- Email: [email protected]
Large scale production of d -allose from d -psicose using continuous bioreactor and separation system
Enzyme and Microbial Technology 38 (2006) 855–859
Large scale production of d-allose from d-psicose using continuous bioreactor and separation system Kenji Morimoto a , Chang-Su Park b , Motofumi Ozaki b , Kei Takeshita c , Tsuyoshi Shimonishi b , Tom Birger Granstr¨om a , Goro Takata a , Masaaki Tokuda a , Ken Izumori a,∗ b
a Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Kagawa Industry Support Foundation, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japan c Fushimi Pharmaceutical Co., Ltd., Nakatsu-cho Marugame, Kagawa 763-8605, Japan
Received 11 April 2005; received in revised form 9 August 2005; accepted 10 August 2005
Abstract l-Rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172 can convert d-psicose to d-allose. Partially purified recombinant l-RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d-allose. Total 20,000 units of immobilized enzyme converted d-psicose to d-allose without remarkable decrease in the enzyme activity over 17 days. When 50% d-psicose (w/w) was applied to a column with a flow rate of 0.8 ml/min at 42 ◦ C, approximately 30% d-psicose was isomerized to d-allose for 17 days. However, by reducing the flow rate to 0.4 ml/min after 17 days, d-allose was transformed at the same rate for 13 days. The total of 27 l reaction mixture was separated by SimulatedMoving-Bed Chromatograph system. Approximately 2.2 l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d-allose and d-psicose fractions were 3 l of approximately 10% (w/w) with 95% purity and 10 l of approximately 8% (w/w) with 95% purity per day, respectively. The separated d-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals of d-allose were separated from the syrup by filtration and 1.65 kg crystals of 100% purity were obtained. The d-allose crystal yield from the d-psicose substrate was approximately 10%. © 2005 Elsevier Inc. All rights reserved. Keywords: Rare sugars; d-Allose; l-Rhamnose isomerase; Simulated-Moving-Bed Chromatograph
1. Introduction Rare sugars including monosaccharides are rare in nature and knowledge of their biological and physiological functions have consequently been known little so far. We are working on production of various kinds of rare sugars using microbial enzymes and whole cells. It has been proven that some rare hexose sugars have exhibited physiologically active functions [1–3]. Rare sugars have received increasing attention in recent years for a variety of usages, such as low-calorie carbohydrate sweeteners, inhibitor of microbial growth and bulking agents [1,2]. For instance, d-allose, one of rare aldo-hexose, has been reported to have a possibility to be applied in pharmaceutical industry, such as inhibitor of ischemia/reperfusion injury [3].
∗
Corresponding author. Tel.: +81 87 891 3290; fax: +81 87 891 3289. E-mail address: [email protected] (K. Izumori).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.08.014
We established the bioproduction strategy route named as “Izumoring” [4]. To produce d-allose in large quantities, one of the best route is a two step reaction as follows: first d-psicose is produced from inexpensive monosaccharide d-fructose, and in the second step, d-psicose is isomerized to d-allose. We reported that mass production of d-psicose from d-fructose was achieved by using d-tagatose 3-epimerase (d-TE) from Pseudomonas cichorii ST-24 [5], d-psicose was separated from sugar mixture by Simulated-Moving-Bed Chromatograph (JPN Pat. No. P2001-354690A, 2001). l-Rhamnose isomerase (l-RhI), l-rhamnose ketol-isomerase [EC 5.3.1.14], which isomerizes l-rhamnose to l-rhamnulose, has been found in Escherichia coli [6,7], Salmonella and Pseudomonas [8–10]. l-Rhamnose isomerase, which was produced constitutively, from Pseudomonas stutzeri LL172was reported to catalyze isomerizations not only between l-rhamnose–l-rhamnulose but also between d-allose–d-psicose (Fig. 1; [10,11]). We had reported that small scale of d-allose production was successful using this enzyme
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
856
Fig. 1. Schematic diagram of d-allose and d-psicose production from d-fructose catalyzed by l-rhamnose isomerase.
[10]. Moreover, l-rhi gene from this strain was successfully cloned into E. coli and over-expressed [12]. In this paper, we describe continuous d-allose production from d-psicose by use of immobilized enzyme of recombinant l-RhI under the optimized condition. The immobilized enzyme has advantages such as reusability, less by-product formation, and long operation time. Furthermore, we established the separation condition for reaction mixture containing d-allose by using Simulated-Moving-Bed Chromatograph system.
Tris–HCl buffer (pH 9.0), and disrupted by ultrasonicator. The cell free extract was heat-treated at 50 ◦ C for 10 min. The supernatant was collected by centrifugation at 8000 × g for 30 min. The supernatant was slowly added with 1M MnCl2 to a final concentration 10 mM. And polyethylene glycol #6000 was slowly added to a final concentration 5% (w/v) and the mixture was stirred for 1 h at 4 ◦ C. This mixture was centrifuged at 12,000 × g for 30 min and precipitation was discarded. More polyethylene glycol #6000 was added to a final concentration 20% (w/v). After stirring for 1 h, the precipitation was collected by centrifugation at 12,000 × g for 30 min and suspended in a small quantity of 10 mM Tris–HCl buffer (pH 9.0). This solution was used as partially purified enzyme.
2.4. Enzyme and protein assay 2. Materials and methods 2.1. Chemicals All chemical agents were purchased from Wako Pure Chemicals (Tokyo, Japan). Chitopearl BCW-2510 resin was purchased from Fuji Spinning Co., Ltd. (Tokyo, Japan). It has primary, secondary and quaternary amine groups and binds to proteins with electrovalent mode. d-Psicose was prepared by our laboratory as described previously [5].
2.2. Microorganism culture condition The l-rhi gene of Pseudomonas stutzeri LL172, which was expressed continuously, was inserted in pQE 60, named as pOI-01. This plasmid was transformed in E. coli JM 109 [12]. The recombinant E. coli JM109 was cultured in a medium composed of 3.5% polypepton, 2.0% yeast extract, 0.5% NaCl and 100 g/ml ampicillin in the four 15 l jar-fermentor at 28 ◦ C for 18 h, and recombinant enzyme was induced by addition 0.5 mM IPTG and 1 mM MnCl2 in the culture medium. After 5 h period, the cells were collected by continuous centrifugation (8000 × g, 4 ◦ C).
2.3. Partial purification of the enzyme
It is difficult to detect an isomerase activity from ketose to aldose, because the amount of reducing ketose is not detected easily. Isomerase activity is generally detected from aldose to ketose, and ketose conversion from aldose is easy to know enzyme activity using cysteine–carbazole method [13]. Appropriately, diluted partially purified enzyme, 50 l, was incubated with 50 l of 50 mM d-allose as a substrate at 30 ◦ C for 10 min. Accumulation of product, d-psicose, was measured by cysteine-carbazole method. One unit of l-RhI is defined as the amount of enzyme that converts 1 mol of d-allose to d-psicose in 1 min. The reaction mixture was assayed by the HITACHI High-performanceliquid-chromatography (HPLC), which is constructed by L-7490 refractive index detector, D-2500 chromato-integrator and Hitachi HPLC column GL-C611. Chromatography was carried out at 60 ◦ C using 10−4 M NaOH solution at a flow rate of 1.0 ml/min. Protein concentration was measured according to the method of Bradford [14] with bovine serum albumin as a standard.
2.5. Optimization of l-RhI immobilization condition The partially purified enzyme (100 units) was immobilized on 1, 2, 3 and 5 g of BCW-2510 Chitopearl beads at 4 ◦ C for 1 day. Immobilization efficiency was calculated as follows: residual activity in the supernatant was analyzed with d-allose as a substrate and then d-psicose amount was measured by cysteine–carbazole method as described above.
2.6. d-Allose mass production When l-RhI was completely purified, it became to be unstable according to formation of aggregation. Therefore, we used stable partially purified enzyme which was prepared as follows. The collected cells were suspended in 10 mM
The immobilized enzyme, which was prepared under the optimized condition, was charged in a column (4 cm × 40 cm) tempered at 42 ◦ C. Substrate 50%
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
857
(w/w) d-psicose dissolved in 10 mM Tris–HCl buffer (pH 9.0) was applied by flow rate of 0.8 ml/min. When the enzyme activity was decreased, the flow rate was reduced to 0.4 ml/min.
2.7. Separation of d-psicose and d-allose Before separation, reaction mixture was desalted by passing through Amberlite IRA-411 (CO3 2− form) and DIAION SK1B (H+ form) ion-exchanged resin. After desalting, the reaction mixture was separated to obtain mainly d-allose and d-psicose by TREZONE system (Organo Corp.) which can operate the Simulated-Moving-Bed Chromatograph. It consisted of 2 pumps, 26 valves and 4 columns. Each column was filled with 750 ml of ion exchanged resin (Ca2+ form). This system can separate a reaction mixture which is composed of two main components.
2.8. Crystallization of d-allose After separation of reaction mixture, d-allose fraction was evaporated to the concentration of 50% (w/w). The syrup was kept at room temperature to allow d-allose crystals to grow without adding seed crystals.
3. Results 3.1. Immobilization of l-RhI When 100 U of the enzyme was immobilized, not less than 90% of l-RhI was bound to 2, 3, or 5 g of BCW-2510 Chitopearl beads. By contrast, only approximately 50% of enzyme was bound to 1 g beads (Table 1). In this study, we found that the maximum equilibrium point (33% d-allose) was achieved after 5 h in all conditions (Fig. 2). The conversion efficiency was, however, the lowest among all in the case of using of 1 g beads. The best condition for immobilization of l-RhI on Chitopearl beads was achieved when 2 or 3 g of beads were used for 100 U of enzyme. Based on this result and cost-effectiveness, we determined that we utilized 2 g beads for 100 U of l-RhI.
Fig. 2. Effect of the Chitopearl BCW-2510 beads volume on d-allose production from d-psicose. d-Psicose in 50 mM Tris–HCl (pH 9.0) was used as a substrate at 42 ◦ C. After incubation, converted d-allose was measured by HPLC as described in Section 2. Symbols: open circles, 1 g beads; open squares, 2 g beads; close squares, 3 g beads; open triangles, 5 g beads.
inactivated in the column and enzyme was gradually detached it from resin, it became impossible to keep an equilibrium point. We made preliminary tests whether bioreactor could be used continuously after enzyme activity was decreased. As a result, the flow rate of 0.4 ml/min was able to maintain the optimal conversion rate. When the flow rate was dropped to 0.4 ml/min after 17 days, d-allose was produced with the some ratio for 13 days more in this study (Fig. 4). Eventually, bioreactor was able to maintain 1 month for continuous operation and to produce reaction mixture containing 5.02 kg of d-allose from 27 l (16.6 kg) of 50% (w/w) d-psicose syrup for 1 month (Table 2). When the same amount of l-RhI, which was used for bioreactor construction in this study, was let to react as soluble enzyme, the d-allose yield was approximately 30%.
3.2. Continuous conversion from d-psicose to d-allose The partially purified l-rhamnose isomerase which was prepared from 40 l culture has 20,000 U of enzyme activity. Based on the result of the optimized immobilization condition, we prepared a bioreactor using 400 g beads. As shown in Figs. 2 and 3, reaction equilibration was 30 (d-allose): 70 (d-psicose) toward 50% (w/w) d-psicose as a substrate, although a small amount of some by-products detected. The immobilized l-rhamnose isomerase was able to convert d-psicose to d-allose in the flow rate of 0.8 ml/min without significant decrease in the enzyme activity over the first 17 days. Since immobilized enzyme was gradually Table 1 Immobilization efficiency of l-Rhl on Chitopearl BCW-2510 Chitopearl resin volume (g)
Residual activity in supernatant (U)
Efficiency (%)
1 2 3 5
42.7 5.98 3.19 3.78
51.3 94.0 96.8 96.2
Fig. 3. HPLC analysis of d-allose from d-psicose by immobilized l-rhamnose isomerase.
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
858
3.3. Separation of d-psicose and d-allose As a result of the optimized separation condition, continuously the 50% (w/w) mixture of a maximum approximately 2.2 l in 1 day could be processed. The separated d-allose fraction volume was 3 l/d containing 10% (w/w) d-allose with 95% purity and a minor amount of by-products (Fig. 5). d-Psicose fraction volume was 10 l/d containing 8% (w/w) d-psicose with 95% purity (Fig. 5). 3.4. Crystallization of d-allose Fig. 4. Bioconversion of d-allose from d-psicose by immobilized l-rhamnose isomerase. Bold arrow and broken arrow indicate a period of flow rate of 0.8 and 0.4 ml/min, respectively.
The separated d-allose solution was concentrated up to about 50% (w/w) and crystallized gradually at room temperature. The crystals of d-allose were separated from the solution by filtration. Finally, we could obtain approximately 1.65 kg d-allose crystal with 100% purity (Fig. 5).
Table 2 The yield of d-allose mass production
Total sugarc d-Allosec d-Psicose By-productsc
Reacted mixture (kg)a
Separated d-allose (kg)a
Separated d-psicose (kg)a
Loss of sugars (kg)a
16.6 5.02 11.36 0.22
3.50 3.33 0.00 0.17
9.61 0.64 8.96 0.00
3.49 1.05 2.40 0.05
Crystallized d-allose (kg)b 1.65
a Reaction mixture indicates total weight after enzyme reaction. “Separated d-allose” and “separated d-psicose” indicate total weights after separation, respectively. “Loss of sugars” indicates total weight of Loss by desalting and separation processes. b “Crystallized d-allose” indicates obtained weight of d-allose crystal from separated d-allose fraction. c “Total sugar”, “d-allose”, “d-psicose” and “by-product” indicate sugar weights contained in “reacted mixture”, “separated d-allose”, “separated d-psicose” and “loss of sugars”, respectively.
Fig. 5. HPLC analysis of reaction mixture before and after separation and d-allose crystal: (A) indicates a deionized solution mixture; (B and C) indicate fractions separated d-allose and d-psicose by TREZONE system, respectively; (D) indicates finally obtained d-allose crystal.
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
4. Discussions We previously reported d-psicose mass production from dfructose by d-tagatose 3-epimerase from Pseudomonas cichorii ST-24 [5], and separation of d-psicose and d-fructose sugar mixture by Simulated-Moving-Bed Chromatograph (Organo Corp, JPN Pat. No. P2001-354690A 2001). In this paper, we have reported d-allose mass production from d-psicose by lrhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172. P. stutzeri LL172 expressed constitutively l-rhi gene. However, expression level was too low to construct an efficient bioreactor. In order to increase the l-RhI expression level and construct the bioreactor, we carried out the over-expression of l-rhi gene in E. coli JM109 [12]. This recombinant could produce l-RhI of 20-fold toward the volumetric yield in comparison with expression level of wild type P. stutzeri LL172 [12]. Partially purified enzyme prepared from recombinant was able to produce mainly d-allose from d-psicose, although it produced some d-fructose and other by-product (Fig. 3). In the previous report [10], d-allose was produced by batch reaction using l-RhI immobilized on Chitopearl beads. The batch reaction has two problems as follows: (1) we have to separate reaction mixture from immobilized enzyme after reaction; (2) l-RhI was damaged by drop of pH due to high concentration d-psicose. Consequently, it was not possible to use it for a long period. Therefore, we constructed a continuous bioreactor where substrate was injected by a pump as described in Section 2. As a result, declining rate of immobilized enzyme activity was decreased even under high substrate condition and immobilized enzyme was able to produce d-allose continuously. The solution after the reaction was very clean, thereby eliminating the separation step to recover the immobilized enzyme for further reactions. The reason why the substrate concentration was 50% (w/w) of d-psicose was that it is the best condition to reduce the formation of by-products as much as possible. Although we tried to use 20% (w/w) d-psicose, much more by-products were generated as compared with 50% (w/w) d-psicose (data not shown). The bioreactor constructed in this study was able to convert dpsicose to d-allose to the equilibrium point at the flow rate of 0.8 ml/min at 42 ◦ C, however, conversion ability was remarkably decreased after 17 days (Fig. 4), suggesting that the half life of this bioreactor was approximately a half month in these conditions. After 17 days, the bioreactor could convert d-psicose into d-allose until equilibrium point for more 13 days, when the flow rate was decreased down to 0.4 ml/min. Although, it was possible to use for 10 more days, we did not try to use more 30 days to consider productivity. We tried to separate the reaction mixture to obtain pure dallose crystal. The TREZONE system could separate 2.2 l of this reaction mixture per day. By this system, we can separate reaction mixture continuously for 10 days and obtained 3.6 l of purity 95% d-allose solution, although it contained d-psicose
859
and some by-product. However, crystallization of d-allose was performed in advance of d-psicose in the syrup mixture because d-allose has a property to crystallize easier than d-psicose. Consequently, d-allose is crystallized specifically in the mixture. The crystallized d-allose was able to be harvested easily by filtration using 5-m filter paper, and the purity was 100% as determined by HPLC analysis (Fig. 5). The yield of d-allose in this study was summarized in Table 2. Finally the crystal of d-allose was obtained 10% efficiency of reacted d-psicose weight. In this study, we established the mass production of d-allose by continuous bioreactor and Simulated-Moving-Bed Chromatograph. From now on, we will carry out improvements of bioreactor performance and Simulated-Moving-Bed Chromatograph in order to increase reaction efficiency and d-allose yield from the mixture of d-allose and d-psicose. References [1] Livesey G, Brown JC. d-Tagatose is a bulk sweetener with zero energy determined in rats. J Nutr 1996;126:1601–9. [2] Levin GV, Zehner LR, Sanders JP, Beadle JR. Sugar substitutes: their energy values, bulk characteristics, and potential health benefits. Am J Clin Nutr 1995;62:1161–8. [3] Hossain MA, Izuishi K, Tokuda M, Izumori K, Maeta H. d-Allose has a strong suppressive effect against ischemia/reperfusion injury: a comparative study with allopurinol and superoxide dismutase. J Hepatobiliary Pancreat Surg 2004;11:181–9. [4] Granstr¨om TB, Takata G, Tokuda M, Izumori K. Izumoring: a novel and complete strategy for bioproduction of rare sugars. J Biosci Bioeng 2004;97:89–94. [5] Takeshita K, Suga A, Takada G, Izumori K. Mass production of dpsicose from d-fructose by a continuous bioreactor system using immobilized d-tagatose 3-epimerase. J Biosci Bioeng 2000;90:453–5. [6] Wilson DM, Ajl S. Metabolism of l-rhamnose by Escherichia coli. I. l-Rhamnose isomerase. J Bateriol 1956;73:410–4. [7] Power J. The l-rhamnose genetic system in Escherichia coli K-12. Genetics 1967;55:557–68. [8] Zarban AL, Heffernan S, Nishitani L, Ransone J, Wilcox G. Positive control of the l-rhamnose genetic system in Selmonella typhimurium LT2. J Bacteriol 1984;158:603–8. [9] Bhuiyan HS, Itami Y, Izumori K. Isolation of an l-rhamnose isomeraseconstitutive mutant of Pseudomonas sp. strain LL172: purification and characterization of the enzyme. J Ferment Bioeng 1997;84:319–23. [10] Bhuiyan HS, Itami Y, Rokui Y, Katayama T, Izumori K. d-Allose production from d-psicose using immobilized l-rhamnose isomerase. J Ferment Bioeng 1998;85:539–41. [11] Leang K, Takada G, Fukai Y, Morimoto K, Granstr¨om TB, Izumori K. Novel reactions of l-rhamnose isomerase from Pseudomonas stutzeri and its relation with d-xylose isomerase via substrate specificity. Biochim Biophys Acta 2004;1674:68–77. [12] Leang K, Ishimura A, Okita M, Takada G, Izumori K. Cloning, nucleotide sequence and overexpression of l-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli. Appl Environ Microbiol 2004;70:3298–304. [13] Dische Z, Borenfreund E. A new spectrophotometric method for the detection of keto sugars and trioses. J Biol Chem 1951;192:583–7. [14] Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54.
Large scale production of d-allose from d-psicose using continuous bioreactor and separation system Kenji Morimoto a , Chang-Su Park b , Motofumi Ozaki b , Kei Takeshita c , Tsuyoshi Shimonishi b , Tom Birger Granstr¨om a , Goro Takata a , Masaaki Tokuda a , Ken Izumori a,∗ b
a Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Kagawa Industry Support Foundation, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japan c Fushimi Pharmaceutical Co., Ltd., Nakatsu-cho Marugame, Kagawa 763-8605, Japan
Received 11 April 2005; received in revised form 9 August 2005; accepted 10 August 2005
Abstract l-Rhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172 can convert d-psicose to d-allose. Partially purified recombinant l-RhI from Escherichia coli was immobilized on BCW-2510 Chitopearl beads and utilized to produce d-allose. Total 20,000 units of immobilized enzyme converted d-psicose to d-allose without remarkable decrease in the enzyme activity over 17 days. When 50% d-psicose (w/w) was applied to a column with a flow rate of 0.8 ml/min at 42 ◦ C, approximately 30% d-psicose was isomerized to d-allose for 17 days. However, by reducing the flow rate to 0.4 ml/min after 17 days, d-allose was transformed at the same rate for 13 days. The total of 27 l reaction mixture was separated by SimulatedMoving-Bed Chromatograph system. Approximately 2.2 l/d of 50% (w/w) reaction mixture was separated continuously. After separation, d-allose and d-psicose fractions were 3 l of approximately 10% (w/w) with 95% purity and 10 l of approximately 8% (w/w) with 95% purity per day, respectively. The separated d-allose solution was concentrated up to about 50% and crystallized gradually by being kept at room temperature. Crystals of d-allose were separated from the syrup by filtration and 1.65 kg crystals of 100% purity were obtained. The d-allose crystal yield from the d-psicose substrate was approximately 10%. © 2005 Elsevier Inc. All rights reserved. Keywords: Rare sugars; d-Allose; l-Rhamnose isomerase; Simulated-Moving-Bed Chromatograph
1. Introduction Rare sugars including monosaccharides are rare in nature and knowledge of their biological and physiological functions have consequently been known little so far. We are working on production of various kinds of rare sugars using microbial enzymes and whole cells. It has been proven that some rare hexose sugars have exhibited physiologically active functions [1–3]. Rare sugars have received increasing attention in recent years for a variety of usages, such as low-calorie carbohydrate sweeteners, inhibitor of microbial growth and bulking agents [1,2]. For instance, d-allose, one of rare aldo-hexose, has been reported to have a possibility to be applied in pharmaceutical industry, such as inhibitor of ischemia/reperfusion injury [3].
∗
Corresponding author. Tel.: +81 87 891 3290; fax: +81 87 891 3289. E-mail address: [email protected] (K. Izumori).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.08.014
We established the bioproduction strategy route named as “Izumoring” [4]. To produce d-allose in large quantities, one of the best route is a two step reaction as follows: first d-psicose is produced from inexpensive monosaccharide d-fructose, and in the second step, d-psicose is isomerized to d-allose. We reported that mass production of d-psicose from d-fructose was achieved by using d-tagatose 3-epimerase (d-TE) from Pseudomonas cichorii ST-24 [5], d-psicose was separated from sugar mixture by Simulated-Moving-Bed Chromatograph (JPN Pat. No. P2001-354690A, 2001). l-Rhamnose isomerase (l-RhI), l-rhamnose ketol-isomerase [EC 5.3.1.14], which isomerizes l-rhamnose to l-rhamnulose, has been found in Escherichia coli [6,7], Salmonella and Pseudomonas [8–10]. l-Rhamnose isomerase, which was produced constitutively, from Pseudomonas stutzeri LL172was reported to catalyze isomerizations not only between l-rhamnose–l-rhamnulose but also between d-allose–d-psicose (Fig. 1; [10,11]). We had reported that small scale of d-allose production was successful using this enzyme
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
856
Fig. 1. Schematic diagram of d-allose and d-psicose production from d-fructose catalyzed by l-rhamnose isomerase.
[10]. Moreover, l-rhi gene from this strain was successfully cloned into E. coli and over-expressed [12]. In this paper, we describe continuous d-allose production from d-psicose by use of immobilized enzyme of recombinant l-RhI under the optimized condition. The immobilized enzyme has advantages such as reusability, less by-product formation, and long operation time. Furthermore, we established the separation condition for reaction mixture containing d-allose by using Simulated-Moving-Bed Chromatograph system.
Tris–HCl buffer (pH 9.0), and disrupted by ultrasonicator. The cell free extract was heat-treated at 50 ◦ C for 10 min. The supernatant was collected by centrifugation at 8000 × g for 30 min. The supernatant was slowly added with 1M MnCl2 to a final concentration 10 mM. And polyethylene glycol #6000 was slowly added to a final concentration 5% (w/v) and the mixture was stirred for 1 h at 4 ◦ C. This mixture was centrifuged at 12,000 × g for 30 min and precipitation was discarded. More polyethylene glycol #6000 was added to a final concentration 20% (w/v). After stirring for 1 h, the precipitation was collected by centrifugation at 12,000 × g for 30 min and suspended in a small quantity of 10 mM Tris–HCl buffer (pH 9.0). This solution was used as partially purified enzyme.
2.4. Enzyme and protein assay 2. Materials and methods 2.1. Chemicals All chemical agents were purchased from Wako Pure Chemicals (Tokyo, Japan). Chitopearl BCW-2510 resin was purchased from Fuji Spinning Co., Ltd. (Tokyo, Japan). It has primary, secondary and quaternary amine groups and binds to proteins with electrovalent mode. d-Psicose was prepared by our laboratory as described previously [5].
2.2. Microorganism culture condition The l-rhi gene of Pseudomonas stutzeri LL172, which was expressed continuously, was inserted in pQE 60, named as pOI-01. This plasmid was transformed in E. coli JM 109 [12]. The recombinant E. coli JM109 was cultured in a medium composed of 3.5% polypepton, 2.0% yeast extract, 0.5% NaCl and 100 g/ml ampicillin in the four 15 l jar-fermentor at 28 ◦ C for 18 h, and recombinant enzyme was induced by addition 0.5 mM IPTG and 1 mM MnCl2 in the culture medium. After 5 h period, the cells were collected by continuous centrifugation (8000 × g, 4 ◦ C).
2.3. Partial purification of the enzyme
It is difficult to detect an isomerase activity from ketose to aldose, because the amount of reducing ketose is not detected easily. Isomerase activity is generally detected from aldose to ketose, and ketose conversion from aldose is easy to know enzyme activity using cysteine–carbazole method [13]. Appropriately, diluted partially purified enzyme, 50 l, was incubated with 50 l of 50 mM d-allose as a substrate at 30 ◦ C for 10 min. Accumulation of product, d-psicose, was measured by cysteine-carbazole method. One unit of l-RhI is defined as the amount of enzyme that converts 1 mol of d-allose to d-psicose in 1 min. The reaction mixture was assayed by the HITACHI High-performanceliquid-chromatography (HPLC), which is constructed by L-7490 refractive index detector, D-2500 chromato-integrator and Hitachi HPLC column GL-C611. Chromatography was carried out at 60 ◦ C using 10−4 M NaOH solution at a flow rate of 1.0 ml/min. Protein concentration was measured according to the method of Bradford [14] with bovine serum albumin as a standard.
2.5. Optimization of l-RhI immobilization condition The partially purified enzyme (100 units) was immobilized on 1, 2, 3 and 5 g of BCW-2510 Chitopearl beads at 4 ◦ C for 1 day. Immobilization efficiency was calculated as follows: residual activity in the supernatant was analyzed with d-allose as a substrate and then d-psicose amount was measured by cysteine–carbazole method as described above.
2.6. d-Allose mass production When l-RhI was completely purified, it became to be unstable according to formation of aggregation. Therefore, we used stable partially purified enzyme which was prepared as follows. The collected cells were suspended in 10 mM
The immobilized enzyme, which was prepared under the optimized condition, was charged in a column (4 cm × 40 cm) tempered at 42 ◦ C. Substrate 50%
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
857
(w/w) d-psicose dissolved in 10 mM Tris–HCl buffer (pH 9.0) was applied by flow rate of 0.8 ml/min. When the enzyme activity was decreased, the flow rate was reduced to 0.4 ml/min.
2.7. Separation of d-psicose and d-allose Before separation, reaction mixture was desalted by passing through Amberlite IRA-411 (CO3 2− form) and DIAION SK1B (H+ form) ion-exchanged resin. After desalting, the reaction mixture was separated to obtain mainly d-allose and d-psicose by TREZONE system (Organo Corp.) which can operate the Simulated-Moving-Bed Chromatograph. It consisted of 2 pumps, 26 valves and 4 columns. Each column was filled with 750 ml of ion exchanged resin (Ca2+ form). This system can separate a reaction mixture which is composed of two main components.
2.8. Crystallization of d-allose After separation of reaction mixture, d-allose fraction was evaporated to the concentration of 50% (w/w). The syrup was kept at room temperature to allow d-allose crystals to grow without adding seed crystals.
3. Results 3.1. Immobilization of l-RhI When 100 U of the enzyme was immobilized, not less than 90% of l-RhI was bound to 2, 3, or 5 g of BCW-2510 Chitopearl beads. By contrast, only approximately 50% of enzyme was bound to 1 g beads (Table 1). In this study, we found that the maximum equilibrium point (33% d-allose) was achieved after 5 h in all conditions (Fig. 2). The conversion efficiency was, however, the lowest among all in the case of using of 1 g beads. The best condition for immobilization of l-RhI on Chitopearl beads was achieved when 2 or 3 g of beads were used for 100 U of enzyme. Based on this result and cost-effectiveness, we determined that we utilized 2 g beads for 100 U of l-RhI.
Fig. 2. Effect of the Chitopearl BCW-2510 beads volume on d-allose production from d-psicose. d-Psicose in 50 mM Tris–HCl (pH 9.0) was used as a substrate at 42 ◦ C. After incubation, converted d-allose was measured by HPLC as described in Section 2. Symbols: open circles, 1 g beads; open squares, 2 g beads; close squares, 3 g beads; open triangles, 5 g beads.
inactivated in the column and enzyme was gradually detached it from resin, it became impossible to keep an equilibrium point. We made preliminary tests whether bioreactor could be used continuously after enzyme activity was decreased. As a result, the flow rate of 0.4 ml/min was able to maintain the optimal conversion rate. When the flow rate was dropped to 0.4 ml/min after 17 days, d-allose was produced with the some ratio for 13 days more in this study (Fig. 4). Eventually, bioreactor was able to maintain 1 month for continuous operation and to produce reaction mixture containing 5.02 kg of d-allose from 27 l (16.6 kg) of 50% (w/w) d-psicose syrup for 1 month (Table 2). When the same amount of l-RhI, which was used for bioreactor construction in this study, was let to react as soluble enzyme, the d-allose yield was approximately 30%.
3.2. Continuous conversion from d-psicose to d-allose The partially purified l-rhamnose isomerase which was prepared from 40 l culture has 20,000 U of enzyme activity. Based on the result of the optimized immobilization condition, we prepared a bioreactor using 400 g beads. As shown in Figs. 2 and 3, reaction equilibration was 30 (d-allose): 70 (d-psicose) toward 50% (w/w) d-psicose as a substrate, although a small amount of some by-products detected. The immobilized l-rhamnose isomerase was able to convert d-psicose to d-allose in the flow rate of 0.8 ml/min without significant decrease in the enzyme activity over the first 17 days. Since immobilized enzyme was gradually Table 1 Immobilization efficiency of l-Rhl on Chitopearl BCW-2510 Chitopearl resin volume (g)
Residual activity in supernatant (U)
Efficiency (%)
1 2 3 5
42.7 5.98 3.19 3.78
51.3 94.0 96.8 96.2
Fig. 3. HPLC analysis of d-allose from d-psicose by immobilized l-rhamnose isomerase.
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
858
3.3. Separation of d-psicose and d-allose As a result of the optimized separation condition, continuously the 50% (w/w) mixture of a maximum approximately 2.2 l in 1 day could be processed. The separated d-allose fraction volume was 3 l/d containing 10% (w/w) d-allose with 95% purity and a minor amount of by-products (Fig. 5). d-Psicose fraction volume was 10 l/d containing 8% (w/w) d-psicose with 95% purity (Fig. 5). 3.4. Crystallization of d-allose Fig. 4. Bioconversion of d-allose from d-psicose by immobilized l-rhamnose isomerase. Bold arrow and broken arrow indicate a period of flow rate of 0.8 and 0.4 ml/min, respectively.
The separated d-allose solution was concentrated up to about 50% (w/w) and crystallized gradually at room temperature. The crystals of d-allose were separated from the solution by filtration. Finally, we could obtain approximately 1.65 kg d-allose crystal with 100% purity (Fig. 5).
Table 2 The yield of d-allose mass production
Total sugarc d-Allosec d-Psicose By-productsc
Reacted mixture (kg)a
Separated d-allose (kg)a
Separated d-psicose (kg)a
Loss of sugars (kg)a
16.6 5.02 11.36 0.22
3.50 3.33 0.00 0.17
9.61 0.64 8.96 0.00
3.49 1.05 2.40 0.05
Crystallized d-allose (kg)b 1.65
a Reaction mixture indicates total weight after enzyme reaction. “Separated d-allose” and “separated d-psicose” indicate total weights after separation, respectively. “Loss of sugars” indicates total weight of Loss by desalting and separation processes. b “Crystallized d-allose” indicates obtained weight of d-allose crystal from separated d-allose fraction. c “Total sugar”, “d-allose”, “d-psicose” and “by-product” indicate sugar weights contained in “reacted mixture”, “separated d-allose”, “separated d-psicose” and “loss of sugars”, respectively.
Fig. 5. HPLC analysis of reaction mixture before and after separation and d-allose crystal: (A) indicates a deionized solution mixture; (B and C) indicate fractions separated d-allose and d-psicose by TREZONE system, respectively; (D) indicates finally obtained d-allose crystal.
K. Morimoto et al. / Enzyme and Microbial Technology 38 (2006) 855–859
4. Discussions We previously reported d-psicose mass production from dfructose by d-tagatose 3-epimerase from Pseudomonas cichorii ST-24 [5], and separation of d-psicose and d-fructose sugar mixture by Simulated-Moving-Bed Chromatograph (Organo Corp, JPN Pat. No. P2001-354690A 2001). In this paper, we have reported d-allose mass production from d-psicose by lrhamnose isomerase (l-RhI) from Pseudomonas stutzeri LL172. P. stutzeri LL172 expressed constitutively l-rhi gene. However, expression level was too low to construct an efficient bioreactor. In order to increase the l-RhI expression level and construct the bioreactor, we carried out the over-expression of l-rhi gene in E. coli JM109 [12]. This recombinant could produce l-RhI of 20-fold toward the volumetric yield in comparison with expression level of wild type P. stutzeri LL172 [12]. Partially purified enzyme prepared from recombinant was able to produce mainly d-allose from d-psicose, although it produced some d-fructose and other by-product (Fig. 3). In the previous report [10], d-allose was produced by batch reaction using l-RhI immobilized on Chitopearl beads. The batch reaction has two problems as follows: (1) we have to separate reaction mixture from immobilized enzyme after reaction; (2) l-RhI was damaged by drop of pH due to high concentration d-psicose. Consequently, it was not possible to use it for a long period. Therefore, we constructed a continuous bioreactor where substrate was injected by a pump as described in Section 2. As a result, declining rate of immobilized enzyme activity was decreased even under high substrate condition and immobilized enzyme was able to produce d-allose continuously. The solution after the reaction was very clean, thereby eliminating the separation step to recover the immobilized enzyme for further reactions. The reason why the substrate concentration was 50% (w/w) of d-psicose was that it is the best condition to reduce the formation of by-products as much as possible. Although we tried to use 20% (w/w) d-psicose, much more by-products were generated as compared with 50% (w/w) d-psicose (data not shown). The bioreactor constructed in this study was able to convert dpsicose to d-allose to the equilibrium point at the flow rate of 0.8 ml/min at 42 ◦ C, however, conversion ability was remarkably decreased after 17 days (Fig. 4), suggesting that the half life of this bioreactor was approximately a half month in these conditions. After 17 days, the bioreactor could convert d-psicose into d-allose until equilibrium point for more 13 days, when the flow rate was decreased down to 0.4 ml/min. Although, it was possible to use for 10 more days, we did not try to use more 30 days to consider productivity. We tried to separate the reaction mixture to obtain pure dallose crystal. The TREZONE system could separate 2.2 l of this reaction mixture per day. By this system, we can separate reaction mixture continuously for 10 days and obtained 3.6 l of purity 95% d-allose solution, although it contained d-psicose
859
and some by-product. However, crystallization of d-allose was performed in advance of d-psicose in the syrup mixture because d-allose has a property to crystallize easier than d-psicose. Consequently, d-allose is crystallized specifically in the mixture. The crystallized d-allose was able to be harvested easily by filtration using 5-m filter paper, and the purity was 100% as determined by HPLC analysis (Fig. 5). The yield of d-allose in this study was summarized in Table 2. Finally the crystal of d-allose was obtained 10% efficiency of reacted d-psicose weight. In this study, we established the mass production of d-allose by continuous bioreactor and Simulated-Moving-Bed Chromatograph. From now on, we will carry out improvements of bioreactor performance and Simulated-Moving-Bed Chromatograph in order to increase reaction efficiency and d-allose yield from the mixture of d-allose and d-psicose. References [1] Livesey G, Brown JC. d-Tagatose is a bulk sweetener with zero energy determined in rats. J Nutr 1996;126:1601–9. [2] Levin GV, Zehner LR, Sanders JP, Beadle JR. Sugar substitutes: their energy values, bulk characteristics, and potential health benefits. Am J Clin Nutr 1995;62:1161–8. [3] Hossain MA, Izuishi K, Tokuda M, Izumori K, Maeta H. d-Allose has a strong suppressive effect against ischemia/reperfusion injury: a comparative study with allopurinol and superoxide dismutase. J Hepatobiliary Pancreat Surg 2004;11:181–9. [4] Granstr¨om TB, Takata G, Tokuda M, Izumori K. Izumoring: a novel and complete strategy for bioproduction of rare sugars. J Biosci Bioeng 2004;97:89–94. [5] Takeshita K, Suga A, Takada G, Izumori K. Mass production of dpsicose from d-fructose by a continuous bioreactor system using immobilized d-tagatose 3-epimerase. J Biosci Bioeng 2000;90:453–5. [6] Wilson DM, Ajl S. Metabolism of l-rhamnose by Escherichia coli. I. l-Rhamnose isomerase. J Bateriol 1956;73:410–4. [7] Power J. The l-rhamnose genetic system in Escherichia coli K-12. Genetics 1967;55:557–68. [8] Zarban AL, Heffernan S, Nishitani L, Ransone J, Wilcox G. Positive control of the l-rhamnose genetic system in Selmonella typhimurium LT2. J Bacteriol 1984;158:603–8. [9] Bhuiyan HS, Itami Y, Izumori K. Isolation of an l-rhamnose isomeraseconstitutive mutant of Pseudomonas sp. strain LL172: purification and characterization of the enzyme. J Ferment Bioeng 1997;84:319–23. [10] Bhuiyan HS, Itami Y, Rokui Y, Katayama T, Izumori K. d-Allose production from d-psicose using immobilized l-rhamnose isomerase. J Ferment Bioeng 1998;85:539–41. [11] Leang K, Takada G, Fukai Y, Morimoto K, Granstr¨om TB, Izumori K. Novel reactions of l-rhamnose isomerase from Pseudomonas stutzeri and its relation with d-xylose isomerase via substrate specificity. Biochim Biophys Acta 2004;1674:68–77. [12] Leang K, Ishimura A, Okita M, Takada G, Izumori K. Cloning, nucleotide sequence and overexpression of l-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli. Appl Environ Microbiol 2004;70:3298–304. [13] Dische Z, Borenfreund E. A new spectrophotometric method for the detection of keto sugars and trioses. J Biol Chem 1951;192:583–7. [14] Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54.