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Production and separation of exo- and endoinulinase from Aspergillus ficuum
Process Biochemistry 39 (2003) 5 /11 www.elsevier.com/locate/procbio
Production and separation of exo- and endoinulinase from Aspergillus ficuum Wang Jing, Jin Zhengyu *, Jiang Bo, Adamu Augustine School of Food Science and Technology, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, JiangSu Province, People’s Republic of China Received 12 June 2002; received in revised form 12 August 2002; accepted 28 August 2002
Abstract The production of both exo- and endoinulinase by Aspergillus ficuum JNSP5-06 was investigated. Optimum fermentation conditions were found to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4 ×/7H2O, 0.05%; ZnSO4 ×/7H2O, 0.01%; initial pH 6.5. A new and convenient method was developed to separate the inulinases by native-polyacrylamide gel electrophoresis (PAGE). Eight protein bands were obtained. Three bands were identified as exoinulinase and two bands were endoinulinase using TLC and HPLC. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus ficuum ; Inulinase; Exoinulinase; Endoinulinase; Native-PAGE; Production; Separation
1. Introduction Inulin, a fructose polymer found as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory and dahlia, represents a potential source of fructose and inulooligosaccharides that can be used as sweeteners and functional food additives. This fructan consists of a linear chain of fructose (b-2,1-link) with a terminal glucose unit [1]. Inulin is depolymerized by two types of inulinase: exoinulinase (b-D-fructan fructohydrolase, EC 3.2.1.80), endoinulinase (2,1-b-D-fructan fructanohydrolase, ECb-3.2.1.7). Exoinulinase can successively release fructose from the non-reducing b-2,1 end of inulin. The complete hydrolysis of inulin by this enzyme yields 95% fructose syrup under optimized condition [2]. Endoinulinase acts on the internal linkage of inulin randomly to release inulo-triose, -tetraose and -pentaose as the major products [3]. Most microbial inulinases are exo-acting enzymes. Nakamura et al. [4] first reported that a strain of Aspergillus niger excreted two distinct inulin-hydrolys-
* Corresponding author. Tel.: /86-510-5879711; fax: /86-5105811950. E-mail address: [email protected] (J. Zhengyu).
ing enzymes, endo- and exoinulinase. Thereafter, Penicillium purpurogenum and Chrysosporium pannorum were reported to produce both endo- and exoinulinase [5,6]. A. ficuum , one of the industrially important fungi, also excreted endoinulinase as well as exoinulinase [7]. Because of the synergistic action of the two enzymes [8], fructose is easily obtained; however, it is difficult to determine whether the enzymes coexist. Similarly, it is difficult to separate the two enzymes completely by conventional methods as they are very similar in properties [9]. This paper describes the production of inulinase by an inulinase-producing strain, A. ficuum , and the development of a new and convenient method to separate and identify exo- and endoinulinases.
2. Materials and methods 2.1. Materials 2.1.1. Micro-organisms and culture conditions The strain isolated from a soil sample was identified as A. ficuum JNSP5-06. The strain was maintained on solid medium at 4 8C. The basal medium contained the following: 2% inulin, 2% peptone, 1.2% (NH4)H2PO4, 0.5% NaCl, 0.05% MgSO4 ×/7H2O, initial pH 5.5.
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W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Table 1 Effect of different carbon sources on the production of inulinase Carbon source (2%, w/v)
Inulinase activity (U/ml)
Invertase activity (U/ml)
I/S
Inulin Glucose Fructose Sucrose Lactose Raffinose Soluble starch
11.02 3.85 4.52 5.21 3.22 7.02 3.81
9.63 3.51 3.31 4.07 1.30 5.80 3.44
1.14 1.10 1.37 1.28 2.47 1.21 1.11
Table 2 Effect of different nitrogen sources on the production of inulinase Nitrogen source
Inulinase activity (U/ml)
Invertase activity (U/ml)
I/S
Organic nitrogen (2%) Peptone Yeast extract Corn steep liquor Urea
11.02 21.54 6.88 1.60
9.63 17.14 5.81 1.35
1.14 1.26 1.19 1.18
Inorganic nitrogen (0.5%) (NH4)H2PO4 NH4Cl (NH4)2SO4
11.02 8.62 3.16
9.63 7.03 2.96
1.14 1.23 1.07
2.1.2. Chemicals Inulin purchased from Orafiti Company. Other chemicals were all of analytical grade. 2.2. Methods 2.2.1. Assay of enzyme activities Enzymes were assayed by measuring the concentration of reducing sugars released from inulin and sucrose. The reaction mixture containing 1 ml of diluted crude enzyme and 4 ml of 2% inulin or 2% sucrose (dissolved in 0.1 M acetate buffer, pH 5.0) was incubated at 50 8C. After incubating for 30 min, aliquots of 0.5 ml were withdrawn and increase in reducing sugar was estimated by a 3,5-dinitrosalicylic acid method [10]. Absorbance was read at 575 nm. A higher absorbance indicated a high level of reducing sugar produced and consequently, a high enzyme activity. One inulinase unit is the amount of enzyme which forms 1 mmol fructose per min. One invertase unit is the amount of enzyme which hydrolyses 1 mmol sucrose per min under the same conditions. The ratio between these two activities is commonly expressed as inulin/sucrose (I/S) [11].
2.2.3. Thin-layer chromatography Thin-layer chromatography was carried out on silica gel 60 H plates. Plates were developed at room temperature for 24 h with a solvent system of chloroform /acetic acid /water (30:35:5, v/v/v). Sugar spots were visualized by spraying the plates with 0.1 anaphthol (containing 10% phosphoric acid) and heating at 120 8C for 10 min [12]. 2.2.4. HPLC An APS Hypersil 4.6 /100 mm column was used. Acetonitrile /water (7:3) served as the mobile phase. Eight bands were cut down from the PAGE plates and macerated in tubes into which 5% inulin (dissolved in 0.1 M acetate buffer, pH 5.0) was added. After incubated on a rotary shaker at 50 8C for 24 h, the hydrolysate was analysed. A sample volume of 10 ml was run at a flow rate of 1 ml/min. The detector signal was electronically monitored with a Waters 2401 RI detector [12].
3. Results 3.1. Production of inulinase of A. ficuum JNSP5-06
2.2.2. Native-polyacrylamide gel electrophoresis (PAGE) Native-PAGE was performed on a 12% gel at a constant current of 10 mA with Tris /glycine (pH 8.3) as the running buffer. Protein was stained with Coomassie Brilliant Blue G-250.
3.1.1. Effect of different carbon sources on the production of inulinase On growing A. ficuum in media containing different carbon sources, the maximum inulinase production was observed with medium containing inulin (Table 1).
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11 Table 3 Effect of addition of different inorganic salt on the production of inulinase Inorganic salt
Relative activity (%)
Control KCl (0.5%) CaCl2 (0.5%) MgSO4 ×/7H2O (0.05%) CuSO4 ×/5H2O (0.01%) FeSO4 ×/7H2O (0.01%) MnSO4 ×/H2O (0.01%) ZnSO4 ×/7H2O (0.01%)
100 58 64 138 79 92 48 128
7
repression and consequently less enzyme activity was observed. Two percent inulin and 2% yeast extract gave the best inulinase production.
3.1.4. Effect of different initial pH on the production of inulinase The initial pH was adjusted by adding 0.2 M HCl or NaOH. Inulinase productivity was optimal at initial pH 6.5, which indicated that this strain is acidophilic.
Fructose, which is believed to be the primary inducer of inulinase [13], produced an inulinase yield that was about 30% of that observed with inulin. This is an indication that inulin is a potential inducer of inulinase. 3.1.2. Effect of different nitrogen sources on the production of inulinase Inulinase activity increased following the replacement of peptone by yeast extract (Table 2). Penicillium sp. TN-88 was also reported to have high inulinase activity with yeast extract as the organic nitrogen source. This may be due to growth factors in the yeast extract [14]. Among the inorganic nitrogen tested, 0.5% (NH4)H2PO4 gave the highest level of inulinase activity. 3.1.3. Effect of different ratio of carbon and nitrogen sources on the production of inulinase Inulin concentrations of 1/4% were evaluated. Inulinase activity was observed to decrease at inulin concentration above 2% (w/v). With high inulin concentrations, accumulation of free reducing sugars at the initial hours of fermentation probably caused catabolite
3.1.5. Effect of addition of different inorganic salt on the production of inulinase Results (Table 3) showed that Mg2 and Zn2 favoured inulinase production. However, K , Ca2, Cu2, Fe2 and Mn2 inhibited inulinase production. Based on the analysis of the results, the optimum fermentation condition was estimated to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4 ×/7H2O, 0.05%; ZnSO4 ×/7H2O, 0.01%; initial pH 6.5.
3.1.6. Time course of inulinase production A. ficuum JNSP5-06 was grown under optimal fermentation conditions at 30 8C up to 6 days. The concentration of reducing sugars liberated from inulin increased during the first 24 h because of the inulinase activity, but the sugars were completely consumed in 72 h (Fig. 1). Inulinase activity reached maximum of 25 U/ ml after 5 days, and invertase activity increased to 20 U/ ml. The ratio of inulinase/invertase activity showed no significant change during this time. The cell density increased to the maximum in 3 days and then declined gradually.
Fig. 1. Time course of inulase production.
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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3.2. Separation of A. ficuum inulinase by native-PAGE The ionic strength of buffer and pH is the main factor influencing native-PAGE. In order to establish an efficient PAGE system, the effect of the concentration of separating gel, ionic strength and pH value of separation buffer and running buffer on the resolution was studied based on the Laemmli system [15]. For the A. ficuum JNSP5-06 inulinase system, a gel concentration of 12%, 0.75 M Tris/HCl (pH 8.9) as separation buffer and 0.05 M Tris/0.384 M glycine (pH 8.3) as running buffer provided a satisfactory separation effect. Following the evaluation by the native-PAGE, eight clear bands were obtained for the culture filtrate of A. ficuum JNSP5-06 (Fig. 2). 3.3. Enzyme assays PAGE bands were cut and macerated in tubes, into which 2% inulin and 2% sucrose (dissolved in 0.1 M acetate buffer, pH 5.0) were added. After incubating at 50 8C for 1, 3 and 11 h, respectively, 0.5 ml reaction mixture was withdrawn to determine reducing sugar
Fig. 2. Native-PAGE for A. ficuum inulinases.
Table 4 Absorbance at 575 nm of eight bands reaction for different period Bands
Reaction time 1h
1 2 3 4 5 6 7 8
3h
11 h
Inulin
Sucrose
Inulin
Sucrose
Inulin
Sucrose
0.153 0.157 0.225 0.159 0.152 0.158 0.153 0.158
0.000 0.046 0.015 0.002 0.001 0.005 0.001 0.005
0.168 0.181 0.330 0.176 0.160 0.162 0.230 0.178
0.01 0.123 0.137 0.452 0.003 0.012 0.102 0.014
0.201 0.353 0.614 0.307 0.173 0.179 0.346 0.303
0.059 0.413 0.359 0.503 0.010 0.026 0.216 0.124
Fig. 3. Thin-layer chromatography of the inulin hydrolysate from enzyme bands.
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Fig. 4. HPLC of the inulin hydrolysate from exoinulinase.
Fig. 5. HPLC of the inulin hydrolysate from endoinulinase.
produced. After addition of 1.5 ml of water and 3,5dinitrosalicylic acid, samples were heated in boiling water for 5 min and cooled to room temperature instantly. Water was added to 25 ml and the absorbance read at 575 nm (Table 4). A higher absorbance indicated a high level of reducing sugar produced and consequently, a high enzyme activity. Of the eight bands, bands 2, 3, 4, 7 and 8 showed enzyme activities towards
inulin and sucrose, which demonstrated that these five bands were inulinases. 3.4. Thin-layer chromatography The products of the enzyme reaction in time course were analysed by TLC. The results showed two characteristic patterns of inulin hydrolysis (Fig. 3). Bands 2
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Table 5 Comparison of the different inulinase-producing strains Strains
Inulinase activity (U/ml)
I/S
Reference
A. niger mutant 817 Kluyveromyces fragilis A. niger -12 Penicillium sp. TN-88
17.5 6.2 4.6 9.9
880 / 53 11.2
[15] [11] [4] [12]
and 3 liberated fructose as the major product, which indicated that these two enzymes were exoinulinases. The product of band 4 showed a little increase in fructose and was ascribed as exoinulinase. The major products of bands 7 and 8 were inulooligofructoses, which suggested that the two enzymes showed endo-type depolymerization activity, which is typical of endoinulinases. 3.5. High-performance liquid chromatography The inulin hydrolysate from bands 2, 3 and 4 had an obvious effect on fructose increase and bands 7 and 8 increased inulooligosaccharides (F2 /F7). Bands 1, 5 and 6 had no noticeable effect on inulin. It is therefore suggested that bands 2, 3 and 4 were exoinulinases and bands 7 and 8 were endoinulinases. HPLC of the inulin hydrolysate from exo- and endoinulinase are shown in Fig. 4 and Fig. 5, respectively. The two peaks of one DP may be due to the two kinds of sugars with the same DP (Fn and G-Fn 1).
4. Discussions The medium composition used for inulinase production depends on the type of micro-organism involved. A. ficuum , a thermostable industrial strain, produced inulinase in response to the presence of inulin. Yeast extract and (NH4)H2PO4 in the medium were suitable organic and inorganic nitrogen sources, respectively, for the synthesis of inulinase while Mg2 and Zn2 further increased the inulinase level. After 5 days fermentation under optimal conditions inulinase activity attained 25 U/ml, which was greater than inulinase from other strains (Table 5). Due to the similarity between the exo- and endoinulinase, there are some difficulties in separating and identifying the two enzymes by conventional methods. A new and convenient method is reported in this paper. Native-PAGE is an effective method to separate enzymes with identical properties. An effective nativePAGE system was established and this can provide higher resolution. Eight enzyme bands were obtained by the system. After assay of the enzyme activity, it was shown that five bands had both inulinase activity and
invertase activity. It was demonstrated by TLC and HPLC that three bands were exoinulinase and two bands were endoinulinase. This showed that A. ficuum could produce endoinulinase as well as exoinulinase. It was suggested that the ratio of the activity on I/S can be used as the criteria to characterize the enzymes. For exoinulinase, the I/S ratio is lower than 10 while for endoinulinase it is higher than 10 [11]. A. ficuum JNSP506 inulinase with an I/S ratio of 1.18 in this work should be ascribed to exoinulinase, but after separation of the inulinase system by native-PAGE and analysis of the hydrolysate by TLC and HPLC it was observed that this inulinase system also contained endoinulinase. It has been reported that purified exoinulinase has both inulinase and invertase activity, while purified endoinulinase has inulinase activity without invertase activity [16]. However, in this work, it was observed that endoinulinase had invertase activity as well as inulinase activity. This is in agreement with the findings of Moussa and Jacques [7]. The mechanism and further characterization of these enzymes is under investigation in this laboratory.
References [1] Takao U. Metabolism in microorganisms: biosynthesis and degradation of fructans by microbial enzymes other than levansucrase. Sci Technol Fructans 1993:P169 /90. [2] Kumiko K, Toshihiro A, Tae K. Purification and properties of a thermostable inulinase (b-D-fructan fructohydrolase) from Bacillus stearothermophilus KP1289. Starch 1999;51:253 /8. [3] Nakamura T, Kurokawa T, Nakatsu S, Ueda S. Crystallization and general properties of an extracellular inulinase from Aspergillus sp. Nippon Nogeikagaku Kaishi 1978;52:159 /66. [4] Nakamura T, Hoashi S, Nakatsu S. Culture conditions for inulinase production by Aspergillus . Nippon Nogeikagaku Kaishi 1978;52:105 /10. [5] Onodera S, Shiomi N. Purification and substrate specificity of endo-type inulinase from Penicillium purpurogenum . Agric Biol Chem 1988;52:2569 /76. [6] Xiao R, Tanida M, Takao S. Purification and some properties of endoinulinase from Chrysosporium pannorum . J Ferment Bioeng 1989;67:244 /8. [7] Moussa E, Jacques CB. Purification, properties and comparison of invertase, exoinulinases and endoinulinases of Aspergillus ficuum . Appl Microbiol Biotechnol 1987;26:13 /20. [8] Moussa E, Jacques CB. Molecular and kinetic properties of Aspergillus ficuum inulinases. Agric Biol Chem 1990;54(1):61 /8. [9] Tai-Boong U, Mi SC, Sun HL. Purification and characterization of Aspergillus ficuum endoinulinase. Biosci Biotechnol Biochem 1999;63(1):146 /51. [10] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 /8. [11] Vandamme EJ, Derycke DG. Microbial inulinases: fermentation process, properties, and applications. Adv Appl Microbiol 1983;29:139 /76. [12] Azharl R, Szlak AM, Ilan E. Purification and characterization of endo- and exo-inulinase. Biotechnol Appl Biochem 1989;11:105 / 17. [13] Grootwassink JWD, Hewitt GM. Inducible and constitutive formation of b-fructofuranosidase (inulinase) in batch and con-
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11 tinuous cultures of the yeast Kluyveromyces fragilis . J Gen Microbiol 1970;129:31 /41. [14] Toyohiko N, Akichika S, Shusaku M. Production, purification and properties of an endoinulinase of Penicillium sp. TN-88 that liberates inulotriose. J Ferment Bioeng 1997;84(4):313 /8.
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[15] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680 /5. [16] Toyohiko N, Yuhi N, Shigeyuki H. Occurrence of two forms of extracellular endoinulinase from Aspergillus niger mutant 817. J Ferment Bioeng 1994;78(2):134 /9.
Production and separation of exo- and endoinulinase from Aspergillus ficuum Wang Jing, Jin Zhengyu *, Jiang Bo, Adamu Augustine School of Food Science and Technology, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, JiangSu Province, People’s Republic of China Received 12 June 2002; received in revised form 12 August 2002; accepted 28 August 2002
Abstract The production of both exo- and endoinulinase by Aspergillus ficuum JNSP5-06 was investigated. Optimum fermentation conditions were found to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4 ×/7H2O, 0.05%; ZnSO4 ×/7H2O, 0.01%; initial pH 6.5. A new and convenient method was developed to separate the inulinases by native-polyacrylamide gel electrophoresis (PAGE). Eight protein bands were obtained. Three bands were identified as exoinulinase and two bands were endoinulinase using TLC and HPLC. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Aspergillus ficuum ; Inulinase; Exoinulinase; Endoinulinase; Native-PAGE; Production; Separation
1. Introduction Inulin, a fructose polymer found as a reserve carbohydrate in the roots and tubers of plants such as Jerusalem artichoke, chicory and dahlia, represents a potential source of fructose and inulooligosaccharides that can be used as sweeteners and functional food additives. This fructan consists of a linear chain of fructose (b-2,1-link) with a terminal glucose unit [1]. Inulin is depolymerized by two types of inulinase: exoinulinase (b-D-fructan fructohydrolase, EC 3.2.1.80), endoinulinase (2,1-b-D-fructan fructanohydrolase, ECb-3.2.1.7). Exoinulinase can successively release fructose from the non-reducing b-2,1 end of inulin. The complete hydrolysis of inulin by this enzyme yields 95% fructose syrup under optimized condition [2]. Endoinulinase acts on the internal linkage of inulin randomly to release inulo-triose, -tetraose and -pentaose as the major products [3]. Most microbial inulinases are exo-acting enzymes. Nakamura et al. [4] first reported that a strain of Aspergillus niger excreted two distinct inulin-hydrolys-
* Corresponding author. Tel.: /86-510-5879711; fax: /86-5105811950. E-mail address: [email protected] (J. Zhengyu).
ing enzymes, endo- and exoinulinase. Thereafter, Penicillium purpurogenum and Chrysosporium pannorum were reported to produce both endo- and exoinulinase [5,6]. A. ficuum , one of the industrially important fungi, also excreted endoinulinase as well as exoinulinase [7]. Because of the synergistic action of the two enzymes [8], fructose is easily obtained; however, it is difficult to determine whether the enzymes coexist. Similarly, it is difficult to separate the two enzymes completely by conventional methods as they are very similar in properties [9]. This paper describes the production of inulinase by an inulinase-producing strain, A. ficuum , and the development of a new and convenient method to separate and identify exo- and endoinulinases.
2. Materials and methods 2.1. Materials 2.1.1. Micro-organisms and culture conditions The strain isolated from a soil sample was identified as A. ficuum JNSP5-06. The strain was maintained on solid medium at 4 8C. The basal medium contained the following: 2% inulin, 2% peptone, 1.2% (NH4)H2PO4, 0.5% NaCl, 0.05% MgSO4 ×/7H2O, initial pH 5.5.
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 6 4 - 9
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Table 1 Effect of different carbon sources on the production of inulinase Carbon source (2%, w/v)
Inulinase activity (U/ml)
Invertase activity (U/ml)
I/S
Inulin Glucose Fructose Sucrose Lactose Raffinose Soluble starch
11.02 3.85 4.52 5.21 3.22 7.02 3.81
9.63 3.51 3.31 4.07 1.30 5.80 3.44
1.14 1.10 1.37 1.28 2.47 1.21 1.11
Table 2 Effect of different nitrogen sources on the production of inulinase Nitrogen source
Inulinase activity (U/ml)
Invertase activity (U/ml)
I/S
Organic nitrogen (2%) Peptone Yeast extract Corn steep liquor Urea
11.02 21.54 6.88 1.60
9.63 17.14 5.81 1.35
1.14 1.26 1.19 1.18
Inorganic nitrogen (0.5%) (NH4)H2PO4 NH4Cl (NH4)2SO4
11.02 8.62 3.16
9.63 7.03 2.96
1.14 1.23 1.07
2.1.2. Chemicals Inulin purchased from Orafiti Company. Other chemicals were all of analytical grade. 2.2. Methods 2.2.1. Assay of enzyme activities Enzymes were assayed by measuring the concentration of reducing sugars released from inulin and sucrose. The reaction mixture containing 1 ml of diluted crude enzyme and 4 ml of 2% inulin or 2% sucrose (dissolved in 0.1 M acetate buffer, pH 5.0) was incubated at 50 8C. After incubating for 30 min, aliquots of 0.5 ml were withdrawn and increase in reducing sugar was estimated by a 3,5-dinitrosalicylic acid method [10]. Absorbance was read at 575 nm. A higher absorbance indicated a high level of reducing sugar produced and consequently, a high enzyme activity. One inulinase unit is the amount of enzyme which forms 1 mmol fructose per min. One invertase unit is the amount of enzyme which hydrolyses 1 mmol sucrose per min under the same conditions. The ratio between these two activities is commonly expressed as inulin/sucrose (I/S) [11].
2.2.3. Thin-layer chromatography Thin-layer chromatography was carried out on silica gel 60 H plates. Plates were developed at room temperature for 24 h with a solvent system of chloroform /acetic acid /water (30:35:5, v/v/v). Sugar spots were visualized by spraying the plates with 0.1 anaphthol (containing 10% phosphoric acid) and heating at 120 8C for 10 min [12]. 2.2.4. HPLC An APS Hypersil 4.6 /100 mm column was used. Acetonitrile /water (7:3) served as the mobile phase. Eight bands were cut down from the PAGE plates and macerated in tubes into which 5% inulin (dissolved in 0.1 M acetate buffer, pH 5.0) was added. After incubated on a rotary shaker at 50 8C for 24 h, the hydrolysate was analysed. A sample volume of 10 ml was run at a flow rate of 1 ml/min. The detector signal was electronically monitored with a Waters 2401 RI detector [12].
3. Results 3.1. Production of inulinase of A. ficuum JNSP5-06
2.2.2. Native-polyacrylamide gel electrophoresis (PAGE) Native-PAGE was performed on a 12% gel at a constant current of 10 mA with Tris /glycine (pH 8.3) as the running buffer. Protein was stained with Coomassie Brilliant Blue G-250.
3.1.1. Effect of different carbon sources on the production of inulinase On growing A. ficuum in media containing different carbon sources, the maximum inulinase production was observed with medium containing inulin (Table 1).
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11 Table 3 Effect of addition of different inorganic salt on the production of inulinase Inorganic salt
Relative activity (%)
Control KCl (0.5%) CaCl2 (0.5%) MgSO4 ×/7H2O (0.05%) CuSO4 ×/5H2O (0.01%) FeSO4 ×/7H2O (0.01%) MnSO4 ×/H2O (0.01%) ZnSO4 ×/7H2O (0.01%)
100 58 64 138 79 92 48 128
7
repression and consequently less enzyme activity was observed. Two percent inulin and 2% yeast extract gave the best inulinase production.
3.1.4. Effect of different initial pH on the production of inulinase The initial pH was adjusted by adding 0.2 M HCl or NaOH. Inulinase productivity was optimal at initial pH 6.5, which indicated that this strain is acidophilic.
Fructose, which is believed to be the primary inducer of inulinase [13], produced an inulinase yield that was about 30% of that observed with inulin. This is an indication that inulin is a potential inducer of inulinase. 3.1.2. Effect of different nitrogen sources on the production of inulinase Inulinase activity increased following the replacement of peptone by yeast extract (Table 2). Penicillium sp. TN-88 was also reported to have high inulinase activity with yeast extract as the organic nitrogen source. This may be due to growth factors in the yeast extract [14]. Among the inorganic nitrogen tested, 0.5% (NH4)H2PO4 gave the highest level of inulinase activity. 3.1.3. Effect of different ratio of carbon and nitrogen sources on the production of inulinase Inulin concentrations of 1/4% were evaluated. Inulinase activity was observed to decrease at inulin concentration above 2% (w/v). With high inulin concentrations, accumulation of free reducing sugars at the initial hours of fermentation probably caused catabolite
3.1.5. Effect of addition of different inorganic salt on the production of inulinase Results (Table 3) showed that Mg2 and Zn2 favoured inulinase production. However, K , Ca2, Cu2, Fe2 and Mn2 inhibited inulinase production. Based on the analysis of the results, the optimum fermentation condition was estimated to be: inulin, 2%; yeast extract, 2%; (NH4)H2PO4, 0.5%; NaCl, 0.5%; MgSO4 ×/7H2O, 0.05%; ZnSO4 ×/7H2O, 0.01%; initial pH 6.5.
3.1.6. Time course of inulinase production A. ficuum JNSP5-06 was grown under optimal fermentation conditions at 30 8C up to 6 days. The concentration of reducing sugars liberated from inulin increased during the first 24 h because of the inulinase activity, but the sugars were completely consumed in 72 h (Fig. 1). Inulinase activity reached maximum of 25 U/ ml after 5 days, and invertase activity increased to 20 U/ ml. The ratio of inulinase/invertase activity showed no significant change during this time. The cell density increased to the maximum in 3 days and then declined gradually.
Fig. 1. Time course of inulase production.
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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3.2. Separation of A. ficuum inulinase by native-PAGE The ionic strength of buffer and pH is the main factor influencing native-PAGE. In order to establish an efficient PAGE system, the effect of the concentration of separating gel, ionic strength and pH value of separation buffer and running buffer on the resolution was studied based on the Laemmli system [15]. For the A. ficuum JNSP5-06 inulinase system, a gel concentration of 12%, 0.75 M Tris/HCl (pH 8.9) as separation buffer and 0.05 M Tris/0.384 M glycine (pH 8.3) as running buffer provided a satisfactory separation effect. Following the evaluation by the native-PAGE, eight clear bands were obtained for the culture filtrate of A. ficuum JNSP5-06 (Fig. 2). 3.3. Enzyme assays PAGE bands were cut and macerated in tubes, into which 2% inulin and 2% sucrose (dissolved in 0.1 M acetate buffer, pH 5.0) were added. After incubating at 50 8C for 1, 3 and 11 h, respectively, 0.5 ml reaction mixture was withdrawn to determine reducing sugar
Fig. 2. Native-PAGE for A. ficuum inulinases.
Table 4 Absorbance at 575 nm of eight bands reaction for different period Bands
Reaction time 1h
1 2 3 4 5 6 7 8
3h
11 h
Inulin
Sucrose
Inulin
Sucrose
Inulin
Sucrose
0.153 0.157 0.225 0.159 0.152 0.158 0.153 0.158
0.000 0.046 0.015 0.002 0.001 0.005 0.001 0.005
0.168 0.181 0.330 0.176 0.160 0.162 0.230 0.178
0.01 0.123 0.137 0.452 0.003 0.012 0.102 0.014
0.201 0.353 0.614 0.307 0.173 0.179 0.346 0.303
0.059 0.413 0.359 0.503 0.010 0.026 0.216 0.124
Fig. 3. Thin-layer chromatography of the inulin hydrolysate from enzyme bands.
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Fig. 4. HPLC of the inulin hydrolysate from exoinulinase.
Fig. 5. HPLC of the inulin hydrolysate from endoinulinase.
produced. After addition of 1.5 ml of water and 3,5dinitrosalicylic acid, samples were heated in boiling water for 5 min and cooled to room temperature instantly. Water was added to 25 ml and the absorbance read at 575 nm (Table 4). A higher absorbance indicated a high level of reducing sugar produced and consequently, a high enzyme activity. Of the eight bands, bands 2, 3, 4, 7 and 8 showed enzyme activities towards
inulin and sucrose, which demonstrated that these five bands were inulinases. 3.4. Thin-layer chromatography The products of the enzyme reaction in time course were analysed by TLC. The results showed two characteristic patterns of inulin hydrolysis (Fig. 3). Bands 2
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11
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Table 5 Comparison of the different inulinase-producing strains Strains
Inulinase activity (U/ml)
I/S
Reference
A. niger mutant 817 Kluyveromyces fragilis A. niger -12 Penicillium sp. TN-88
17.5 6.2 4.6 9.9
880 / 53 11.2
[15] [11] [4] [12]
and 3 liberated fructose as the major product, which indicated that these two enzymes were exoinulinases. The product of band 4 showed a little increase in fructose and was ascribed as exoinulinase. The major products of bands 7 and 8 were inulooligofructoses, which suggested that the two enzymes showed endo-type depolymerization activity, which is typical of endoinulinases. 3.5. High-performance liquid chromatography The inulin hydrolysate from bands 2, 3 and 4 had an obvious effect on fructose increase and bands 7 and 8 increased inulooligosaccharides (F2 /F7). Bands 1, 5 and 6 had no noticeable effect on inulin. It is therefore suggested that bands 2, 3 and 4 were exoinulinases and bands 7 and 8 were endoinulinases. HPLC of the inulin hydrolysate from exo- and endoinulinase are shown in Fig. 4 and Fig. 5, respectively. The two peaks of one DP may be due to the two kinds of sugars with the same DP (Fn and G-Fn 1).
4. Discussions The medium composition used for inulinase production depends on the type of micro-organism involved. A. ficuum , a thermostable industrial strain, produced inulinase in response to the presence of inulin. Yeast extract and (NH4)H2PO4 in the medium were suitable organic and inorganic nitrogen sources, respectively, for the synthesis of inulinase while Mg2 and Zn2 further increased the inulinase level. After 5 days fermentation under optimal conditions inulinase activity attained 25 U/ml, which was greater than inulinase from other strains (Table 5). Due to the similarity between the exo- and endoinulinase, there are some difficulties in separating and identifying the two enzymes by conventional methods. A new and convenient method is reported in this paper. Native-PAGE is an effective method to separate enzymes with identical properties. An effective nativePAGE system was established and this can provide higher resolution. Eight enzyme bands were obtained by the system. After assay of the enzyme activity, it was shown that five bands had both inulinase activity and
invertase activity. It was demonstrated by TLC and HPLC that three bands were exoinulinase and two bands were endoinulinase. This showed that A. ficuum could produce endoinulinase as well as exoinulinase. It was suggested that the ratio of the activity on I/S can be used as the criteria to characterize the enzymes. For exoinulinase, the I/S ratio is lower than 10 while for endoinulinase it is higher than 10 [11]. A. ficuum JNSP506 inulinase with an I/S ratio of 1.18 in this work should be ascribed to exoinulinase, but after separation of the inulinase system by native-PAGE and analysis of the hydrolysate by TLC and HPLC it was observed that this inulinase system also contained endoinulinase. It has been reported that purified exoinulinase has both inulinase and invertase activity, while purified endoinulinase has inulinase activity without invertase activity [16]. However, in this work, it was observed that endoinulinase had invertase activity as well as inulinase activity. This is in agreement with the findings of Moussa and Jacques [7]. The mechanism and further characterization of these enzymes is under investigation in this laboratory.
References [1] Takao U. Metabolism in microorganisms: biosynthesis and degradation of fructans by microbial enzymes other than levansucrase. Sci Technol Fructans 1993:P169 /90. [2] Kumiko K, Toshihiro A, Tae K. Purification and properties of a thermostable inulinase (b-D-fructan fructohydrolase) from Bacillus stearothermophilus KP1289. Starch 1999;51:253 /8. [3] Nakamura T, Kurokawa T, Nakatsu S, Ueda S. Crystallization and general properties of an extracellular inulinase from Aspergillus sp. Nippon Nogeikagaku Kaishi 1978;52:159 /66. [4] Nakamura T, Hoashi S, Nakatsu S. Culture conditions for inulinase production by Aspergillus . Nippon Nogeikagaku Kaishi 1978;52:105 /10. [5] Onodera S, Shiomi N. Purification and substrate specificity of endo-type inulinase from Penicillium purpurogenum . Agric Biol Chem 1988;52:2569 /76. [6] Xiao R, Tanida M, Takao S. Purification and some properties of endoinulinase from Chrysosporium pannorum . J Ferment Bioeng 1989;67:244 /8. [7] Moussa E, Jacques CB. Purification, properties and comparison of invertase, exoinulinases and endoinulinases of Aspergillus ficuum . Appl Microbiol Biotechnol 1987;26:13 /20. [8] Moussa E, Jacques CB. Molecular and kinetic properties of Aspergillus ficuum inulinases. Agric Biol Chem 1990;54(1):61 /8. [9] Tai-Boong U, Mi SC, Sun HL. Purification and characterization of Aspergillus ficuum endoinulinase. Biosci Biotechnol Biochem 1999;63(1):146 /51. [10] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 /8. [11] Vandamme EJ, Derycke DG. Microbial inulinases: fermentation process, properties, and applications. Adv Appl Microbiol 1983;29:139 /76. [12] Azharl R, Szlak AM, Ilan E. Purification and characterization of endo- and exo-inulinase. Biotechnol Appl Biochem 1989;11:105 / 17. [13] Grootwassink JWD, Hewitt GM. Inducible and constitutive formation of b-fructofuranosidase (inulinase) in batch and con-
W. Jing et al. / Process Biochemistry 39 (2003) 5 /11 tinuous cultures of the yeast Kluyveromyces fragilis . J Gen Microbiol 1970;129:31 /41. [14] Toyohiko N, Akichika S, Shusaku M. Production, purification and properties of an endoinulinase of Penicillium sp. TN-88 that liberates inulotriose. J Ferment Bioeng 1997;84(4):313 /8.
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[15] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680 /5. [16] Toyohiko N, Yuhi N, Shigeyuki H. Occurrence of two forms of extracellular endoinulinase from Aspergillus niger mutant 817. J Ferment Bioeng 1994;78(2):134 /9.