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Immobilization and stabilization of pullulanase from Klebsiella pneumoniae by a multipoint attachment method using activated agar gel supports
Process Biochemistry 40 (2005) 2637–2642 www.elsevier.com/locate/procbio
Immobilization and stabilization of pullulanase from Klebsiella pneumoniae by a multipoint attachment method using activated agar gel supports Takashi Kuroiwa, Hiroyuki Shoda, Sosaku Ichikawa, Seigo Sato, Sukekuni Mukataka * Department of Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Received 16 July 2004; accepted 20 October 2004
Abstract The immobilization and stabilization of pullulanase from Klebsiella pneumoniae were investigated. Pullulanases immobilized on activated agar gel (glyoxyl-agar gel) via multipoint attachment between amino groups of pullulanase and aldehyde groups on the gel surface were significantly stable compared with free pullulanase and pullulanase adsorbed on chitosan beads. The immobilization yield, thermal stability and activity of immobilized pullulanase greatly depended on the surface density of aldehyde groups on the support gel. The optimum temperature and pH of immobilized pullulanases prepared by the multipoint attachment method using activated agar gels were 50 8C and 6.0, respectively. These values were the same as those of free pullulanase. Pullulanase immobilized on agar gel activated with 4.8 M glycidol retained 60% of its initial activity after a 250-h incubation at 45 8C, while free pullulanase completely lost its activity after a 72-h incubation at the same temperature. # 2004 Elsevier Ltd. All rights reserved. Keywords: Immobilized enzyme; Pullulanase; Thermal stability; Multipoint attachment; Activated agar gel; Surface aldehyde density
1. Introduction Pullulanase (EC 3.2.1.41) is an enzyme whose primary specificity is to hydrolyze the (1 ! 6) a-D-glucosidic linkages in pullulan and amylopectin. This enzyme improves the saccharification of starch to produce glucose, maltose and malto-oligosaccharides using glucoamylase (EC 3.2.1.3), beta-amylase (EC 3.2.1.3) or alpha-amylase (EC 3.2.1.1). The utilization of pullulanases is essential for efficient processing of starch in food industries. However, the thermal stability of commercial pullulanases is not high and a large portion of the initial pullulanase activity is lost within a few hours even at a low temperature (60 8C) compared with that in liquefication processes of starch by alpha-amylases (105 8C). Because of these facts, the application of pullulanases for a wide range of industrial processes is limited. * Corresponding author. Tel.: +81 29 853 4607; fax: +81 29 853 4605. E-mail address: [email protected] (S. Mukataka). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.10.002
Immobilization of enzymes is one of the effective methods to improve the thermal stability of enzymes. Furthermore, continuous operation of industrial enzymatic processes can be realized using immobilized enzymes. To date, many investigators have reported the immobilization methods of pullulanases [1–10], but sufficient improvement of their thermal stability has not been achieved. In previous studies [11–13], the authors developed immobilized chitosanase from Bacillus pumilus by the multipoint attachment method [14] using activated agar gel support. This chitosanase lost 80% of its initial activity during a 1-h incubation at 50 8C in a native form, while immobilized chitosanases retained 95% of their initial activity for over 250 h at 50 8C [11] and continuous production of physiologically active chitosan oligosaccharides was carried out stably for a month [13]. In this method, amino groups of lysine residue and N-terminals of protein are used for immobilization to form multipoint covalent bonds between enzymes and support materials. Since these
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amino groups mainly exist on the outer surface of protein molecules, this immobilization method is potentially applicable for a wide range of enzymes. In this study, a commercial pullulanase from Klebsiella pneumoniae was immobilized onto activated agar gel supports by the multipoint attachment method. The thermal stability and activity of immobilized pullulanase were investigated under various conditions and the relationship between the properties of immobilized pullulanase and the conditions in the immobilization procedure are discussed.
2. Materials and methods 2.1. Enzyme and chemicals Pullulanase ‘‘Amano’’ (type ‘‘3’’, from K. pneumoniae) was kindly donated by Amano Enzyme, Inc. (Nagoya, Japan). This was supplied as brown liquid and contained 32.3 mg of protein per 1 ml of solution. This was determined by the Bradford method [15] using bovine serum albumin as the standard. Pullulan (average molecular weight: 73,000) was purchased from Seikagaku Corporation (Tokyo, Japan). Modified chitosan beads (Chitopearl BCW-3510) were purchased from Fuji Spinning Co. Ltd. (Tokyo, Japan). Agar powder, glycidol (2,3-epoxypropanol), sodium borohydrate and other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 2.2. Preparation of immobilized pullulanases by the physical adsorption method using chitosan beads Immobilization of pullulanase onto chitosan beads was carried out by the adsorption method reported by Yoshida et al. [16]. Chitosan beads were pre-equilibrated in 0.1 M phosphate buffer (pH 7.0) overnight at 4 8C. Five grams of pre-equilibrated chitosan beads was suspended into 0.1 M phosphate buffer (pH 7.0) containing a prescribed amount of pullulanase. Then the suspension was stirred gently at 25 8C for 1 h. The beads were recovered by filtration and washed with the same buffer and deionized water. The obtained immobilized pullulanases were stored in deionized water at 4 8C until used. The yield of immobilized protein was determined from the amount of protein remaining in the supernatant after 1 h of stirring. The amount of protein was determined by the Bradford method [15]. 2.3. Preparation of immobilized pullulanases by the multipoint attachment method using activated agar gel supports 2.3.1. Preparation of activated agar gels Twenty grams of 6% (w/w) agar gel cut into 1-mm cubes was suspended in 100 ml of 0.16 M NaOH containing 630 mg of NaBH4. Then a given amount of glycidol was added to the suspension. The mixture was gently stirred for
18 h at 25 8C. The gels were recovered by filtration and washed with deionized water and were then suspended in 100 ml of 35 mM NaIO4 solution with gentle stirring for 1 h at 25 8C. After filtration, the recovered activated gels were washed with deionized water. The aldehyde content of the activated agar gel supports was determined to be equivalent to the amount of periodate consumed [14]. The periodate that was not consumed in the activation of the gels was measured by titration with KI. 2.3.2. Immobilization of pullulanase Fifteen grams of the activated agar gel was suspended in 120 ml of 0.2 M borate buffer (pH 10.0) containing pullulanase. The suspension was gently stirred for 24 h at 25 8C, followed by the addition of 240 mg of NaBH4, and the suspension was stirred for a further 30 min at 25 8C. The gels were recovered from the suspension by filtration and washed with deionized water and 0.1 M phosphate buffer (pH 7.0). The immobilized pullulanases obtained were stored in deionized water at 4 8C until used. The yield of immobilized protein was determined from the amount of protein remaining in the borate buffer solution after 24 h of stirring. 2.4. Assay of pullulanase activity Pullulan was used as the substrate to measure the pullulanase activity. Pullulan was dissolved in 0.1 M phosphate buffer (pH 6.0) or 0.1 M McIlvaine buffer (pH 3.5–7.5) at a concentration of 0.5% (w/v). Free or immobilized pullulanases were added into the Monod-type test tube containing 10 ml of pullulan solution and the reaction mixture was shaken with a shaking speed of 50 strokes/min by the Monod-type shaker (model MD-100, TAITEC Corporation, Koshigaya, Japan) at 50 8C. Aliquots were periodically taken from the reaction mixture through a steel filter to separate support particles and mixed with the same volume of a 0.4 M trichloroacetic acid solution to terminate the reaction. The pullulanase activity was determined from the amount of liberated reducing sugars by the modified Schales method [17] with maltose as a reference compound. One unit of pullulanase activity was defined as the amount of enzyme that produced 1 mmol of maltose equivalent in 1 min at 50 8C using pullulan dissolved in 0.1 M phosphate buffer (pH 6.0) at a concentration of 0.5% (w/v).
3. Results and discussion 3.1. Pullulanase immobilization onto modified chitosan beads and activated agar gels In order to increase the stability of pullulanases, immobilization them onto chitosan beads and activated agar gels was attempted. The chitosan beads used here can
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Table 1 Activated agar gels used as supports for immobilization
Fig. 1. Thermal stability of free and immobilized pullulanases at 50 8C. Pullulanases were incubated in 0.1 M phosphate buffer (pH 6.0) at 50 8C. Open circles: free pullulanase; closed circles: pullulanase immobilized on activated agar gel; closed squares: pullulanase immobilized on chitosan beads.
adsorb proteins mainly via hydrophobic and electrostatic interactions. The activated agar gels can immobilize proteins onto its surface via multipoint covalent bonds. In our previous studies, a thermally unstable chitosanase was dramatically stabilized by immobilization onto adequately activated agar supports [11–13]. Fig. 1 shows the time courses of the residual activity of the different pullulanase preparations at 50 8C. Free pullulanase was completely inactivated after an 8-h incubation. The stability of pullulanase adsorbed on chitosan beads was decreased compared to that of free pullulanase, as were pullulanases previously immobilized on chitosan beads by crosslinking treatment following physical adsorption previously [18]. On the other hand, pullulanase immobilized on activated agar gels was significantly stabilized compared with other preparations and about 30% of its initial activity was retained after a 48-h incubation. Based on these results, activated agar gels were used for pullulanase immobilization in subsequent studies. 3.2. Effect of activation degree of agar gel supports In a previous study [11], the thermal stability and the activity of immobilized chitosanases prepared by the multipoint attachment method greatly depended on the surface density of the aldehyde group on the activated gel support. Therefore, in order to optimize the support activation condition, five kinds of activated agar gels having aldehyde groups with different surface densities were tested (Table 1). The surface density of the aldehyde groups was ˚2 represented as the number of aldehyde residues per 3600 A 2 ˚ corresponds to that of a of gel surface. The area of 3600 A cross-section of a pullulanase molecule calculated from its ˚ , estimated by Young’s correlation Stokes’ radius (ca. 68 A [19] and the Stokes–Einstein equation using the molecular weight of pullulanase determined by the sedimentation equilibrium method [20]). Therefore, the surface aldehyde density of each gel listed in Table 1 roughly corresponds to the number of aldehyde residues with which one pullulanase molecule can interact. The specific surface area of 6% agar gels was assumed to be the same as that of sepharose 6B (6%
Activated agar preparation
Glycidol concentration (M)
Density of aldehyde group ˚ 2 )a (aldehyde/3600 A
A B C D E
0.1 0.7 3.0 4.8 8.6
1.0 8.1 13.5 17.9 23.8
a
This roughly corresponds to the number of aldehyde residues with which one pullulanase molecule can interact.
agarose gel, specific surface area = 25 m2/ml [21]) since agar and agarose gels have almost the same pore structure above a concentration of 4.5% [22]. 3.2.1. Immobilization yield The immobilization yield increased with the increase of surface density of the aldehyde groups (Fig. 2). Since a single bond of Schiff’s base between an amino group of a protein molecule and the support is reversible, pullulanases bound to the support via a one-point interaction could detach easily [23]. From Fig. 2, it was suggested that more than 15 aldehyde groups per pullulanase molecule were necessary to form multipoint covalent bonds between all pullulanase molecules and the supports. Gels D and E were sufficiently activated to immobilize pullulanases completely. 3.2.2. Thermal stability Fig. 3 shows the effect of the activation degree of the support gel on the stability of immobilized pullulanase. Free or immobilized pullulanases were incubated in 0.1 M phosphate buffer (pH 6.0) at 45 8C. At this temperature, free pullulanases were completely inactivated after incubation for 72 h. The stability of pullulanase immobilized on gel A was almost the same as that of a free one. On the other hand, the residual activity of pullulanases immobilized on gel D still retained about 60% of their initial value after incubation for 200 h. First-order inactivation was observed in the case of free pullulanase and an immobilized one on gel
Fig. 2. Effect of surface aldehyde density on the immobilization yield of pullulanase by the multipoint attachment method using activated agar gels. Values of surface aldehyde density correspond to the number of aldehyde residues that can interact with one pullulanase molecule, approximately. For details see text.
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covalent bonds between one pullulanase and the surface aldehyde groups increased the thermal stability of immobilized pullulanases significantly. With an increase in the population of pullulanase immobilized by multiple bonds, the ‘‘plateau’’ level of the residual activity in a later phase in Fig. 3, i.e., 18 and 60% for gels B and D, respectively, would become high. This result suggests that the surface density of aldehyde groups is a very important factor for the stabilization of immobilized pullulanase. Fig. 3. Effect of surface aldehyde density on the stability of immobilized pullulanase prepared by the multipoint attachment method using activated agar gels. Each pullulanase preparation was incubated in 0.1 M phosphate buffer at 45 8C. Open circles: free pullulanase; closed circles: pullulanase immobilized on gel A; closed squares: pullulanase immobilized on gel B: closed triangles: pullulanase immobilized on gel D. Surface aldehyde density of each gel support is listed in Table 1.
A, while in the cases of pullulanases on the other gels the inactivation kinetics could not be described by a simple firstorder model. In those cases, the decrease of residual activity in an initial phase of incubation was relatively quick compared with that in a later phase. This would be attributed to the distribution of the number of covalent bonds between one pullulanase molecule and the support surface. A simple geometrical model of the interaction between one pullulanase molecule and surface aldehyde groups on each gel is depicted in Fig. 4. It can be presumed that this pullulanase molecule has about 17 amino groups, based on the results of the amino acid analysis [24] and SDS-PAGE analysis [20]. On the assumption of a uniform distribution of amino groups on the protein surface, maximally 8–9 amino groups could bind to the surface aldehyde groups. In the case of gel A, i.e., low aldehyde density, pullulanase could not make multiple bonds (Fig. 4a). On the other hand, aldehyde groups with higher densities could make multiple covalent bonds possible, as schematically shown in Fig. 4b and c. Therefore, the results in Fig. 3 indicate that pullulanase molecules could be stabilized by the formation of multipoint binding with the support. An increase in the number of
3.2.3. Activity The activity of an immobilized enzyme is important for the development of an efficient biocatalyst as well as its stability. The effect of the surface aldehyde density on the activity and the specific activity of immobilized pullulanase was examined (Fig. 5). At a low surface aldehyde density, the exhibited activity was low due to the decrease of the amount of immobilized pullulanase, as shown in Fig. 2. On the other hand, an overly high surface aldehyde density led to the decrease of specific activity. The highest activity, 5.61 U/g support, was observed when gel D was used as the support. The specific activities of all immobilized preparations were low (only 8.2–11.3% of that of a free enzyme). The activity loss of free pullulanase during incubation in 0.2 M borate buffer (pH 10.0) at 25 8C for 24 h and in the same buffer containing NaBH4 (2.0 mg/l) at 25 8C for 30 min (these conditions corresponded to that in the immobilization procedure – see Section 2) was substantially low (less than 4% of initial activity, data not shown). Therefore the low specific activity of the immobilized pullulanases seems to be due to the covalent bonds between the pullulanase and the support. One of the possible factors responsible for the activity loss is the steric hindrance that decreases the substrate accessibility to a catalytic domain of a pullulanase covered with a gel support surface. Another factor would be the conformational change of pullulanase molecules caused by immobilization. The formation of multipoint covalent bonds would change the conformation of the enzyme. As shown in Fig. 5, the specific activity of
Fig. 4. Simple geometrical model of the interaction between one pullulanase molecule and surface aldehyde residues on the activated agar gel support.
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Fig. 5. Effects of surface aldehyde density on the activity and the specific activity of immobilized pullulanase prepared by the multipoint attachment method using activated agar gels. Circles: activity; squares: specific activity.
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Fig. 7. Effect of temperature on the stability of immobilized pullulanase prepared by the multipoint attachment method using gel D. Pullulanases were incubated at 40 8C (circles), 45 8C (squares) and 50 8C (triangles).
immobilized pullulanase decreased with an increase of the surface aldehyde density. However, this decrease is relatively small compared to the total decrease of specific activity. On the other hand, a significant decrease of the specific activity of immobilized pullulanase compared with that of free pullulanase was observed even when only one covalent bond was formed, as in the case of gel A. Thus, the major factor for the decrease of the specific activity of immobilized pullulanase might be steric hindrance. This would be unavoidable when a substrate having a high molecular weight is used. From the results of Fig. 5, it was concluded that the optimum support for pullulanase immobilization was gel D in this system.
were still active at 70 8C, while free pullulanases had no activity at that temperature. This would be due to the stabilization by the multipoint binding between one pullulanase molecule and the support gel, and even at a higher temperature the immobilized pullulanase could retain its active structure compared to free enzymes. The effect of pH on the activities of free and immobilized pullulanases was almost identical. The optimum temperature and pH of immobilized pullulanases bound to gel D were 50 8C and 6.0, respectively, so they were the same as those of free pullulanase.
3.3. Temperature and pH dependencies of immobilized pullulanase
The time courses of the residual activity of pullulanase immobilized on gel D at various temperatures are shown in Fig. 7. The residual activity decreased to 70 and 60% during a 250-h incubation at 40 and 45 8C, respectively, while it quickly dropped to 40% after 24 h at 50 8C. When a substrate (30% soluble starch) co-existed, immobilized pullulanases were more stable (84% of initial activity was retained after 216 h at 45 8C). This fact is an advantage for continuous use of immobilized pullulanases, such as in a packed-bed-type reactor.
The effects of temperature and pH on the activity of immobilized pullulanase prepared using gel D are shown in Fig. 6. The temperature dependence of the activity of immobilized pullulanase was almost identical to that of free pullulanases up to 50 8C, but immobilized pullulanase exhibited a higher relative activity than that of free pullulanase over 60 8C (Fig. 6a). Immobilized pullulanases
3.4. Effect of temperature on the stability of immobilized pullulanases
4. Conclusions
Fig. 6. Effects of temperature (a) and pH (b) on the activity of free and immobilized pullulanases. Effects of temperature and pH were assayed at pH 6.0 and 50 8C, respectively. Open circles: free pullulanase; closed circles: immobilized pullulanase prepared by the multipoint attachment method using gel D.
The immobilization of pullulanase by multipoint attachment to activated agar gel supports was investigated in order to prepare stable immobilized pullulanase. Pullulanases immobilized on activated agar gels by the multipoint attachment method were significantly stabilized compared with free pullulanase, while no stabilizing effect was observed when chitosan beads were used as the support material. The effect of surface aldehyde density on the activated agar gel support was examined. The immobilization yield increased with increasing surface aldehyde density and 100% of applied protein was immobilized for the activated
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agar support having a surface aldehyde density above 15 ˚ 2 (this value corresponds to 15 aldehyde residues per 3600 A residues per one pullulanase molecule, approximately). The thermal stability of pullulanase was significantly improved by increasing the surface aldehyde density. The effect of surface aldehyde density on the stability of pullulanase was discussed in relation to the number of covalent bonds between one pullulanase molecule and the support. It was suggested that the formation of multipoint covalent bonds between the pullulanase molecule and the support was essential for the stabilization of pullulanase. The maximal activity of immobilized pullulanase was 5.61 U/g support at a surface aldehyde density of 17.9 residues ˚ 2. The optimum temperature and pH of pullper 3600 A ulanases immobilized on the activated agar support were the same as those of free pullulanase. Immobilized pullulanase was more stable under incubation with a substrate (lowmolecular-weight soluble starch) than without a substrate. The method for pullulanase immobilization described here can contribute to the development of continuous processes of starch saccharification and various sugar productions. The application of immobilized pullulanase for the continuous production of useful oligosaccharide by combining it with other amylolytic enzymes is under investigation.
Acknowledgement The authors express their sincere appreciation to Amano Enzyme, Inc. for the supply of pullulanase.
References [1] Ma˚ rtensson K, Mosbach K. Covalent coupling of pullulanase to an acrylic copolymer using a water soluble carbodiimide. Biotechnol Bioeng 1972;14:715–24. [2] Ma˚ rtensson K. Preparation of an immobilized two-enzyme system, bamylase-pullulanase, to an acrylic copolymer for the conversion of starch to maltose. II. Co-coupling of the enzymes and use in a packed bed column. Biotechnol Bioeng 1974;16:579–91. [3] Ma˚ rtensson K. Preparation of an immobilized two-enzyme system, bamylase-pullulanase, to an acrylic copolymer for the conversion of starch to maltose. III. Process kinetic studies on continuous reactors. Biotechnol Bioeng 1974;16:1567–87. [4] Lenders JP, Crichton RR. Thermal stabilization of amylolytic enzymes by covalent coupling to soluble polysaccharides. Biotechnol Bioeng 1984;26:1343–51. [5] Kusano S, Shiraishi T, Takahashi S, Fujimoto D, Sakano Y. Immobilization of Bacillus acidopullulyticus pullulanase and properties of the immobilized pullulanases. J Fermentation Bioeng 1989;68:233–7.
[6] Chakrabarti AC, Storey KB. Co-immobilization of amyloglucosidase and pullulanase for enhanced starch hydrolysis. Appl Microbiol Biotechnol 1990;33:48–50. [7] Manolov RJ, Kambourova MS, Emanuilova EI. Immobilization and properties of Bacillus stearothermophilus pullulanase. Biotechnol Appl Biochem 1993;18:409–15. [8] Diaz T, Sta˚ hl U, Batista-Viera F, Carlsson J. Reversible immobilization of chemically modified pullulanase. Biotechnol Tech 1995;9: 533–8. [9] Dessouki AM, Issa G, Atia KS. Pullulanase immobilization on natural and synthetic polymers. J Chem Technol Biotechnol 2001;76:700–6. [10] Atia KS, Ismail SA, El-Arnaouty MB, Dessouki AM. Use of coimmobilized b-amylase and pullulanase in reduction of saccharification time of starch and increase in maltose yield. Biotechnol Prog 2003;19:853–7. [11] Ichikawa S, Takano K, Kuroiwa T, Hiruta O, Sato S, Mukataka S. Immobilization and stabilization of chitosanase by multipoint attachment to agar gel support. J Biosci Bioeng 2002;93:201–6. [12] Kuroiwa T, Ichikawa S, Hiruta O, Sato S, Mukataka S. Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases. Biotechnol Prog 2002;18:969–74. [13] Kuroiwa T, Ichikawa S, Sato S, Mukataka S. Improvement of the yield of physiologically active oligosaccharides produced in continuous hydrolysis of chitosan using immobilized chitosanases. Biotechnol Bioeng 2003;84:121–7. [14] Guisan JM. Aldehyde gels as activated support for immobilization of enzymes. Enzyme Microb Technol 1988;10:375–82. [15] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of the proteindye binding. Anal Biochem 1976;72:248–54. [16] Yoshida M, Kimura T, Ogata M, Nakakuki T. Immobilization of the exo-maltotetraohydrolase and some properties of the enzyme. J Jpn Soc Starch Sci 1988;35:245–52. [17] Imoto T, Yagishita K. A simple activity measurement of lysozyme. Agric Biol Chem 1971;35:1154–6. [18] Kusano S, Takahashi S, Fujimoto D, Sakano Y. Effects of reduced malto-oligosaccharides on the thermal stability of pullulanase from Bacillus acidopullulyticus. Carbohydrate Res 1990;199:83–9. [19] Young ME, Carroad PA, Bell RL. Estimation of diffusion coefficients of proteins. Biotechnol Bioeng 1980;22:947–55. [20] Ueda S, Ohba R. Purification, crystallization and some properties of extracellular pullulanase from Aerobacter aerogenes. Agric Biol Chem 1972;36:2381–91. [21] Guisan JM, Blanco RM. Stabilization of trypsin by multiple-point attachment to aldehyde-agarose gels. Ann NY Acad Sci 1987;501:67– 72. [22] Chui MM, Phillips RJ, McCarthy MJ. Measurement of the porous microstructure of hydrogels by nuclear magnetic resonance. J Colloid Interf Sci 1995;174:336–44. [23] Blanco RM, Calvete JJ, Guisan JM. Immobilization-stabilization of enzymes: variables that control the intensity of the trypsin (amine)agarose (aldehyde) multipoint attachment. Enzyme Microb Technol 1989;11:353–9. [24] Ohba R, Ueda S. Some properties of extra- and intracellular pullulanases from Aerobacter aerogenes. Agric Biol Chem 1975;39:967– 72.
Immobilization and stabilization of pullulanase from Klebsiella pneumoniae by a multipoint attachment method using activated agar gel supports Takashi Kuroiwa, Hiroyuki Shoda, Sosaku Ichikawa, Seigo Sato, Sukekuni Mukataka * Department of Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Received 16 July 2004; accepted 20 October 2004
Abstract The immobilization and stabilization of pullulanase from Klebsiella pneumoniae were investigated. Pullulanases immobilized on activated agar gel (glyoxyl-agar gel) via multipoint attachment between amino groups of pullulanase and aldehyde groups on the gel surface were significantly stable compared with free pullulanase and pullulanase adsorbed on chitosan beads. The immobilization yield, thermal stability and activity of immobilized pullulanase greatly depended on the surface density of aldehyde groups on the support gel. The optimum temperature and pH of immobilized pullulanases prepared by the multipoint attachment method using activated agar gels were 50 8C and 6.0, respectively. These values were the same as those of free pullulanase. Pullulanase immobilized on agar gel activated with 4.8 M glycidol retained 60% of its initial activity after a 250-h incubation at 45 8C, while free pullulanase completely lost its activity after a 72-h incubation at the same temperature. # 2004 Elsevier Ltd. All rights reserved. Keywords: Immobilized enzyme; Pullulanase; Thermal stability; Multipoint attachment; Activated agar gel; Surface aldehyde density
1. Introduction Pullulanase (EC 3.2.1.41) is an enzyme whose primary specificity is to hydrolyze the (1 ! 6) a-D-glucosidic linkages in pullulan and amylopectin. This enzyme improves the saccharification of starch to produce glucose, maltose and malto-oligosaccharides using glucoamylase (EC 3.2.1.3), beta-amylase (EC 3.2.1.3) or alpha-amylase (EC 3.2.1.1). The utilization of pullulanases is essential for efficient processing of starch in food industries. However, the thermal stability of commercial pullulanases is not high and a large portion of the initial pullulanase activity is lost within a few hours even at a low temperature (60 8C) compared with that in liquefication processes of starch by alpha-amylases (105 8C). Because of these facts, the application of pullulanases for a wide range of industrial processes is limited. * Corresponding author. Tel.: +81 29 853 4607; fax: +81 29 853 4605. E-mail address: [email protected] (S. Mukataka). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.10.002
Immobilization of enzymes is one of the effective methods to improve the thermal stability of enzymes. Furthermore, continuous operation of industrial enzymatic processes can be realized using immobilized enzymes. To date, many investigators have reported the immobilization methods of pullulanases [1–10], but sufficient improvement of their thermal stability has not been achieved. In previous studies [11–13], the authors developed immobilized chitosanase from Bacillus pumilus by the multipoint attachment method [14] using activated agar gel support. This chitosanase lost 80% of its initial activity during a 1-h incubation at 50 8C in a native form, while immobilized chitosanases retained 95% of their initial activity for over 250 h at 50 8C [11] and continuous production of physiologically active chitosan oligosaccharides was carried out stably for a month [13]. In this method, amino groups of lysine residue and N-terminals of protein are used for immobilization to form multipoint covalent bonds between enzymes and support materials. Since these
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amino groups mainly exist on the outer surface of protein molecules, this immobilization method is potentially applicable for a wide range of enzymes. In this study, a commercial pullulanase from Klebsiella pneumoniae was immobilized onto activated agar gel supports by the multipoint attachment method. The thermal stability and activity of immobilized pullulanase were investigated under various conditions and the relationship between the properties of immobilized pullulanase and the conditions in the immobilization procedure are discussed.
2. Materials and methods 2.1. Enzyme and chemicals Pullulanase ‘‘Amano’’ (type ‘‘3’’, from K. pneumoniae) was kindly donated by Amano Enzyme, Inc. (Nagoya, Japan). This was supplied as brown liquid and contained 32.3 mg of protein per 1 ml of solution. This was determined by the Bradford method [15] using bovine serum albumin as the standard. Pullulan (average molecular weight: 73,000) was purchased from Seikagaku Corporation (Tokyo, Japan). Modified chitosan beads (Chitopearl BCW-3510) were purchased from Fuji Spinning Co. Ltd. (Tokyo, Japan). Agar powder, glycidol (2,3-epoxypropanol), sodium borohydrate and other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). 2.2. Preparation of immobilized pullulanases by the physical adsorption method using chitosan beads Immobilization of pullulanase onto chitosan beads was carried out by the adsorption method reported by Yoshida et al. [16]. Chitosan beads were pre-equilibrated in 0.1 M phosphate buffer (pH 7.0) overnight at 4 8C. Five grams of pre-equilibrated chitosan beads was suspended into 0.1 M phosphate buffer (pH 7.0) containing a prescribed amount of pullulanase. Then the suspension was stirred gently at 25 8C for 1 h. The beads were recovered by filtration and washed with the same buffer and deionized water. The obtained immobilized pullulanases were stored in deionized water at 4 8C until used. The yield of immobilized protein was determined from the amount of protein remaining in the supernatant after 1 h of stirring. The amount of protein was determined by the Bradford method [15]. 2.3. Preparation of immobilized pullulanases by the multipoint attachment method using activated agar gel supports 2.3.1. Preparation of activated agar gels Twenty grams of 6% (w/w) agar gel cut into 1-mm cubes was suspended in 100 ml of 0.16 M NaOH containing 630 mg of NaBH4. Then a given amount of glycidol was added to the suspension. The mixture was gently stirred for
18 h at 25 8C. The gels were recovered by filtration and washed with deionized water and were then suspended in 100 ml of 35 mM NaIO4 solution with gentle stirring for 1 h at 25 8C. After filtration, the recovered activated gels were washed with deionized water. The aldehyde content of the activated agar gel supports was determined to be equivalent to the amount of periodate consumed [14]. The periodate that was not consumed in the activation of the gels was measured by titration with KI. 2.3.2. Immobilization of pullulanase Fifteen grams of the activated agar gel was suspended in 120 ml of 0.2 M borate buffer (pH 10.0) containing pullulanase. The suspension was gently stirred for 24 h at 25 8C, followed by the addition of 240 mg of NaBH4, and the suspension was stirred for a further 30 min at 25 8C. The gels were recovered from the suspension by filtration and washed with deionized water and 0.1 M phosphate buffer (pH 7.0). The immobilized pullulanases obtained were stored in deionized water at 4 8C until used. The yield of immobilized protein was determined from the amount of protein remaining in the borate buffer solution after 24 h of stirring. 2.4. Assay of pullulanase activity Pullulan was used as the substrate to measure the pullulanase activity. Pullulan was dissolved in 0.1 M phosphate buffer (pH 6.0) or 0.1 M McIlvaine buffer (pH 3.5–7.5) at a concentration of 0.5% (w/v). Free or immobilized pullulanases were added into the Monod-type test tube containing 10 ml of pullulan solution and the reaction mixture was shaken with a shaking speed of 50 strokes/min by the Monod-type shaker (model MD-100, TAITEC Corporation, Koshigaya, Japan) at 50 8C. Aliquots were periodically taken from the reaction mixture through a steel filter to separate support particles and mixed with the same volume of a 0.4 M trichloroacetic acid solution to terminate the reaction. The pullulanase activity was determined from the amount of liberated reducing sugars by the modified Schales method [17] with maltose as a reference compound. One unit of pullulanase activity was defined as the amount of enzyme that produced 1 mmol of maltose equivalent in 1 min at 50 8C using pullulan dissolved in 0.1 M phosphate buffer (pH 6.0) at a concentration of 0.5% (w/v).
3. Results and discussion 3.1. Pullulanase immobilization onto modified chitosan beads and activated agar gels In order to increase the stability of pullulanases, immobilization them onto chitosan beads and activated agar gels was attempted. The chitosan beads used here can
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Table 1 Activated agar gels used as supports for immobilization
Fig. 1. Thermal stability of free and immobilized pullulanases at 50 8C. Pullulanases were incubated in 0.1 M phosphate buffer (pH 6.0) at 50 8C. Open circles: free pullulanase; closed circles: pullulanase immobilized on activated agar gel; closed squares: pullulanase immobilized on chitosan beads.
adsorb proteins mainly via hydrophobic and electrostatic interactions. The activated agar gels can immobilize proteins onto its surface via multipoint covalent bonds. In our previous studies, a thermally unstable chitosanase was dramatically stabilized by immobilization onto adequately activated agar supports [11–13]. Fig. 1 shows the time courses of the residual activity of the different pullulanase preparations at 50 8C. Free pullulanase was completely inactivated after an 8-h incubation. The stability of pullulanase adsorbed on chitosan beads was decreased compared to that of free pullulanase, as were pullulanases previously immobilized on chitosan beads by crosslinking treatment following physical adsorption previously [18]. On the other hand, pullulanase immobilized on activated agar gels was significantly stabilized compared with other preparations and about 30% of its initial activity was retained after a 48-h incubation. Based on these results, activated agar gels were used for pullulanase immobilization in subsequent studies. 3.2. Effect of activation degree of agar gel supports In a previous study [11], the thermal stability and the activity of immobilized chitosanases prepared by the multipoint attachment method greatly depended on the surface density of the aldehyde group on the activated gel support. Therefore, in order to optimize the support activation condition, five kinds of activated agar gels having aldehyde groups with different surface densities were tested (Table 1). The surface density of the aldehyde groups was ˚2 represented as the number of aldehyde residues per 3600 A 2 ˚ corresponds to that of a of gel surface. The area of 3600 A cross-section of a pullulanase molecule calculated from its ˚ , estimated by Young’s correlation Stokes’ radius (ca. 68 A [19] and the Stokes–Einstein equation using the molecular weight of pullulanase determined by the sedimentation equilibrium method [20]). Therefore, the surface aldehyde density of each gel listed in Table 1 roughly corresponds to the number of aldehyde residues with which one pullulanase molecule can interact. The specific surface area of 6% agar gels was assumed to be the same as that of sepharose 6B (6%
Activated agar preparation
Glycidol concentration (M)
Density of aldehyde group ˚ 2 )a (aldehyde/3600 A
A B C D E
0.1 0.7 3.0 4.8 8.6
1.0 8.1 13.5 17.9 23.8
a
This roughly corresponds to the number of aldehyde residues with which one pullulanase molecule can interact.
agarose gel, specific surface area = 25 m2/ml [21]) since agar and agarose gels have almost the same pore structure above a concentration of 4.5% [22]. 3.2.1. Immobilization yield The immobilization yield increased with the increase of surface density of the aldehyde groups (Fig. 2). Since a single bond of Schiff’s base between an amino group of a protein molecule and the support is reversible, pullulanases bound to the support via a one-point interaction could detach easily [23]. From Fig. 2, it was suggested that more than 15 aldehyde groups per pullulanase molecule were necessary to form multipoint covalent bonds between all pullulanase molecules and the supports. Gels D and E were sufficiently activated to immobilize pullulanases completely. 3.2.2. Thermal stability Fig. 3 shows the effect of the activation degree of the support gel on the stability of immobilized pullulanase. Free or immobilized pullulanases were incubated in 0.1 M phosphate buffer (pH 6.0) at 45 8C. At this temperature, free pullulanases were completely inactivated after incubation for 72 h. The stability of pullulanase immobilized on gel A was almost the same as that of a free one. On the other hand, the residual activity of pullulanases immobilized on gel D still retained about 60% of their initial value after incubation for 200 h. First-order inactivation was observed in the case of free pullulanase and an immobilized one on gel
Fig. 2. Effect of surface aldehyde density on the immobilization yield of pullulanase by the multipoint attachment method using activated agar gels. Values of surface aldehyde density correspond to the number of aldehyde residues that can interact with one pullulanase molecule, approximately. For details see text.
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covalent bonds between one pullulanase and the surface aldehyde groups increased the thermal stability of immobilized pullulanases significantly. With an increase in the population of pullulanase immobilized by multiple bonds, the ‘‘plateau’’ level of the residual activity in a later phase in Fig. 3, i.e., 18 and 60% for gels B and D, respectively, would become high. This result suggests that the surface density of aldehyde groups is a very important factor for the stabilization of immobilized pullulanase. Fig. 3. Effect of surface aldehyde density on the stability of immobilized pullulanase prepared by the multipoint attachment method using activated agar gels. Each pullulanase preparation was incubated in 0.1 M phosphate buffer at 45 8C. Open circles: free pullulanase; closed circles: pullulanase immobilized on gel A; closed squares: pullulanase immobilized on gel B: closed triangles: pullulanase immobilized on gel D. Surface aldehyde density of each gel support is listed in Table 1.
A, while in the cases of pullulanases on the other gels the inactivation kinetics could not be described by a simple firstorder model. In those cases, the decrease of residual activity in an initial phase of incubation was relatively quick compared with that in a later phase. This would be attributed to the distribution of the number of covalent bonds between one pullulanase molecule and the support surface. A simple geometrical model of the interaction between one pullulanase molecule and surface aldehyde groups on each gel is depicted in Fig. 4. It can be presumed that this pullulanase molecule has about 17 amino groups, based on the results of the amino acid analysis [24] and SDS-PAGE analysis [20]. On the assumption of a uniform distribution of amino groups on the protein surface, maximally 8–9 amino groups could bind to the surface aldehyde groups. In the case of gel A, i.e., low aldehyde density, pullulanase could not make multiple bonds (Fig. 4a). On the other hand, aldehyde groups with higher densities could make multiple covalent bonds possible, as schematically shown in Fig. 4b and c. Therefore, the results in Fig. 3 indicate that pullulanase molecules could be stabilized by the formation of multipoint binding with the support. An increase in the number of
3.2.3. Activity The activity of an immobilized enzyme is important for the development of an efficient biocatalyst as well as its stability. The effect of the surface aldehyde density on the activity and the specific activity of immobilized pullulanase was examined (Fig. 5). At a low surface aldehyde density, the exhibited activity was low due to the decrease of the amount of immobilized pullulanase, as shown in Fig. 2. On the other hand, an overly high surface aldehyde density led to the decrease of specific activity. The highest activity, 5.61 U/g support, was observed when gel D was used as the support. The specific activities of all immobilized preparations were low (only 8.2–11.3% of that of a free enzyme). The activity loss of free pullulanase during incubation in 0.2 M borate buffer (pH 10.0) at 25 8C for 24 h and in the same buffer containing NaBH4 (2.0 mg/l) at 25 8C for 30 min (these conditions corresponded to that in the immobilization procedure – see Section 2) was substantially low (less than 4% of initial activity, data not shown). Therefore the low specific activity of the immobilized pullulanases seems to be due to the covalent bonds between the pullulanase and the support. One of the possible factors responsible for the activity loss is the steric hindrance that decreases the substrate accessibility to a catalytic domain of a pullulanase covered with a gel support surface. Another factor would be the conformational change of pullulanase molecules caused by immobilization. The formation of multipoint covalent bonds would change the conformation of the enzyme. As shown in Fig. 5, the specific activity of
Fig. 4. Simple geometrical model of the interaction between one pullulanase molecule and surface aldehyde residues on the activated agar gel support.
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Fig. 5. Effects of surface aldehyde density on the activity and the specific activity of immobilized pullulanase prepared by the multipoint attachment method using activated agar gels. Circles: activity; squares: specific activity.
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Fig. 7. Effect of temperature on the stability of immobilized pullulanase prepared by the multipoint attachment method using gel D. Pullulanases were incubated at 40 8C (circles), 45 8C (squares) and 50 8C (triangles).
immobilized pullulanase decreased with an increase of the surface aldehyde density. However, this decrease is relatively small compared to the total decrease of specific activity. On the other hand, a significant decrease of the specific activity of immobilized pullulanase compared with that of free pullulanase was observed even when only one covalent bond was formed, as in the case of gel A. Thus, the major factor for the decrease of the specific activity of immobilized pullulanase might be steric hindrance. This would be unavoidable when a substrate having a high molecular weight is used. From the results of Fig. 5, it was concluded that the optimum support for pullulanase immobilization was gel D in this system.
were still active at 70 8C, while free pullulanases had no activity at that temperature. This would be due to the stabilization by the multipoint binding between one pullulanase molecule and the support gel, and even at a higher temperature the immobilized pullulanase could retain its active structure compared to free enzymes. The effect of pH on the activities of free and immobilized pullulanases was almost identical. The optimum temperature and pH of immobilized pullulanases bound to gel D were 50 8C and 6.0, respectively, so they were the same as those of free pullulanase.
3.3. Temperature and pH dependencies of immobilized pullulanase
The time courses of the residual activity of pullulanase immobilized on gel D at various temperatures are shown in Fig. 7. The residual activity decreased to 70 and 60% during a 250-h incubation at 40 and 45 8C, respectively, while it quickly dropped to 40% after 24 h at 50 8C. When a substrate (30% soluble starch) co-existed, immobilized pullulanases were more stable (84% of initial activity was retained after 216 h at 45 8C). This fact is an advantage for continuous use of immobilized pullulanases, such as in a packed-bed-type reactor.
The effects of temperature and pH on the activity of immobilized pullulanase prepared using gel D are shown in Fig. 6. The temperature dependence of the activity of immobilized pullulanase was almost identical to that of free pullulanases up to 50 8C, but immobilized pullulanase exhibited a higher relative activity than that of free pullulanase over 60 8C (Fig. 6a). Immobilized pullulanases
3.4. Effect of temperature on the stability of immobilized pullulanases
4. Conclusions
Fig. 6. Effects of temperature (a) and pH (b) on the activity of free and immobilized pullulanases. Effects of temperature and pH were assayed at pH 6.0 and 50 8C, respectively. Open circles: free pullulanase; closed circles: immobilized pullulanase prepared by the multipoint attachment method using gel D.
The immobilization of pullulanase by multipoint attachment to activated agar gel supports was investigated in order to prepare stable immobilized pullulanase. Pullulanases immobilized on activated agar gels by the multipoint attachment method were significantly stabilized compared with free pullulanase, while no stabilizing effect was observed when chitosan beads were used as the support material. The effect of surface aldehyde density on the activated agar gel support was examined. The immobilization yield increased with increasing surface aldehyde density and 100% of applied protein was immobilized for the activated
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agar support having a surface aldehyde density above 15 ˚ 2 (this value corresponds to 15 aldehyde residues per 3600 A residues per one pullulanase molecule, approximately). The thermal stability of pullulanase was significantly improved by increasing the surface aldehyde density. The effect of surface aldehyde density on the stability of pullulanase was discussed in relation to the number of covalent bonds between one pullulanase molecule and the support. It was suggested that the formation of multipoint covalent bonds between the pullulanase molecule and the support was essential for the stabilization of pullulanase. The maximal activity of immobilized pullulanase was 5.61 U/g support at a surface aldehyde density of 17.9 residues ˚ 2. The optimum temperature and pH of pullper 3600 A ulanases immobilized on the activated agar support were the same as those of free pullulanase. Immobilized pullulanase was more stable under incubation with a substrate (lowmolecular-weight soluble starch) than without a substrate. The method for pullulanase immobilization described here can contribute to the development of continuous processes of starch saccharification and various sugar productions. The application of immobilized pullulanase for the continuous production of useful oligosaccharide by combining it with other amylolytic enzymes is under investigation.
Acknowledgement The authors express their sincere appreciation to Amano Enzyme, Inc. for the supply of pullulanase.
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