC. Quando tratada por 30 minutos nesta temperatura nenhuma perda de atividade foi observada. A enzima mostrou capacidade de hidrolisar sacarose, rafinose e inulina, da qual liberou apenas unidades de frutose, mostrando, portanto, um mecanismo de exo-ação. Atuando sobre inulinas de diversas fontes, a enzima mostrou maior velocidade de hidrólise sobre o polissacarídeo da chicória, comparativamente, às inulinas de raízes de dalia e alcachofra.
Palavras chaves: inulinase, Aspergillus niger, inulina, xarope de frutose
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