THE OBTAINING AND CHARACTERIZATION OF A FUNGAL INULINASE

Transcription

1 ORIGINAL ARTICLES THE OBTAINING AND CHARACTERIZATION OF A FUNGAL INULINASE LUMINIŢA GEORGESCU 1, ANCA NICOLAU 1, IRINA STOICA 2 1 University Dunărea de Jos, Str. Domnească 47, Galaţi, Romania 2 Rompak-Pakmaya, Str. Grădiniţei 1, Paşcani, Romania (Received September 9, 2006) This study was determined by the need to produce fructooligosaccharides, which act as prebiotics and whose utilization as functional food ingredients significantly increased in the latest years. Fructooligosaccharide production needs an inulinase preparation that can be obtained from Aspergillus niger strains. We obtained an inulinase preparation by cultivating A. niger MIUG 1.15 strain on a liquid medium. Mycelium was removed by centrifugation and the enzyme was semi-purified by (NH 4 ) 2 SO 4 precipitation. The semi-purified inulinase was characterized by determining its optimum activity parameters: ph = 4.5, temperature = 60 C, thermal stability (30 min at 60 C) and stability in freezethaw cycles (a loss of 5.9% of the inulinase activity after 6 cycles). Also, the kinetic parameters of this inulinase were established: V max = µmol fructose per min and K m = 7.64 mm inulin. The spectrum of the inulinase hydrolysis products was determined by HPLC analysis. It was revealed that the enzyme preparation acted as an exoinulinase that had a better affinity for sucrose than for inulin. Key words: inulinase, Aspergillus niger, HPLC analysis. INTRODUCTION Inulin is a widespread polyfructan in plants, which is hydrolysed by two types of inulinases: endoinulinase (2,1-beta-D-fructan fructanohydrolase, EC ) and exoinulinase (beta-d-fructan fructanohydrolase, EC ). While an exoinulinase hydrolyses terminal fructose units from inulin, endoinulinase splits inulin into fructooligosaccharides (FOS) (1 3). A number of fungal and bacterial strains have been used for the production of FOS. Fungal inulinases are frequently composed of a mixture of fructanohydrolases displaying high activity and stability. The best known fungal inulinases are those produced by species of Aspergillus, Penicillium and Kluyveromyces (2 4). According to their mechanism of action, there are different uses of the inulinase preparations in the food industry. Thus, the exoinulinase is used to obtain fructose syrup and ethanol from inulin-rich plant sources and the endoinulinase is used to obtain FOS, which are functional food ingredients (5, 6). ROM. J. BIOCHEM., 43 44, 3 12 ( )

2 4 Luminiţa Georgescu, Anca Nicolau, Irina Stoica 2 FOS are used as hypocaloric sweeteners when light variants of some food products are obtained. The energetic value of FOS varies between 1.5 and 2 kcal/g, while their sweetness is strictly connected to the polymerization level. Furthermore, FOS are functional ingredients that positively modulate the consumers health. They are included in the category of prebiotic dietary fibers, which are able to stimulate the growth and/or activity of colonic bacteria, especially of the probiotic ones. Other benefic physiological effects associated to FOS consumption are the enhancement of calcium bioavailability, the decrease of colon cancer risk, and the decrease of the colesterolemia, triglyceridemia, insulinemia and uremia levels (5 7). MATERIAL AND METHODS MATERIALS Inulinase preparations were obtained from A. niger MIUG 1.15, a strain selected from the aspergilli included in the MIUG Collection of the Food Science and Engineering Faculty, University Dunărea de Jos, Galaţi. The substrates used in the selection stages of the aspergilli and to produce and characterize the enzyme were prepared on the basis of: Czapeck salt solution (1% KH 2 PO 4, 0.5% MgSO4 7H 2 O, 0.01% FeSO 4, 0.5% KCl), casein peptone (Merck), fibruline LC (artichoke inulin, medium grade of polymerization GP m = 20, and mono- and diglucide content = 1%) (Warcoing Industrie SA - Belgium), fibruline instant (artichoke inulin, GP m = 8, monoand diglucide content = 10%) (Warcoing Industrie SA Belgium), turnip inulin (purified, GP = 15) (Merck), sucrose p.a. (Merck). Obtaining of the variants A and B of raw inulinase. Using A. niger MIUG 1.15, we obtained two variants of raw inulinase preparation (culture liquid obtained after filtration and centrifugation). Variant A was produced on a medium containing 3% fibruline LC, 1.5% peptone and Czapeck salts, while variant B was produced on a medium containing 3% turnip inulin, 0.23% NH 4 NO 3, 0.37% (NH 4 ) 2 HPO 4, 0.1% K 2 HPO 4, 0.05% MgSO 4 and 0.15% peptone. Characterization of raw inulinase. The inulinase activity was spectrophotometrically assayed (λ=535 nm), based on the reducing sugars determination by the 3,5 dinitrosalicylic acid method, and was expressed as fructose. A standard curve was established for fructose using concentrations between 0 and 1.1 mg/ml. One unit of inulinase activity (U) was defined as the amount of enzyme which produced 1µmol of reducing sugars per minute at ph 4.5 and 60 C. To establish the optimum temperature, reaction mixtures of 2 ml fibruline LC buffered solution (0.5 M phosphate-citrate buffer, ph 4.7) and 2 ml inulinase preparation were incubated for 15 min at various temperatures, from 20 to 80ºC.

3 3 Fungal inulinase 5 To determine the optimum ph, reaction mixtures of 2 ml inulinase preparation, 1 ml 2% fibruline LC and 1 ml buffer solution (0.5 M phosphate-citrate buffer with ph varying from 2.5 to 8) were incubated for 15 min at 60ºC. The inulinase stability during six freezing/thawing cycles was determined just for the A variant of the inulinase preparation. The inulinase activity determined at each two cycles was compared to the initial one. The inulinase stability at the optimum temperature was tested for both variants by incubating the substrate with enzyme at 60ºC for 120 min and determining the activity at certain time intervals. To put into evidence the hydrolysis dynamics, the ratio between the substrate solution (turnip inulin) and the raw inulinase (variants A or B) was 1:4 by volume. Inulin hydrolysis was performed at 60ºC, in stationary conditions. Samples were taken each 15 min during a period of 75 min and resulted fructose was spectrophotometrically assayed. Preparation A showed a higher inulinase activity as compared to preparation B and was used in subsequent experiments concerning enzyme characterization. Partial purification and characterization of the variant A of raw inulinase. Purification was made using the salting-out effect of (NH 4 ) 2 SO 4. The concentration of (NH 4 ) 2 SO 4 was expressed in saturation per cents and solid salt was used in order to not increase the volume of the inulinase preparation. Following precipitation two proteic fractions showing inulinase activity were obtained. They corresponded to 80% and 90% (NH 4 ) 2 SO 4 and were named inulinase-80 and inulinase-90, respectively. A vacuum concentration of the inulinase preparation was performed at 50 55ºC in a rotary concentrator coupled to a vacuum pump, before starting purification. The efficiency of the purification process was determined based on the specific activity (U/mg protein). Protein was assayed through the Lowry method (8). Sediments resulted from (NH 4 ) 2 SO 4 precipitation were dissolved in 2 ml buffer solution (0.5 M phosphate-citrate ph 4.5), submitted to dialysis and assayed for specific activity. Dialysis was performed against distilled water (+4ºC) using a carboxymethylcellulose membrane. The dialysis end was appreciated with Nessler reactive, which identified the ammonium ions in the dialysis medium (9, 10). Reverse phase HPLC was used to determine the spectrum of the hydrolysis products of both purified inulinases. The system was an isocratic system containing a HPLC Jasco PU-980 pump, a Rheodyne injector with a 20 µl sample loop, a Nucleosil 100 NH 2 column, 5µ, cm, and a detector based on the refraction index ERC-7515A. A mixture of acetonitril:water (75:25) was used as eluent at a rate of 1 ml/min and 30ºC. Calibration curves were automatically built as well as the peak integration and concentration measurement, using the Borwin 1.20 programme. The kinetic parameters (V max and K m ) of the inulinase-90 were determined by the Lineweaver-Burk method (11). The results were processed using Microsoft Excel and Statistica for Windows version 4.3.

4 6 Luminiţa Georgescu, Anca Nicolau, Irina Stoica 4 RESULTS AND DISCUSSION A. niger inulinase properties (optimum temperature, optimum ph, thermal stability), the hydrolysis dynamics and kinetic parameters were determined only for variant A of the raw preparation, the one which showed the best inulinase activity. THE OPTIMUM TEMPERATURE AND OPTIMUM ph OF INULINASE The optimum temperature for the A. niger MIUG 1.15 inulinase was 60 C (Figure 1). Because temperatures higher than 60ºC did not determine the rapid loss of inulinase activity, it could be said that the A. niger MIUG 1.15 inulinase is a thermostable enzyme y = 6E-06x x x x x R 2 = Relative activity, % Temperature, C Fig. 1. The optimum temperature for the A. niger MIUG 1.15 inulinase. The optimum ph value for the A. niger MIUG 1.15 inulinase was 4.5 (Figure 2). It is interesting to note that the regression equation indicated an optimum value of 4.3. THE STABILITY OF INULINASE AT THE OPTIMUM TEMPERATURE The thermal stability of A. niger MIUG 1.15 inulinase was studied at 60 C, value that represents its optimum temperature. It was noticed that no activity loss was registered after 30 min of maintenance at 60 C and a small loss was registered after 1 h of maintenance at this temperature.

5 5 Fungal inulinase Relative activity, % y = x x 4-251x x x R 2 = ph Fig. 2. The optimum ph for the A. niger MIUG 1.15 inulinase. THE STABILITY OF INULINASE DURING REPEATED FREEZING/THAWING CYCLES The results concerning the stability of the inulinase preparation during repeated freezing/thawing cycles are presented in Figure 3. The enzyme had a high stability, a loss of activity of 5.9% being registered after 6 freezing/thawing cycles Relative activity, % Initial freezing/thawing cycles Fig. 3. The stability of inulinase preparation at repeated freezing/thawing cycles.

6 8 Luminiţa Georgescu, Anca Nicolau, Irina Stoica 6 7 THE HYDROLYSIS DYNAMICS OF INULINASE ON TURNIP INULIN To establish the hydrolysis dynamics of the raw inulinase, the reducing sugars released into the medium were assayed and expressed as fructose (Figure 4). One could observe that the reaction rate linearly increased during the first 30 min and then had a constant value. For fructose concentrations higher than 6%, the inulinase activity was inhibited by the final product. The higher hydrolysis yield was attained during the first 45 min. 6 5 mg fructose/ml Time, min Fig The hydrolysis dynamics of inulinase on turnip inulin. THE PARTIAL PURIFICATION OF RAW INULINASE The enzymatic activity of the two precipitated fractions (inulinase-80 and inulinase-90) was determined on two substrates: pure inulin and sucrose. The obtained results, the purification factor and recovery rate are presented in Table 1. Due to advanced concentration (concentration factor = 20.5), the inulinase activity decreased by 49.3%. The higher salt concentration modified the ionic strength of the preparation and affected the enzyme structure and its catalytic activity. Following (NH 4 ) 2 SO 4 precipitation, two inulinase fractions were separated and characterized based on their I/Z ratios (the ratio between the enzymatic activity determined on inulin and the one determined on sucrose). Practically, the I/Z ratio expressed the affinity of the enzyme for inulin as compared to that for sucrose. This ratio has a value of 0.186/3.908 = for inulinase-80 and of 0.740/0.977 = for inulinase-90. Although both fractions have shown inulinase activity, they

7 7 Fungal inulinase 9 behave as different enzymes. Thus, inulinase-80 resembles to invertase because it presents a higher affinity for sucrose than for inulin. Table 1 Fraction Partial purification of the A. niger MIUG 1.15 inulinase Enzymatic activity (U/ml) Specific activity (U/mg protein) Purification factor Recovery rate (%) I Z I Z I Z I Z Raw inulinase, variant A Concentrated raw inulinase Inulinase Inulinase Note: inulin (I) or sucrose (Z) was used as substrate for assaying the inulinase activity. As inulinase-80, inulinase-90 also showed a higher affinity for sucrose than for inulin, but its affinity for inulin is higher than that of inulinase-80. This is in accordance to the fact that the enzymes having group specificity present a higher affinity for low molecular weight substrates. We considered inulinase-90 as an exoinulinase and characterized it from the kinetic point of view. THE KINETIC PARAMETERS V max AND K m OF INULINASE-90 The kinetic parameters were determined on pure turnip inulin and the hydrolysis products were determined after 30 min and were expressed as fructose. To obtain V max and K m values, the reaction rate values were plotted versus substrate concentrations by the Lineweaver-Burk method (Figure 5). The maximum velocity rate (V max ) was µmols fructose/min and the Michaelis constant (K m ) was 7.64 mm, considering that 2700 Da is the molecular weight of an inulin having a GP of 15. HPLC ANALYSIS OF THE HYDROLYSIS PRODUCTS The enzymatic activity of inulinase-80 was evidenced chromatographically. Fructose was the major hydrolysis product resulted from the action of this enzyme fraction on inulin (Figure 6). Also a fructosyltransferase secondary activity could be noted, which was certified by the fact that when inulinase-80 acted on sucrose, it did not form an equimolecular mixture of glucose and fructose. At the same time, the chromatogram presented in Figure 6 shows a peak at min that undoubtedly corresponded to a fructooligosaccharide. The chromatogram presented in Figure 7 sustains the supposition that inulinase-90 is an exoinulinase.

8 10 Luminiţa Georgescu, Anca Nicolau, Irina Stoica /reaction velocity (as micromols fructose/cc) y = x R 2 = /inuline concentration, mm -1 Fig. 5. The influence of substrate concentration upon the reaction rate of partially purified inulinase (inulinase-90). glucose unknown Fig. 6. The HPLC chromatogram presenting the composition of the hydrolysis products obtained by inulinase-80 action on 2% sucrose.

9 9 Fungal inulinase 11 Fig. 7. The HPLC chromatogram presenting the composition of the hydrolysis products obtained by inulinase-90 action on 2% fibruline LC. CONCLUSIONS Having in view to obtain FOS by fructan hydrolysis, these experiments were designed in order to obtain and characterize a fungal endoinulinase. After the partial purification of the raw inulinase preparation, we obtained two proteic fractions showing inulinase activity, but acting as exoinulinases (fact proved by the spectrum of hydrolysis product). Both inulinase fractions had a higher affinity for sucrose than for inulin, but just one preparation (inulinase-80) behaved as invertase. This fraction also exhibited a fructosyltransferase activity. This secondary activity, clearly evidenced by HPLC analysis, allows the biosynthesis of FOS having a certain molecular weight, but this needs further studies on inulinase-80 preparation.

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