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Purification and characterization of an endoinulinase from Xanthomonas oryzae No.5
Process Biochemistry 37 (2002) 1325– 1331 www.elsevier.com/locate/procbio
Purification and characterization of an endoinulinase from Xanthomonas oryzae No.5 Youn Jeung Cho, Jong Won Yun * Department of Biotechnology, Taegu Uni6ersity, Kyungsan, Kyungbuk 712 -714, South Korea Received 31 October 2001; received in revised form 8 January 2002; accepted 18 January 2002
Abstract The extracellular endoinulinase from Xanthomonas oryzae No. 5 which converts inulin into inulooligosaccharides was purified from the culture broth by ammonium sulphate precipitation, followed by column chromatography on Phenyl-Sepharose and DEAE-Sephacel. The enzyme was purified 29-fold with a yield of 5.5% from the starting culture broth. The purified enzyme gave a single band on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 139 kDa. The specific activity of the purified enzyme was 1372 U/mg. The enzyme activity was highest at pH 7.5 and 50 °C, and stable over a pH range of 6.0–9.0 and up to 45 °C. The reaction rates declined at inulin concentrations over 125 g/l indicating occurrence of substrate inhibition. The Michaelis constant (Km) and maximum reaction velocity (Vmax) of the endoinulinase were 16.7 g/l and 12.1 g/l·h, respectively. There was no significant difference in the product distribution from the reaction between crude and purified enzyme, where major product was penta-inulooligosaccharides. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Inulooligosaccharides; Endoinulinase; Enzyme purification; Xanthomonas oryzae
1. Introduction Inulin is a linear b-2,1 linked fructan terminated by a sucrose residue, which is usually found as reserve cabohydrate in various plants such as chicory, dahlia, and Jerusalem artichoke. Inulinases are fructofuranosyl hydrolases, which are produced by bacteria and plants as well as moulds [1 – 3]. Three types of inulinase have been described, including exoinulinase, endoinulinase, and fructotransferase which is not a common catabolic carbohydratase. The general enzymic reaction mainly occurs by two enzymes: exoinulinase (EC 3.2.1.80) which splits off the terminal fructose units from inulin and endoinulinase (EC 3.2.1.7) which hydrolyses inulin into inulooligosaccharides. Inulooligosaccharides produced from inulin have been suggested to have similar physiological
* Corresponding author. Tel.: + 82-53-850-6556; fax: +82-53-8506559. E-mail address: [email protected] (J.W. Yun).
functions to fructooligosaccharides [4]. Many kinds of oligosaccharides have been of increasing importance because of their favourable functionalities such as being low caloric, non-cariogenic and acting as a growth factor for beneficial microorganisms in the intestinal flora [5–10]. Inulin can be hydrolyzed by either sole action of exoinulinase or synergistic action of exo- and endoinulinase. More recent investigations have revealed that endoinulinase free from invertase or exoinulinase activity, hydrolyses the internal linkages of inulin to yield several oligosaccharides which are soluble dietary fibres and/or a functional sweeteners [11 –13]. Endoinulinases have been produced from several microorganisms such as Aspergillus niger [13,14], Aspergillus ficuum [2], Arthrobacter sp. [15,16], and Penicillium purpurogenum [17]. In previous articles [1,18,19], we have reported on the production of inulo-oligosaccharides from inulin or chicory extract using crude endoinulinase preparations from Xanthomonas sp. In the present study, we have purified the endoinulinase and investigated the kinetic properties.
0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0032-9592(02)00018-3
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Y.J. Cho, J.W. Yun / Process Biochemistry 37 (2002) 1325–1331
2. Materials and methods
2.1. Microorganism and culture conditions Xanthomonas oryzae No. 5 was grown aerobically in a suspension culture. The medium contained (g/l) inulin 20, yeast extract 20, (NH4)2HPO4 5, NH4H2PO4 2, MnCl2 ·4H2O 0.5, KCl 0.5, MgSO4 ·7H2O 0.5, FeSO4 ·7H2O 0.01, and pH was adjusted to 7.0 before sterilization. The pre-culture was carried out in 250 ml flasks contained 50 ml medium at 37 °C for 24 h on a rotary shaker at 150 rpm. The main culture for the production of enzyme was carried out in a 5 l fermenter (KoBioTech Co., Seoul, Korea) at 37 °C for 48 h. The working volume and inoculum size were 3 l and 2.0% (v/v), respectively. Agitation speed and aeration rate were controlled to 150 rpm and 2.0 vvm, respectively. After removal of cells by centrifugation at 10000×g for 20 min, the supernatant was used as a starting enzyme solution throughout the purification processes.
2.2. Chemicals The inulin used as substrate was purchased from Sigma Chemical Co. (MO, USA) (prepared from chicory root). 1-Kestose (GF2), nystose (GF3), 1F-bfructofuranosyl nystose (GF4) were purchased from Wako Pure Chemical, Ltd. (Osaka, Japan). DEAESephacel was the product of Sigma. Phenyl-Sepharose High Performance was purchased from Pharmacia (Uppsala, Sweden). Protein assay kit and standard markers of molecular weights for SDS-PAGE were purchased from Bio-rad Laboratories (California, USA). TLC plates (Silica gel 60 F254) were purchased from Merck (Darmstdt, Germany). All other chemicals were of reagent grade.
with Phenyl-Sepharose (Pharmacia). The column was pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 1.5 M (NH4)2SO4 and eluted with a decreasing linear gradient of ammonium sulphate from 1.5 to 0 M in the same buffer. The resulting enzyme solution was concentrated and dialyzed against 50 mM sodium phosphate buffer (pH 7.0). Proteins were analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE).
2.4. Enzyme assays Endoinulinase activity was assayed by incubating 1 ml enzyme solution with 5% (w/v) inulin dissolved in 50 mM sodium phosphate buffer (pH 6.0) at 45 °C for 1 h. The reaction was terminated by heating at 100 °C for 10 min. One enzyme unit was defined as the amount of enzyme responsible for hydrolysis of 1 mmol inulin per min under the above conditions. Where, the mean molecular weight of inulin was estimated as 5000 Da after acid-hydrolysis analysis. Proteins were measured by the Bradford method [20], using bovine serum albumin as the standard protein.
2.5. Gel electrophoresis SDS-PAGE was performed at a concentration of 12% polyacrylamide according to the method of Laemmli [21]. The gel was stained with Coomassie Brilliant Blue R-250. The standard proteins used were aprotinin (6.6 kDa), lysozyme (20.6 kDa), soybean trypsin inhibitor (29.8 kDa), carbonic anhydrase (36.4 kDa), ovalbumin (48.6 kDa), bovine serum albumin (94 kDa), b-galactosidase (130 kDa), and myosin (201 kDa).
2.3. Enzyme purification
2.6. Analytical methods
All of the operations for enzyme purification were done at 4 °C, and centrifugation was conducted at 7000×g for 15 min. Crude enzyme solution was first concentrated by ammonium sulphate precipitation (40–80% saturation) followed by dialysis against 50 mM sodium phosphate buffer (pH 6.0) at 4 °C and then applied on a DEAE-Sephacel column (2.5× 30 cm) pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.0). Absorbed proteins were eluted in salt gradients (with 600 ml of 0 to 1 M NaCl) at a flow rate of 0.3 ml/min (3 ml per tube). The fractions showing inulinase activity were pooled and dialyzed overnight against 50 mM sodium phosphate buffer (pH 7.0) containing 1.5 M (NH4)2SO4 at 4 °C and subjected to the hydrophobic interaction chromatography (HIC) using a column (1.5×20 cm) packed
Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were simultaneously used for quantitative analysis of the reaction products. Pre-coated TLC plate (Merck) spotted with samples were developed with the solvent systems; isoprophyl alcohol: ethyl acetate: water (2:2:1 by volume). The carbohydrates loaded in the plates were visualized by heating the plates after spraying solvent containing 0.5% a-naphtol and 5% sulfuric acid in absolute ethanol. The quantitative analyses of the carbohydrates were carried out by HPLC using an Aminex HPX-42C column (0.78× 30 cm, Biorad) and a refractive index detector (Shimadzu Co, Kyoto, Japan). The column temperature was maintained at 85 °C and water was used as a mobile phase at a flow rate of 0.6 ml/min.
Y.J. Cho, J.W. Yun / Process Biochemistry 37 (2002) 1325–1331
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Fig. 1 shows the typical time profiles for endoinulinase production from X. oryzae No. 5 in a 5 l stirredtank fermentor. The cell growth increased with time and reached a maximum value at 30 h and enzyme activity indicated 77 U/ml at 30 h of fermentation, thereafter enzyme activity began to decrease rapidly. Similar trends in decrease of enzyme production at prolonged fermentation period have been reported in inulinases production from Bacillus sphaericus and transfructosylating enzyme production from Bacillus macerans [22,23].
summarizes results of the three-step purification of endoinulinase. The supernatant obtained from ammonium sulphate precipitates were first loaded on an anion exchange column, DEAE-Sephacel. The elution profile in DEAE-Sephacel chromatography showed that single active peak appeared (fraction No. 106 145) during washing with a linear gradient of 1 M NaCl (Fig. 2). The active fractions were consecutively applied on a hydrophobic column, Phenyl-Sepharose. The elution was done with a linear gradient from 1.5 to 0 M (NH4)2SO4 in same buffer after adsorbed protein (Fig. 3). The active fractions of each purification step were analyzed by SDS-PAGE. The final enzyme was purified 28.7-fold with a yield of 5.5% from the starting culture broth.
3.2. Purification of endoinulinase
3.3. Molecular mass of the purified enzyme
Endoinulinase was obtained as an extracellular enzyme in the culture broth of X. oryzae No. 5. Table 1
The molecular weight of the purified enzyme was estimated to be approximately 139 kDa by SDS-poly-
3. Results and discussion
3.1. Fermentation for enzyme production
Fig. 1. Typical profiles of bacterial cell growth and enzyme production from X. oryzae No. 5 in a 5-l fermentor. ( ), cell growth; (), endoinulinase activity; ("), protein content; ( ), total sugar content; (), pH. Table 1 Summary of purification of endoinulinase from X. oryzae No.5 Step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Crude enzyme (NH4)2SO4 (40–80%) DEAE-Sephacel Phenyl-Sepharose
1568 126 49 3
76 524 40 255 27 931 4222
49 319 570 1407
1 6.5 11.6 28.7
100 52.6 36.5 5.5
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Y.J. Cho, J.W. Yun / Process Biochemistry 37 (2002) 1325–1331
Fig. 2. Elution profile of DEAE-Sephacel column chromatography for endoinulinase purification.
Fig. 3. Elution profile of Phenyl-Sepharose column chromatography for endoinulinase purification. The elution was carried out with a linear gradient from 1.5 M (NH4)2SO4 to 0 in pH 7.0.
acrylamide gel electrophoresis (SDS-PAGE) as shown in Fig. 4. Many investigators have reported molecular mass of microbial endoinulinases. For example, the molecular mass of endoinulinases from Pseudomonas
sp. were 210 and 170 kDa, whereas those of many other microbial endoinulinases were around 75, 68, and 53 kDa from Arthrobacter sp., Penicillium sp. and Aspergillus fungi, respectively [15,24–26].
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Fig. 4. SDS-PAGE of the purified endoinulinase. M: standard marker; lane 1: purified endoinulinase.
Fig. 6. Lineweaver – Burk plot for determination of kinetic parameters of endoinulinase.
3.4. Effect of pH and temperature on the enzyme acti6ity
(Fig. 5B). These results are very similar to those of other microbial endoinulinases [15,25,26]. Thermo-stability of the enzyme was determined by measurement of residual activity after incubation for 1 h at various temperatures. The enzyme were stable up to 45 °C (Fig. 5B).
The effect of pH on endoinulinase activity was investigated by measuring the activity within the pH ranges from 4.0 to 9.0 by incubating the enzyme for 1 h. As shown in Fig. 5A, the maximal activity of the enzyme was observed at pH 7.5. The pH stability of the endoinulinase was determined by incubating the enzyme at 0.05 M buffer of different pH values for 24 h at 20 °C and then remaining activities were measured. The enzyme was stable at the pH range of 6.0– 9.0 (Fig. 5A). To examine the effect of temperature on enzyme activity, enyme reaction was performed at various temperature ranging from 30 to 70 °C for 1 h. The optimum temperature of endoinulinase activity was 50 °C
3.5. Kinetic study To determine the kinetic parameters (Km and Vmax) for the purified endoinulinase, enzyme reactions were carried out at 50 °C and pH 7.5 until the degree of conversion for initial substrates reached 5%. The Lineweaver–Burk plot shows the occurrence of substrate inhibition at inulin concentrations over 125 g/l and the Michaelis constant (Km) and maximum reac-
Fig. 5. (A) Effect of pH on endoinulinase activity () and pH stability ( ). (B) Effect of temperature on endoinulinase activity () and thermal stability ( ).
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tion velocity (Vmax) were found to be 16.7 g/l and 12.1 g/l·h, respectively (Fig. 6).
3.6. Reaction mode of endoinulinase on inulin To compare the product distribution obtained from enzymic reactions between crude and purified endoinulinases, enzyme reactions were performed at 50 °C for 45 h using different enzyme solution. As shown in Table 2 and Fig. 7, There was no significant difference Table 2 Comparison of reaction products produced from crude and purified endoinulinase Carbohydrates
Product composition (%, w/w)a Crude enzyme
Purified enzyme
Inulin Fructose Glucose Sucrose
4.3 ndb nd nd
4.7 nd nd nd
Oligosaccharides Inulobiose DP3 DP4 DP5 DP\5 Total IOS content
2.3 3.8 2.1 64.2 23.3 95.7
2.6 4.0 3.7 58.0 27.0 95.3
Enzyme dosage: 460 U/g inulin. a Compositions were given at the reaction time to reach a maximun oligosacccharide yield. b nd: not detected.
Fig. 8. Thin layer chromatogram of reaction products formed by hydrolysis of inulin by purified endoinulinase in the course of reaction. G, glucose; F, fructose; GF, sucrose; GF2, 1-kestose; GF3, nystose; GF4,1F-b-fructofuranosyl nystose.
in the product distribution from the reaction between crude and purified enzyme, where major products were inulooligosaccharides with DP of 5, 6, and 7. When the time-dependent reaction products were analyzed by TLC, it was observed that higher chain oligosaccharides were degraded again into shorter chain oligosaccharides, increasing the amounts of DP4 and DP5 inulooligosaccharides as the reaction proceeded (Fig. 8). Therefore, it is important to determine the terminating point of reaction to control the product distribution. For both crude and purified endoinulinases, there was no detection of mono- and di-saccharides in the reaction products, which suggests that both of endoinulinase preparations are completely free from exoinulinase and invertase activities.
4. Conclusion
Fig. 7. Typical time profiles of reaction products produced from inulin by purified endoinulinase. ( ) inulin; ( ) DP2; () DP4; () DP5; ()\ DP5; () Total inulooligosaccharides.
Since there was no significant difference in the product composition between crude and purified endoinulinase preparations without detection of monoand di-saccharides, the crude endoinulinase preparation is directly useful for large-scale production of inulooligosaccharides from inulin or inulin-containing agricultural crops.
Y.J. Cho, J.W. Yun / Process Biochemistry 37 (2002) 1325–1331
Acknowledgements This work was supported by the RRC program of MOST and KOSEF.
References [1] Cho YJ, Shinha J, Park JP, Yun JW. Production of inulooligosaccharides from chicory extract by endoinulinase from Xanthomonas oryzae No. 5. Enzyme Microb Technol 2001;28:439 –45. [2] Ettalibi M, Baratti JC. Molecular and kinetic properties of Aspergillus ficuum inulinases. Agric Biol Chem 1990;54:61 – 8. [3] Koops AJ, Jonker HH. Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus colombia. Plant Physiol 1996;110:1167 –75. [4] Roberfroid MB, van-Loo JAE, Gibson GR. The bifidogenic nature of chicory inulin and its hydrolysis products. J Nutr 1998;128:11 – 9. [5] Wang XD, Rakshit SK. Iso-oligosaccharides production by multiple forms of transferase enzymes from Aspersillus foetidus. Proc Biochem 2000;35:771 –5. [6] Shin HJ, Park JM, Yang JW. Continuous production of galactooligosaccharides from lactose by Ballera singularis b-galactosidase immobilized in chitosan beads, Proc Biochem 1998;33:787 – 792. [7] Oku T, Tokunaga T, Hosoya N. Nondigestibility of a new sweetener, ‘Neosugar’ in the rat. J Nutr 1984;114:1574 –81. [8] Roberfroid M. Dietary fiber, inulin, and oligofructose. A review comparing their physiological effects. Crit Rev Food Sci Nutr 1993;33:103 –48. [9] Tomomatsu H. Health effects of oligosaccharides, Food Technol. 1994:61 – 65. [10] Yun JW. Fructooligosaccharides — Occurrence, Preparation, and Application. Enzyme Microb Technol 1996;19:107 –17. [11] Roberfroid MB. Dietary fiber, inulin, and oligofructose: a review comparing their physiological effects. Crit Rev Food Sci Nutr 1993;33:103 – 48. [12] Yun JW, Kim DH, Kim BW, Song SK. Production of inulooligosaccharides from inulin by immobilized endoinulinase from Pseudomonas sp. J Ferment Bioeng 1997;84:369 –71.
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[13] Nakamura T, Nagamoto Y, Hamada S, Nishino Y, Ohta K. Occurrence of two forms of extracellular endoinulinase from Aspergillus niger mutant 817. J Ferment Bioeng 1994;78:134 –9. [14] Hirayama M, Sumi N, Hidaka H. Purification and properties of a fructooligosaccharide —producing fructofuranosidase from Aspersillus niger ATCC 20611. Agric Biol Chem 1989;53:667 –73. [15] Kang SI, Chang YJ, Oh SJ, Kim SI. Purification and properties of an endo-inulinase from an Arthrobacter sp. Biotechnol Lett 1998;20:983 – 6. [16] Fujita K, Hara K, Hashimoto H, Kitahata S. Purification and some properties of b-fructofuranosidase I from Arthrobacter sp. K-1. Agric Biol Chem 1990;54:913 – 9. [17] Onodera S, Shiomi N. Purification and substrate specificity of endo-type inulinase from Penicillium purpurogenum. Agric Biol Chem 1988;52:2569 – 76. [18] Park JP, Bae JT, You DJ, Kim BW, Yun JW. Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp. Biotechnol Lett 1999;21:1043 – 6. [19] Cho YJ, Sinha J, Park JP, Yun JW. Production of inulooligosaccharides from inulin by a dual endoinulinase system. Enzyme Microb Technol 2001;29:428 – 33. [20] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein —dye binding. Anal Biochem 1976;72:248 – 54. [21] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 –5. [22] Park JP, Oh TK, Yun JW. Purification and characterization of a novel transfructosylating enzyme from Bacillus macerans EG-6. Proc Biochem 2001;37:471 – 6. [23] Kwon SJ. Biochemical and biotechnological aspects of intracellular invertase and inulinase. MS thesis, Paichai University, Taejun, Korea, 1998. [24] Azhari R, Szlak AM, Ilan E, Sideman S, Lotan N. Purification and characterization of endo- and exo-inulinase. Biotechnol Appl Biochem 1989;11:105 – 17. [25] Nakamura T, Shitara A, Matsuda S, Matsuo T, Suiko M, Ohta K. Production, purification and properties of an endoinulinase of Penicillum sp. TN-88 that liberates inulotriose. J Ferment Bioeng 1997;84:313 – 8. [26] Lee TK, Shin HC, Choi YJ, Yang HC. Characteristics of extracellular endoinulinase produced by Pseudomonas sp. Kor J Appl Microbiol Bioeng 1998;16:484 – 8.
Purification and characterization of an endoinulinase from Xanthomonas oryzae No.5 Youn Jeung Cho, Jong Won Yun * Department of Biotechnology, Taegu Uni6ersity, Kyungsan, Kyungbuk 712 -714, South Korea Received 31 October 2001; received in revised form 8 January 2002; accepted 18 January 2002
Abstract The extracellular endoinulinase from Xanthomonas oryzae No. 5 which converts inulin into inulooligosaccharides was purified from the culture broth by ammonium sulphate precipitation, followed by column chromatography on Phenyl-Sepharose and DEAE-Sephacel. The enzyme was purified 29-fold with a yield of 5.5% from the starting culture broth. The purified enzyme gave a single band on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 139 kDa. The specific activity of the purified enzyme was 1372 U/mg. The enzyme activity was highest at pH 7.5 and 50 °C, and stable over a pH range of 6.0–9.0 and up to 45 °C. The reaction rates declined at inulin concentrations over 125 g/l indicating occurrence of substrate inhibition. The Michaelis constant (Km) and maximum reaction velocity (Vmax) of the endoinulinase were 16.7 g/l and 12.1 g/l·h, respectively. There was no significant difference in the product distribution from the reaction between crude and purified enzyme, where major product was penta-inulooligosaccharides. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Inulooligosaccharides; Endoinulinase; Enzyme purification; Xanthomonas oryzae
1. Introduction Inulin is a linear b-2,1 linked fructan terminated by a sucrose residue, which is usually found as reserve cabohydrate in various plants such as chicory, dahlia, and Jerusalem artichoke. Inulinases are fructofuranosyl hydrolases, which are produced by bacteria and plants as well as moulds [1 – 3]. Three types of inulinase have been described, including exoinulinase, endoinulinase, and fructotransferase which is not a common catabolic carbohydratase. The general enzymic reaction mainly occurs by two enzymes: exoinulinase (EC 3.2.1.80) which splits off the terminal fructose units from inulin and endoinulinase (EC 3.2.1.7) which hydrolyses inulin into inulooligosaccharides. Inulooligosaccharides produced from inulin have been suggested to have similar physiological
* Corresponding author. Tel.: + 82-53-850-6556; fax: +82-53-8506559. E-mail address: [email protected] (J.W. Yun).
functions to fructooligosaccharides [4]. Many kinds of oligosaccharides have been of increasing importance because of their favourable functionalities such as being low caloric, non-cariogenic and acting as a growth factor for beneficial microorganisms in the intestinal flora [5–10]. Inulin can be hydrolyzed by either sole action of exoinulinase or synergistic action of exo- and endoinulinase. More recent investigations have revealed that endoinulinase free from invertase or exoinulinase activity, hydrolyses the internal linkages of inulin to yield several oligosaccharides which are soluble dietary fibres and/or a functional sweeteners [11 –13]. Endoinulinases have been produced from several microorganisms such as Aspergillus niger [13,14], Aspergillus ficuum [2], Arthrobacter sp. [15,16], and Penicillium purpurogenum [17]. In previous articles [1,18,19], we have reported on the production of inulo-oligosaccharides from inulin or chicory extract using crude endoinulinase preparations from Xanthomonas sp. In the present study, we have purified the endoinulinase and investigated the kinetic properties.
0032-9592/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0032-9592(02)00018-3
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2. Materials and methods
2.1. Microorganism and culture conditions Xanthomonas oryzae No. 5 was grown aerobically in a suspension culture. The medium contained (g/l) inulin 20, yeast extract 20, (NH4)2HPO4 5, NH4H2PO4 2, MnCl2 ·4H2O 0.5, KCl 0.5, MgSO4 ·7H2O 0.5, FeSO4 ·7H2O 0.01, and pH was adjusted to 7.0 before sterilization. The pre-culture was carried out in 250 ml flasks contained 50 ml medium at 37 °C for 24 h on a rotary shaker at 150 rpm. The main culture for the production of enzyme was carried out in a 5 l fermenter (KoBioTech Co., Seoul, Korea) at 37 °C for 48 h. The working volume and inoculum size were 3 l and 2.0% (v/v), respectively. Agitation speed and aeration rate were controlled to 150 rpm and 2.0 vvm, respectively. After removal of cells by centrifugation at 10000×g for 20 min, the supernatant was used as a starting enzyme solution throughout the purification processes.
2.2. Chemicals The inulin used as substrate was purchased from Sigma Chemical Co. (MO, USA) (prepared from chicory root). 1-Kestose (GF2), nystose (GF3), 1F-bfructofuranosyl nystose (GF4) were purchased from Wako Pure Chemical, Ltd. (Osaka, Japan). DEAESephacel was the product of Sigma. Phenyl-Sepharose High Performance was purchased from Pharmacia (Uppsala, Sweden). Protein assay kit and standard markers of molecular weights for SDS-PAGE were purchased from Bio-rad Laboratories (California, USA). TLC plates (Silica gel 60 F254) were purchased from Merck (Darmstdt, Germany). All other chemicals were of reagent grade.
with Phenyl-Sepharose (Pharmacia). The column was pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 1.5 M (NH4)2SO4 and eluted with a decreasing linear gradient of ammonium sulphate from 1.5 to 0 M in the same buffer. The resulting enzyme solution was concentrated and dialyzed against 50 mM sodium phosphate buffer (pH 7.0). Proteins were analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE).
2.4. Enzyme assays Endoinulinase activity was assayed by incubating 1 ml enzyme solution with 5% (w/v) inulin dissolved in 50 mM sodium phosphate buffer (pH 6.0) at 45 °C for 1 h. The reaction was terminated by heating at 100 °C for 10 min. One enzyme unit was defined as the amount of enzyme responsible for hydrolysis of 1 mmol inulin per min under the above conditions. Where, the mean molecular weight of inulin was estimated as 5000 Da after acid-hydrolysis analysis. Proteins were measured by the Bradford method [20], using bovine serum albumin as the standard protein.
2.5. Gel electrophoresis SDS-PAGE was performed at a concentration of 12% polyacrylamide according to the method of Laemmli [21]. The gel was stained with Coomassie Brilliant Blue R-250. The standard proteins used were aprotinin (6.6 kDa), lysozyme (20.6 kDa), soybean trypsin inhibitor (29.8 kDa), carbonic anhydrase (36.4 kDa), ovalbumin (48.6 kDa), bovine serum albumin (94 kDa), b-galactosidase (130 kDa), and myosin (201 kDa).
2.3. Enzyme purification
2.6. Analytical methods
All of the operations for enzyme purification were done at 4 °C, and centrifugation was conducted at 7000×g for 15 min. Crude enzyme solution was first concentrated by ammonium sulphate precipitation (40–80% saturation) followed by dialysis against 50 mM sodium phosphate buffer (pH 6.0) at 4 °C and then applied on a DEAE-Sephacel column (2.5× 30 cm) pre-equilibrated with 50 mM sodium phosphate buffer (pH 7.0). Absorbed proteins were eluted in salt gradients (with 600 ml of 0 to 1 M NaCl) at a flow rate of 0.3 ml/min (3 ml per tube). The fractions showing inulinase activity were pooled and dialyzed overnight against 50 mM sodium phosphate buffer (pH 7.0) containing 1.5 M (NH4)2SO4 at 4 °C and subjected to the hydrophobic interaction chromatography (HIC) using a column (1.5×20 cm) packed
Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were simultaneously used for quantitative analysis of the reaction products. Pre-coated TLC plate (Merck) spotted with samples were developed with the solvent systems; isoprophyl alcohol: ethyl acetate: water (2:2:1 by volume). The carbohydrates loaded in the plates were visualized by heating the plates after spraying solvent containing 0.5% a-naphtol and 5% sulfuric acid in absolute ethanol. The quantitative analyses of the carbohydrates were carried out by HPLC using an Aminex HPX-42C column (0.78× 30 cm, Biorad) and a refractive index detector (Shimadzu Co, Kyoto, Japan). The column temperature was maintained at 85 °C and water was used as a mobile phase at a flow rate of 0.6 ml/min.
Y.J. Cho, J.W. Yun / Process Biochemistry 37 (2002) 1325–1331
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Fig. 1 shows the typical time profiles for endoinulinase production from X. oryzae No. 5 in a 5 l stirredtank fermentor. The cell growth increased with time and reached a maximum value at 30 h and enzyme activity indicated 77 U/ml at 30 h of fermentation, thereafter enzyme activity began to decrease rapidly. Similar trends in decrease of enzyme production at prolonged fermentation period have been reported in inulinases production from Bacillus sphaericus and transfructosylating enzyme production from Bacillus macerans [22,23].
summarizes results of the three-step purification of endoinulinase. The supernatant obtained from ammonium sulphate precipitates were first loaded on an anion exchange column, DEAE-Sephacel. The elution profile in DEAE-Sephacel chromatography showed that single active peak appeared (fraction No. 106 145) during washing with a linear gradient of 1 M NaCl (Fig. 2). The active fractions were consecutively applied on a hydrophobic column, Phenyl-Sepharose. The elution was done with a linear gradient from 1.5 to 0 M (NH4)2SO4 in same buffer after adsorbed protein (Fig. 3). The active fractions of each purification step were analyzed by SDS-PAGE. The final enzyme was purified 28.7-fold with a yield of 5.5% from the starting culture broth.
3.2. Purification of endoinulinase
3.3. Molecular mass of the purified enzyme
Endoinulinase was obtained as an extracellular enzyme in the culture broth of X. oryzae No. 5. Table 1
The molecular weight of the purified enzyme was estimated to be approximately 139 kDa by SDS-poly-
3. Results and discussion
3.1. Fermentation for enzyme production
Fig. 1. Typical profiles of bacterial cell growth and enzyme production from X. oryzae No. 5 in a 5-l fermentor. ( ), cell growth; (), endoinulinase activity; ("), protein content; ( ), total sugar content; (), pH. Table 1 Summary of purification of endoinulinase from X. oryzae No.5 Step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
Crude enzyme (NH4)2SO4 (40–80%) DEAE-Sephacel Phenyl-Sepharose
1568 126 49 3
76 524 40 255 27 931 4222
49 319 570 1407
1 6.5 11.6 28.7
100 52.6 36.5 5.5
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Fig. 2. Elution profile of DEAE-Sephacel column chromatography for endoinulinase purification.
Fig. 3. Elution profile of Phenyl-Sepharose column chromatography for endoinulinase purification. The elution was carried out with a linear gradient from 1.5 M (NH4)2SO4 to 0 in pH 7.0.
acrylamide gel electrophoresis (SDS-PAGE) as shown in Fig. 4. Many investigators have reported molecular mass of microbial endoinulinases. For example, the molecular mass of endoinulinases from Pseudomonas
sp. were 210 and 170 kDa, whereas those of many other microbial endoinulinases were around 75, 68, and 53 kDa from Arthrobacter sp., Penicillium sp. and Aspergillus fungi, respectively [15,24–26].
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Fig. 4. SDS-PAGE of the purified endoinulinase. M: standard marker; lane 1: purified endoinulinase.
Fig. 6. Lineweaver – Burk plot for determination of kinetic parameters of endoinulinase.
3.4. Effect of pH and temperature on the enzyme acti6ity
(Fig. 5B). These results are very similar to those of other microbial endoinulinases [15,25,26]. Thermo-stability of the enzyme was determined by measurement of residual activity after incubation for 1 h at various temperatures. The enzyme were stable up to 45 °C (Fig. 5B).
The effect of pH on endoinulinase activity was investigated by measuring the activity within the pH ranges from 4.0 to 9.0 by incubating the enzyme for 1 h. As shown in Fig. 5A, the maximal activity of the enzyme was observed at pH 7.5. The pH stability of the endoinulinase was determined by incubating the enzyme at 0.05 M buffer of different pH values for 24 h at 20 °C and then remaining activities were measured. The enzyme was stable at the pH range of 6.0– 9.0 (Fig. 5A). To examine the effect of temperature on enzyme activity, enyme reaction was performed at various temperature ranging from 30 to 70 °C for 1 h. The optimum temperature of endoinulinase activity was 50 °C
3.5. Kinetic study To determine the kinetic parameters (Km and Vmax) for the purified endoinulinase, enzyme reactions were carried out at 50 °C and pH 7.5 until the degree of conversion for initial substrates reached 5%. The Lineweaver–Burk plot shows the occurrence of substrate inhibition at inulin concentrations over 125 g/l and the Michaelis constant (Km) and maximum reac-
Fig. 5. (A) Effect of pH on endoinulinase activity () and pH stability ( ). (B) Effect of temperature on endoinulinase activity () and thermal stability ( ).
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tion velocity (Vmax) were found to be 16.7 g/l and 12.1 g/l·h, respectively (Fig. 6).
3.6. Reaction mode of endoinulinase on inulin To compare the product distribution obtained from enzymic reactions between crude and purified endoinulinases, enzyme reactions were performed at 50 °C for 45 h using different enzyme solution. As shown in Table 2 and Fig. 7, There was no significant difference Table 2 Comparison of reaction products produced from crude and purified endoinulinase Carbohydrates
Product composition (%, w/w)a Crude enzyme
Purified enzyme
Inulin Fructose Glucose Sucrose
4.3 ndb nd nd
4.7 nd nd nd
Oligosaccharides Inulobiose DP3 DP4 DP5 DP\5 Total IOS content
2.3 3.8 2.1 64.2 23.3 95.7
2.6 4.0 3.7 58.0 27.0 95.3
Enzyme dosage: 460 U/g inulin. a Compositions were given at the reaction time to reach a maximun oligosacccharide yield. b nd: not detected.
Fig. 8. Thin layer chromatogram of reaction products formed by hydrolysis of inulin by purified endoinulinase in the course of reaction. G, glucose; F, fructose; GF, sucrose; GF2, 1-kestose; GF3, nystose; GF4,1F-b-fructofuranosyl nystose.
in the product distribution from the reaction between crude and purified enzyme, where major products were inulooligosaccharides with DP of 5, 6, and 7. When the time-dependent reaction products were analyzed by TLC, it was observed that higher chain oligosaccharides were degraded again into shorter chain oligosaccharides, increasing the amounts of DP4 and DP5 inulooligosaccharides as the reaction proceeded (Fig. 8). Therefore, it is important to determine the terminating point of reaction to control the product distribution. For both crude and purified endoinulinases, there was no detection of mono- and di-saccharides in the reaction products, which suggests that both of endoinulinase preparations are completely free from exoinulinase and invertase activities.
4. Conclusion
Fig. 7. Typical time profiles of reaction products produced from inulin by purified endoinulinase. ( ) inulin; ( ) DP2; () DP4; () DP5; ()\ DP5; () Total inulooligosaccharides.
Since there was no significant difference in the product composition between crude and purified endoinulinase preparations without detection of monoand di-saccharides, the crude endoinulinase preparation is directly useful for large-scale production of inulooligosaccharides from inulin or inulin-containing agricultural crops.
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Acknowledgements This work was supported by the RRC program of MOST and KOSEF.
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