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Stabilization of free and immobilized enzymes using hyperthermophilic chaperonin
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 101, No. 2, 131–136. 2006 DOI: 10.1263/jbb.101.131
© 2006, The Society for Biotechnology, Japan
Stabilization of Free and Immobilized Enzymes Using Hyperthermophilic Chaperonin Jiro Kohda,1* Hirofumi Kawanishi,1 Ken-Ichiro Suehara,1 Yasuhisa Nakano,1 and Takuo Yano1 Department of Information Machines and Interfaces, Faculty of Information Science, Hiroshima City University, 3-4-1 Ozuka-higashi, Asaminami-ku, Hiroshima 731-3194, Japan1 Received 1 September 2005/Accepted 31 October 2005
Chaperonins suppress the denaturation of proteins and promote protein folding in vivo. Because hyperthermophilic chaperonins are expected to be used as a stabilizer for proteins, the effects of a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 (T. KS-1 cpn), on the stabilization of mesophilic and thermophilic free enzymes and an enzyme co-immobilized with T. KS-1 cpn were studied. T. KS-1 cpn prevented the thermal inactivation of yeast alcohol dehydrogenase (ADH), jack bean urease, and Thermus flavus malate dehydrogenase (MDH) at high temperatures. T. KS-1 cpn also improved the long-term stability of ADH at lower temperatures. Moreover, the residual ADH activity of ADH co-entrapped with T. KS-1 cpn was improved and maintained at a higher level than that of the entrapped ADH without chaperonin. T. KS-1 cpn is useful for the stabilization of free and immobilized enzymes and applicable to various fields of biotechnology. [Key words: archaea, chaperonin, heat stabilization, hyperthermophile, immobilized enzyme]
zymes. In previous studies, a thermophilic group I chaperonin from Bacillus strain MS stabilized enzymes at 30°C and 80°C (9). Mesophilic group I chaperonins, such as GroEL/ES from E. coli, stabilized several proteins in a range of temperatures from 4°C to 48°C (10–12). Among hyperthermophilic group II chaperonins, S. solfataricus chaperonin (13) and recombinant Thermococcus kodakaraensis KOD1 (formerly Pyrococcus sp. KOD1) chaperonin expressed in E. coli (14, 15) stabilized enzymes at 50°C. When enzymes are used in biotechnological processes, they are often immobilized onto insoluble support materials. The advantage of immobilization is not only the fact that enzyme is reusable but it is also capable of stabilizing enzymes. However, because immobilization causes a structural change of enzymes, the activity of immobilized enzymes is generally lower than that of free enzymes. Moreover, despite the increase in their stabilities, immobilized enzymes are gradually inactivated. Therefore, it is desirable that enzymes are immobilized without loss of activity, and that immobilized enzymes maintain their activities for a longer period. The co-immobilization of chaperonin has some advantages compared with small-molecule stabilizing agents, the chemical modification of enzymes and protein engineering such as amino acid replacement. One advantage is the ease of preparation of chaperonin co-immobilized enzymes because chaperonins and target enzymes can be immobilized using the same method simultaneously. Another is that many enzymes can be simultaneously immobilized because of the low specificity of chaperonin to substrate proteins. Hence, the co-immobilization of T. KS-1 cpn is expected to stabilize immobilized enzymes.
The chaperonin family, a group of molecular chaperones, plays a central role in protein folding in vivo (1, 2). Hyperthermophilic chaperonins belong to group II chaperonins, which are found in archaea and eukaryotic cytosol. Group II chaperonins form cylindrical structures made up of two stacked rotationally symmetrical rings consisting of eight or nine subunits, and have no co-chaperonin such as the Escherichia coli GroES. Hyperthermophilic group II chaperonins have one to three distinct subunits, α, β and γ. In previous studies, several thermophilic group II chaperonins, namely Sulfolobus solfataricus chaperonin (3) and recombinant Methanococcus thermolithotrophicus chaperonin expressed in E. coli (4), have been found to promote the refolding of chemically denatured proteins in an ATP-dependent manner. Recombinant group II chaperonin α and β subunit homo-oligomers from a hyperthermophilic archaeum, Thermococcus strain KS-1 expressed in E. coli have been found to form cylindrical structures composed of two stacked 8fold rotational symmetric rings of each subunit. T. KS-1 chaperonin (T. KS-1 cpn) α and β homo-oligomers exhibited ATPase activity, and facilitated the refolding of chemically denatured proteins in the presence of ATP (5–8). Furthermore, T. KS-1 α and β cpn monomers facilitated the refolding of chemically denatured enzymes even in the absence of ATP (8). Thermophilic and hyperthermophilic chaperonins are expected to be used for stabilizing proteins because they are thermostable and suppress the thermal inactivation of en* Corresponding author. e-mail: [email protected] phone: +81-(0)82-830-1869 fax: +81-(0)82-830-1792 131
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To apply hyperthermophilic chaperonins to biotechnological processes, we studied the heat stabilization effect of enzymes using a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. Furthermore, the stability of a T. KS-1 cpn co-immobilized enzyme by an entrapping method was examined. MATERIALS AND METHODS Materials Yeast alcohol dehydrogenase (ADH: EC 1.1.1.1, 37 kDa, tetramer) and ATP were purchased from Oriental Yeast (Tokyo). Thermus flavus malate dehydrogenase (MDH: EC 1.1.1.37, 35 kDa, dimer) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Jack bean urease (EC 3.5.1.5, 60 kDa, trimer) was purchased from Wako Pure Chemical Industries (Osaka). Bovine liver glutamate dehydrogenase (GlDH: EC 1.4.1.3, 56 kDa, hexamer) was purchased from Roche Diagnostics (Basel, Switzerland). The other materials were purchased from Nakalai Tesque (Kyoto). Expression and purification of chaperonin T. KS-1 cpn α and β subunits (T. KS-1 α and β cpns) were overexpressed as described previously (8). Cells were harvested by centrifugation at 5000×g for 10 min at 4°C, resuspended in 50 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)–NaOH buffer (pH 7.5), disrupted by sonication, and recentrifuged at 30,000×g for 60 min at 4°C. Magnesium chloride, dithiothreitol (DTT) and glycerol were added to the recovered supernatant at final concentrations of 25 mM, 1 mM, and 5%, respectively, and the supernatant was heated at 70°C for 30 min to remove most E. coli proteins. After removing denatured proteins by centrifugation at 30,000×g for 60 min at 4°C, the extract was dialyzed against 50 mM HEPES– NaOH buffer (pH 7.5) containing 25 mM MgCl2. The concentrations of T. KS-1 α and β cpns were determined by the method of Bradford using bovine serum albumin (BSA) as a standard (16). The purity of the chaperonins was determined by 12.5% SDS– PAGE and monomeric and oligomeric forms of the chaperonins were confirmed by 6% native PAGE. The percentage of T. KS-1 hexadecamer (16-mer) in the total T. KS-1 cpn was quantified by NIH imaging (ver. 1.62). Stabilization of free enzymes ADH, urease or MDH was mixed with incubation buffer (50 mM Tris–HCl buffer (pH 7.8) containing 300 mM KCl, 50 mM MgCl2, 100 mM guanidine hydrochloride and 83.3 µM DTT) containing T. KS-1 cpn pre-incubated at 50°C, 30°C or 4°C (ADH), 65°C (urease), and 85°C (MDH). The enzyme solutions were incubated at the same temperatures. The stabilization effect was evaluated as the residual activity which is the relative activity of incubated enzymes relative to untreated enzymes. Enzyme assay The activities of ADH, urease and MDH were measured by the following methods. An aliquot of ADH solution was added to 100 mM glycine–KOH buffer (pH 9.5) containing 2 mM NAD+ and 100 mM ethanol. The ADH activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. Urease activity was determined by measuring NADH oxidation rates using NADH-dependent GlDH and ammonia produced by urease (17). An aliquot of urease solution was added to 50 mM Tris–HCl buffer (pH 7.8) containing 0.81 mM 2-oxoglutarate, 0.24 mM NADH, 10 mM urea and 3.0 U/ml GlDH. The urease activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. An aliquot of MDH solution was added to 90 mM Tris–HCl buffer (pH 7.4) containing 0.5 mM oxalacetate and 0.25 mM NADH. The MDH activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. Detailed experimental conditions are described in the figure legends. Stabilization of chaperonin co-immobilized ADH An en-
trapping method, which is one of the immobilization strategies, is routinely used for the preparation of several immobilized enzymes. Entrapment using calcium alginate gel is used for several enzymes (18, 19). In this study, ADH and T. KS-1 cpn were co-entrapped in calcium alginate gel. Sodium alginate (5%) was dissolved by heating using an autoclave (105°C, 1 min). After cooling down to room temperature, ADH and T. KS-1 cpn or BSA were added to the sodium alginate solution. This mixture was dropped into ice-cold buffer A (50 mM Tris–HCl buffer (pH 7.0) containing 1.0 M CaCl2). Four types of gel beads were prepared: entrapped ADH (ADHalone) gel beads, ADH co-entrapped with T. KS-1 α cpn (ADH-T. KS-1 α cpn) gel beads, ADH co-entrapped with T. KS-1 β cpn (ADH-T. KS-1 β cpn) gel beads and ADH co-entrapped with BSA (ADH-BSA) gel beads. These gel beads were stored at 4°C in 50 mM Tris–HCl buffer (pH 7.0) until the measurement of entrapped ADH activity. ADH activity was measured by the following method. Entrapped ADH (0.2 g wet gel) was added to 100 mM glycine– KOH buffer (pH 9.5) containing 2 mM NAD+ and 100 mM ethanol. The total reaction volume was 1.5 ml. After 5 min of stirring, the entrapped ADH activity was determined from the increase in the absorbance at 340 nm as a function of time at 25°C by measuring the absorbance at 340 nm of the reaction solution at intervals of 5 min to 15 min. The stabilization effect was evaluated as the residual activity compared to the entrapped ADH activity stored for 1 d after preparation.
RESULTS Stabilization of free enzymes at high temperatures Hyperthermophilic chaperonins are expected to increase the thermal stability of enzymes because of their high thermostability. Figure 1 shows the thermal inactivation of ADH at 50°C in the presence and absence of T. KS-1 cpn. After a 120-min incubation, ADH activity decreased in the absence of T. KS-1 cpn. A 5-fold excess concentration of T. KS-1
FIG. 1. Thermal inactivation of yeast ADH at 50°C and pH 7.8. The final concentrations of ADH, BSA and T. KS-1 cpn are 50 nM, 250 nM and 250 nM, respectively. The concentration of T. KS-1 cpn was calculated by assuming that all subunits formed a 16-mer. The residual activity was calculated as the percentage of incubated enzyme activity relative to untreated enzyme activity. Closed and open symbols indicate the incubation in the absence and presence of 5 mM ATP, respectively. Circles, Incubation in the absence of T. KS-1 cpn and BSA; diamonds, incubation in the presence of BSA; squares, incubation in the presence of T. KS-1 α cpn; triangles, incubation in the presence of T. KS-1 β cpn.
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TABLE 1. Half-life (t1/2) of free enzymes at indicated temperaturesa
FIG. 2. Thermal inactivation of jack bean urease at 65°C and pH 7.8. The final concentrations of urease, BSA and T. KS-1 cpn are 25 nM, 125 nM and 125 nM, respectively. Symbols, see Fig. 1.
FIG. 3. Thermal inactivation of T. flavus MDH at 85°C and pH 7.8. The final concentrations of MDH, BSA and T. KS-1 cpn are 75 nM, 150 nM and 150 nM, respectively. Symbols, see Fig. 1.
cpn slightly stabilized ADH in the absence of ATP, and there was no difference in the stabilization effect between T. KS-1 α and β cpns. The same concentration of BSA relative to T. KS-1 cpn did not stabilize ADH. T. KS-1 cpn plus ATP significantly increased the stability of ADH. The ADH stabilization effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. Urease, which is relatively stable, was completely inactivated after 3 h at 65°C (Fig. 2). T. KS-1 α and β cpns stabilized urease in the absence of ATP, and the urease stabilization effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. The urease stabilization effect was significantly increased in the presence of ATP, and the stabilization effect of T. KS-1 β cpn was higher than that of T. KS-1 α cpn. A thermophilic enzyme, T. flavus MDH, was also stabilized by T. KS-1 α and β cpns with or without ATP at 85°C (Fig. 3). Not only T. KS-1 cpn but also BSA prevented the inactivation of MDH in the absence of ATP. There was no significant difference in the stabilization effect between T. KS-1 α and β cpns either in the absence or presence of ATP. The stabilization effect in the presence of ATP was a
ADH urease MDH 30°C 4°C 65°C 85°C 50°C Additives t1/2 t1/2 t1/2 t1/2 t1/2 (min) (d) (d) (h) (min) 30.8 0.243 0.505 0.635 9.73 Noneb BSA 34.4 0.227 1.37 0.484 38.1 T. KS-1 α cpn 46.1 0.716 0.693 2.29 39.4 T. KS-1 α cpn+ ATP 199 3.28 1.90 4.17 95.2 T. KS-1 β cpn 42.9 0.554 0.683 1.59 54.3 T. KS-1 β cpn + ATP 123 1.51 1.88 8.19 94.0 ATP 0.504 1.99 BSA+ATP 0.665 6.07 GroEL/ES 0.729 0.821 GroEL/ES + ATP 4.89 3.46 a Half-life was determined from semilogarithmic plots shown in Figs. 1–4. b Incubation in the absence of ATP, T. KS-1 cpn, BSA and GroEL/ES.
little higher than that in the absence of ATP. From the semilogarithmic plots shown in Figs. 1–3, these inactivations can be considered first-order inactivations. Therefore, the half-life (t1/2) of ADH, urease and MDH, indicating the time when the residual activity reaches 50%, was determined from the semilogarithmic plots shown in Figs. 1–3. Table 1 shows the half-life of ADH, urease and MDH at several temperatures. In all cases, T. KS-1 cpn increased the halflife of these enzymes, and the addition of ATP in the presence of T. KS-1 cpn further increased it. These results indicate that T. KS-1 α and β cpns significantly stabilized mesophilic and thermophilic free enzymes in the presence of ATP. Long-term stabilization of free ADH at lower temperatures Considering that most enzymes are used at moderate temperature in bioreactors and stored at 4°C or −20°C, it was considered interesting to determine whether chaperonins have a long-term stabilization effect at the lower temperatures used for reaction and storage rather than the shortterm stabilization effect at high temperatures. Therefore, the ADH stabilization effect of T. KS-1 cpn at lower temperatures was examined. ADH activity was completely lost after a 4-d incubation at 30°C (Fig. 4a). T. KS-1 cpn and E. coli GroEL/ES slightly stabilized ADH in the absence of ATP. ATP also prevented the inactivation of ADH. T. KS-1 cpn significantly stabilized ADH at 30°C in the presence of ATP, and the effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. The stabilization effects of T. KS-1 cpns were lower than that of E. coli GroEL/ES in the presence of ATP. In the case of BSA, the effect of the addition of ATP was not as significant as in the case of chaperonin. When ADH was stored at 4°C (Fig. 4b), the stabilization effect of BSA was higher than those of chaperonins in both the presence and absence of ATP. In the presence of ATP, the stabilization effects of T. KS-1 cpn and GroEL/ES at 4°C were less than those at 30°C. The half-life of ADH at 30°C and 4°C was increased by the addition of T. KS-1 cpn and GroEL/ES, and the addition of ATP in the presence of chaperonin further increased it (Table 1). These results indicate that T. KS-1 cpn has a stabilizing effect on ADH at low and high temperatures.
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FIG. 5. Storage stability of chaperonin co-immobilized ADH at 4°C and pH 7.0. The final concentrations of ADH, BSA, T. KS-1 α cpn and T. KS-1 β cpn are 0.020 µmol/g-gel, 0.040 µmol/g-gel, 0.040 µmol/g-gel and 0.040 µmol/g-gel, respectively. The residual activity is calculated as the percentage of the immobilized enzyme activity stored for the indicated period relative to that stored for 1 d after preparation. Circles, ADH-alone gel beads; diamonds, ADH-BSA gel beads; squares, ADH-T. KS-1 α cpn gel beads; triangles, ADH-T. KS-1 β cpn gel beads.
FIG. 4. Long-term stability of free yeast ADH at 30°C (a) and 4°C (b) at pH 7.8. The final concentrations of ADH, BSA, T. KS-1 cpn and GroEL/ES are 20 nM, 100 nM, 100 nM and 100 nM, respectively. Closed and open symbols indicate the incubation in the absence and presence of 5 mM ATP, respectively. Circles, Incubation in the absence of T. KS-1 cpn, GroEL/ES and BSA; diamonds, incubation in the presence of BSA; squares, incubation in the presence of T. KS-1 α cpn; triangles, incubation in the presence of T. KS-1 β cpn; reversed triangles, incubation in the presence of GroEL/ES.
Stabilization effect of chaperonin for gel-entrapped ADH In this study, four types of calcium alginate-entrapped gel beads were prepared: entrapped ADH (ADHalone) gel beads, ADH co-entrapped with T. KS-1 α cpn (ADH-T. KS-1 α cpn) gel beads, ADH co-entrapped with T. KS-1 β cpn (ADH-T. KS-1 β cpn) gel beads and ADH coentrapped with BSA (ADH-BSA) gel beads. The calcium alginate gel beads prepared in this study were 2.5–3.5 mm in diameter. The leakage of ADH or T. KS-1 cpn from these gel beads was not observed (data not shown). Then, when these gel beads were stored at 4°C, the stabilization effect of T. KS-1 cpn for entrapped ADH was investigated (Fig. 5). The residual ADH activity of ADH-alone gel beads decreased to 15% after 5 d at 4°C. One day following the gel preparation, the ADH activities of ADH-BSA, and ADH-T. KS-1 α or β cpn gel beads were 1.3-fold higher than that of ADH-alone gel beads (data not shown). BSA and T. KS-1 cpn prevented the inactivation of entrapped ADH for 5 d at 4°C. The inactivation rates of ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were lower than that of ADH-BSA gel beads even in the absence of ATP. After 5 d,
the residual ADH activities of ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were maintained at 72% and 77%, respectively. When calcium alginate entrapped ADHalone, ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were used repeatedly for the measurement of ADH activity, the residual ADH activities decreased to 35%, 45% and 35%, respectively, after 5 d (data not shown). This result indicates that T. KS-1 cpn stabilized the entrapped ADH as well as the free ADH. DISCUSSION T. KS-1 α and β cpns expressed in E. coli cells contained a large amount of monomer after the heat treatment purification (8). In this study, the average weight percentage of the T. KS-1 α and β 16-mer were 56.4 and 23.4% (16-mer/ total chaperonin [w/w]), respectively. It has been shown that T. KS-1 cpn 16-mer facilitated the refolding of proteins in an ATP-dependent manner (5, 6, 20), and the T. KS-1 cpn monomer facilitated the refolding of proteins even in the absence of ATP (8). Because the hyperthermophilic group II chaperonin from S. solfataricus as well as the thermophilic group I chaperonin from Bacillus strain MS facilitated protein refolding and prevented the thermal inactivation of enzymes (3, 9, 13), it was expected that T. KS-1 cpn would also be effective for the prevention of the thermal inactivation of enzymes. Therefore, we used T. KS-1 cpn containing the 16-mer and the monomer for the stabilization of enzymes. T. KS-1 cpn prevented the thermal inactivation of mesophilic and thermophilic enzymes at several temperatures higher than 50°C in the presence of ATP (Figs. 1–3). These results are consistent with those of previous studies (13, 15). Although T. KS-1 α cpn stabilized ADH better than T. KS-1 β cpn at 50°C, T. KS-1 β cpn stabilized urease better than T. KS-1 α cpn at 65°C. Moreover, T. KS-1 α cpn stabilized
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MDH to the same degree as T. KS-1 β cpn at 85°C. The ATPase activities of the T. KS-1 α and β cpn 16-mers were almost the same from 40°C to 70°C, and the ATPase activity of the T. KS-1 β cpn 16-mer was higher than that of the T. KS-1 α cpn 16-mer at temperatures higher than 70°C (6, 21). Therefore, the difference in the stabilization effect between the T. KS-1 α and β cpns is probably due to the difference in not only the ATPase activity of T. KS-1 cpn but also the characteristics of target enzymes. T. KS-1 cpn also prevented the thermal inactivation of urease and MDH even in the absence of ATP (Figs. 2 and 3). It was reported that other hyperthermophilic group II chaperonins stabilized enzymes at various temperatures in the absence of ATP (13, 15). Our results are consistent with those of previous studies. Considering that the T. KS-1 cpn 16-mer exhibits ATPase activity at temperatures higher than 50°C, this stabilization effect is probably due to the T. KS-1 cpn monomer. Therefore, it is assumed that the stabilization effect at high temperatures, at which T. KS-1 cpn expresses ATPase activity, is due to both the ATP-dependent effect of the T. KS-1 cpn 16-mer and the ATP-independent effect of the T. KS-1 cpn monomer. It has been shown that some group I chaperonins enhanced the thermal stability of several enzymes at various temperatures (9–12). Our results suggest that group II chaperonins as well as group I chaperonins are effective for the stabilization of enzymes. In particular, it will be of economic advantage not to require ATP for the heat stabilization of enzymes due to the use of T. KS-1 cpn. It should be investigated whether the T. KS-1 cpn 16-mer or monomer is more suitable for the stabilization of enzymes. The stabilization properties were dependent on incubation temperature. T. KS-1 cpn had high stabilization effects at 30°C and 4°C as well as 50°C in the presence of ATP (Figs. 1 and 4). From previous results (6, 21), it is presumed that the T. KS-1 cpn 16-mer has no detectable ATPase activity at 30°C and 4°C. Furthermore, the T. KS-1 cpn monomer had no detectable ATPase activity (21). Therefore, it is assumed that these stabilization effects at 30°C and 4°C are not due to the T. KS-1 cpn 16-mer but due to the T. KS-1 cpn monomer. Although the T. KS-1 cpn 16-mer and monomer have no detectable ATPase activities at 30°C and 4°C, apparent ATP dependency was observed. This is presumed to be caused by the stabilization effect of ATP on ADH, and then ADH was synergistically stabilized by ATP and T. KS-1 cpn, probably by the T. KS-1 cpn monomer. Because GroEL/ES had ATPase activity at 30°C (22), the stabilization effect of GroEL/ES was higher than that of T. KS-1 cpn. It has been reported that the ATPase activity of GroEL at 10°C was lower than at 25°C (23). Therefore, it was assumed that the ADH stabilization effect of T. KS-1 cpn or GroEL/ES at 4°C was lower than that at 30°C. From these results, it is assumed that the stabilization effect at lower temperatures, at which T. KS-1 cpn has no detectable ATPase activity, is mainly due to the ATP-independent effect of the T. KS-1 cpn monomer. BSA or T. KS-1 cpn co-entrapped with ADH increased the ADH activity (data not shown). This is probably because BSA and T. KS-1 cpn prevent the inactivation of ADH during immobilization. Although both BSA and T.
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KS-1 cpn prevented the inactivation of ADH in the gel matrix for 5 d at 4°C, the inactivation rate of ADH-T. KS-1 cpn gel beads was lower than that of ADH-BSA gel beads (Fig. 5). Therefore, T. KS-1 cpn is more efficient for the stabilization of immobilized enzymes than BSA. Although T. KS-1 cpn prevented the inactivation of ADH in the gel matrix in the absence of ATP, this stabilization effect was not observed in free ADH (Fig. 4b). It is thought that the function of T. KS-1 cpn 16-mer is inhibited in the gel matrix at 4°C for the reasons outlined above. Therefore, it is assumed that the T. KS-1 cpn monomer, which locally concentrated in the gel matrix, interacted with ADH more strongly in the gel matrix than in the solution, and then prevented the inactivation of ADH in the gel matrix. Although T. KS-1 cpn was efficient for the stabilization of immobilized enzymes, the ADH activity of ADH-T. KS-1 cpn gel beads decreased during repeated use (data not shown). Immobilized ADH was assayed in 100 mM glycine–KOH buffer (pH 9.5). Although the highest activity of ADH was observed at around pH 9, ADH stability was not very high at around this pH. From these results, it is assumed that ADH was inactivated at this pH and T. KS-1 cpn did not stabilize ADH at this pH. Our results show that T. KS-1 cpn can be used for the stabilization of free and entrapped enzymes. In a previous study, although covalently immobilized recombinant human HSP70 onto surface modified glass with silane was able to reactivate the thermally denatured firefly luciferase, HSP70 co-immobilized luciferase did not exhibit increased thermal stability (24). Therefore, entrapment immobilization is suitable for the stabilization of immobilized enzymes by the co-immobilization of chaperonin and enzymes because the degree of interaction between enzymes and chaperonin is higher than that which occurs in other immobilization strategies and because this method does not involve chemical bonds between proteins and support materials. In particular, calcium alginate gel entrapment is a more suitable method than other entrapment methods, because other gel entrapment methods involve some factors contributing to the inactivation such as high gelation temperatures, the heat of polymerization and radicals. When an entrapped ADH gel was prepared with agar gel, ADH activity was not observed even after 1 d (data not shown). To apply hyperthermophilic chaperonins to various fields of biotechnology, the efficiency of chaperonins in the stabilization of immobilized enzymes should be investigated for other immobilization methods such as covalent immobilization and physical adsorption. ACKNOWLEDGMENTS We are grateful to Dr. T. Yoshida and Dr. T. Maruyama, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), and Prof. M. Yohda, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology for providing the chaperonin expression plasmid and helpful discussion. This work was supported by the Hiroshima City University Grant for Special Academic Research (General Studies).
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2. Hartl, F. U.: Molecular chaperones in cellular protein folding. Nature, 381, 571–579 (1996). 3. Guagliardri, A., Cerchia, L., Bartolucci, S., and Rossi, M.: The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro. Protein Sci., 3, 1436–1443 (1994). 4. Furutani, M., Iida, T., Yoshida, T., and Maruyama, T.: Group II chaperonin in a thermophilic methanogen, Methanococcus thermolithotrophicus. J. Biol. Chem., 273, 28399– 28407 (1998). 5. Yoshida, T., Yohda, M., Iida, T., Maruyama, T., Taguchi, H., Yazaki, K., Ohta, T., Odaka, M., Endo, I., and Kagawa, Y.: Structural and functional characterization of homo-oligomeric complexes of α and β chaperonin subunits from the hyperthermophilic archaeum, Thermococcus strain KS-1. J. Mol. Biol., 273, 635–645 (1997). 6. Yoshida, T., Yohda, M., Iida, T., Maruyama, T., Taguchi, H., Yazaki, K., Ohta, T., Odaka, M., Endo, I., and Kagawa, Y.: Corrigendum. J. Mol. Biol., 299, 1399–1400 (2000). 7. Yoshida, T., Kawaguchi, R., and Maruyama, T.: Nucleotide specificity of an archaeal group II chaperonin from Thermococcus strain KS-1 with reference to the ATP-dependent protein folding cycle. FEBS Lett., 514, 269–274 (2002). 8. Kohda, J., Yamada, T., Yoshida, T., Maruyama, T., Yohda, M., Fukuda, H., and Kondo, A.: Refolding of proteins by hexadecamers and monomers of the α and β subunits of group II chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1. Biochem. Eng. J., 18, 73–79 (2004). 9. Teshima, T., Kohda, J., Kondo, A., Yohda, M., Tamura, A., and Fukuda, H.: Affinity purification of fusion chaperonin cpn60-(His)6 from thermophilic bacteria Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation. Biotechnol. Prog., 16, 442–446 (2000). 10. Kawata, Y., Nosaka, K., Hongo, K., Mizobata, T., and Nagai, J.: Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation. FEBS Lett., 345, 229–232 (1994). 11. Mendoza, J. A., Lorimer, G. H., and Horowitz, P. M.: Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures. J. Biol. Chem., 267, 17631–17634 (1992). 12. Hartman, D. J., Surin, B. P., Dixon, N. E., Hoogenraad, N. J., and Høj, P. B.: Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro. Proc. Natl. Acad. Sci. USA, 90, 2276–
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2280 (1993). 13. Guagliardri, A., Cerchia, L., and Rossi, M.: Prevention of in vitro protein thermal aggregation by the Sulfolobus solfataricus chaperonin. J. Biol. Chem., 270, 28126–28132 (1995). 14. Atomi, H., Fukui, T., Kanai, T., Morikawa, M., and Imanaka, T.: Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1. Archaea, 1, 263–267 (2004). 15. Yan, Z., Fujiwara, S., Kohda, K., Takagi, M., and Imanaka, T.: In vitro stabilization and in vivo solubilization of foreign proteins by the β subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1. Appl. Environ. Microbiol., 63, 785–789 (1997). 16. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 166, 248–254 (1976). 17. Kaltwasser, H. and Schlegel, H. G.: NADH-dependent coupled enzyme assay for urease and other ammonia-producing systems. Anal. Biochem., 16, 132–138 (1966). 18. Tanaka, H., Kurosawa, H., Kokufuta, E., and Veliky, I. A.: Preparation of immobilized glucoamylase using Ca–alginate gel coated with partially quaterized poly(ethyleneimine). Biotechnol. Bioeng., 26, 1393–1394 (1984). 19. Chang, H. N., Joo, I. S., and Ghim, Y. S.: Performance of rotating packed disk reactor with immobilized glucose oxidase. Biotechnol. Lett., 6, 487–492 (1984). 20. Yoshida, T., Kawaguchi, R., Taguchi, H., Yoshida, M., Yasunaga, T., Wakabayashi, T., Yohda, M., and Maruyama, T.: Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin. J. Mol. Biol., 315, 73–85 (2002). 21. Yoshida, T., Ideno, A., Suzuki, R., Yohda, M., and Maruyama, T.: Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1. Mol. Microbiol., 44, 761–769 (2002). 22. Mendoza, J. A., Warren, T., and Dulin, P.: The ATPase activity of chaperonin GroEL is highly stimulated at elevated temperatures. Biochem. Biophys. Res. Commun., 229, 271– 274 (1996). 23. Mendoza, J. A., Dulin, P., and Warren, T.: The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature. Cryobiology, 41, 319–323 (2000). 24. Yang, Y., Zeng, J., Gao, C., and Krull, U. J.: Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones. Biosens. Bioelectron., 18, 311–317 (2003).
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Stabilization of Free and Immobilized Enzymes Using Hyperthermophilic Chaperonin Jiro Kohda,1* Hirofumi Kawanishi,1 Ken-Ichiro Suehara,1 Yasuhisa Nakano,1 and Takuo Yano1 Department of Information Machines and Interfaces, Faculty of Information Science, Hiroshima City University, 3-4-1 Ozuka-higashi, Asaminami-ku, Hiroshima 731-3194, Japan1 Received 1 September 2005/Accepted 31 October 2005
Chaperonins suppress the denaturation of proteins and promote protein folding in vivo. Because hyperthermophilic chaperonins are expected to be used as a stabilizer for proteins, the effects of a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 (T. KS-1 cpn), on the stabilization of mesophilic and thermophilic free enzymes and an enzyme co-immobilized with T. KS-1 cpn were studied. T. KS-1 cpn prevented the thermal inactivation of yeast alcohol dehydrogenase (ADH), jack bean urease, and Thermus flavus malate dehydrogenase (MDH) at high temperatures. T. KS-1 cpn also improved the long-term stability of ADH at lower temperatures. Moreover, the residual ADH activity of ADH co-entrapped with T. KS-1 cpn was improved and maintained at a higher level than that of the entrapped ADH without chaperonin. T. KS-1 cpn is useful for the stabilization of free and immobilized enzymes and applicable to various fields of biotechnology. [Key words: archaea, chaperonin, heat stabilization, hyperthermophile, immobilized enzyme]
zymes. In previous studies, a thermophilic group I chaperonin from Bacillus strain MS stabilized enzymes at 30°C and 80°C (9). Mesophilic group I chaperonins, such as GroEL/ES from E. coli, stabilized several proteins in a range of temperatures from 4°C to 48°C (10–12). Among hyperthermophilic group II chaperonins, S. solfataricus chaperonin (13) and recombinant Thermococcus kodakaraensis KOD1 (formerly Pyrococcus sp. KOD1) chaperonin expressed in E. coli (14, 15) stabilized enzymes at 50°C. When enzymes are used in biotechnological processes, they are often immobilized onto insoluble support materials. The advantage of immobilization is not only the fact that enzyme is reusable but it is also capable of stabilizing enzymes. However, because immobilization causes a structural change of enzymes, the activity of immobilized enzymes is generally lower than that of free enzymes. Moreover, despite the increase in their stabilities, immobilized enzymes are gradually inactivated. Therefore, it is desirable that enzymes are immobilized without loss of activity, and that immobilized enzymes maintain their activities for a longer period. The co-immobilization of chaperonin has some advantages compared with small-molecule stabilizing agents, the chemical modification of enzymes and protein engineering such as amino acid replacement. One advantage is the ease of preparation of chaperonin co-immobilized enzymes because chaperonins and target enzymes can be immobilized using the same method simultaneously. Another is that many enzymes can be simultaneously immobilized because of the low specificity of chaperonin to substrate proteins. Hence, the co-immobilization of T. KS-1 cpn is expected to stabilize immobilized enzymes.
The chaperonin family, a group of molecular chaperones, plays a central role in protein folding in vivo (1, 2). Hyperthermophilic chaperonins belong to group II chaperonins, which are found in archaea and eukaryotic cytosol. Group II chaperonins form cylindrical structures made up of two stacked rotationally symmetrical rings consisting of eight or nine subunits, and have no co-chaperonin such as the Escherichia coli GroES. Hyperthermophilic group II chaperonins have one to three distinct subunits, α, β and γ. In previous studies, several thermophilic group II chaperonins, namely Sulfolobus solfataricus chaperonin (3) and recombinant Methanococcus thermolithotrophicus chaperonin expressed in E. coli (4), have been found to promote the refolding of chemically denatured proteins in an ATP-dependent manner. Recombinant group II chaperonin α and β subunit homo-oligomers from a hyperthermophilic archaeum, Thermococcus strain KS-1 expressed in E. coli have been found to form cylindrical structures composed of two stacked 8fold rotational symmetric rings of each subunit. T. KS-1 chaperonin (T. KS-1 cpn) α and β homo-oligomers exhibited ATPase activity, and facilitated the refolding of chemically denatured proteins in the presence of ATP (5–8). Furthermore, T. KS-1 α and β cpn monomers facilitated the refolding of chemically denatured enzymes even in the absence of ATP (8). Thermophilic and hyperthermophilic chaperonins are expected to be used for stabilizing proteins because they are thermostable and suppress the thermal inactivation of en* Corresponding author. e-mail: [email protected] phone: +81-(0)82-830-1869 fax: +81-(0)82-830-1792 131
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To apply hyperthermophilic chaperonins to biotechnological processes, we studied the heat stabilization effect of enzymes using a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1. Furthermore, the stability of a T. KS-1 cpn co-immobilized enzyme by an entrapping method was examined. MATERIALS AND METHODS Materials Yeast alcohol dehydrogenase (ADH: EC 1.1.1.1, 37 kDa, tetramer) and ATP were purchased from Oriental Yeast (Tokyo). Thermus flavus malate dehydrogenase (MDH: EC 1.1.1.37, 35 kDa, dimer) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Jack bean urease (EC 3.5.1.5, 60 kDa, trimer) was purchased from Wako Pure Chemical Industries (Osaka). Bovine liver glutamate dehydrogenase (GlDH: EC 1.4.1.3, 56 kDa, hexamer) was purchased from Roche Diagnostics (Basel, Switzerland). The other materials were purchased from Nakalai Tesque (Kyoto). Expression and purification of chaperonin T. KS-1 cpn α and β subunits (T. KS-1 α and β cpns) were overexpressed as described previously (8). Cells were harvested by centrifugation at 5000×g for 10 min at 4°C, resuspended in 50 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)–NaOH buffer (pH 7.5), disrupted by sonication, and recentrifuged at 30,000×g for 60 min at 4°C. Magnesium chloride, dithiothreitol (DTT) and glycerol were added to the recovered supernatant at final concentrations of 25 mM, 1 mM, and 5%, respectively, and the supernatant was heated at 70°C for 30 min to remove most E. coli proteins. After removing denatured proteins by centrifugation at 30,000×g for 60 min at 4°C, the extract was dialyzed against 50 mM HEPES– NaOH buffer (pH 7.5) containing 25 mM MgCl2. The concentrations of T. KS-1 α and β cpns were determined by the method of Bradford using bovine serum albumin (BSA) as a standard (16). The purity of the chaperonins was determined by 12.5% SDS– PAGE and monomeric and oligomeric forms of the chaperonins were confirmed by 6% native PAGE. The percentage of T. KS-1 hexadecamer (16-mer) in the total T. KS-1 cpn was quantified by NIH imaging (ver. 1.62). Stabilization of free enzymes ADH, urease or MDH was mixed with incubation buffer (50 mM Tris–HCl buffer (pH 7.8) containing 300 mM KCl, 50 mM MgCl2, 100 mM guanidine hydrochloride and 83.3 µM DTT) containing T. KS-1 cpn pre-incubated at 50°C, 30°C or 4°C (ADH), 65°C (urease), and 85°C (MDH). The enzyme solutions were incubated at the same temperatures. The stabilization effect was evaluated as the residual activity which is the relative activity of incubated enzymes relative to untreated enzymes. Enzyme assay The activities of ADH, urease and MDH were measured by the following methods. An aliquot of ADH solution was added to 100 mM glycine–KOH buffer (pH 9.5) containing 2 mM NAD+ and 100 mM ethanol. The ADH activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. Urease activity was determined by measuring NADH oxidation rates using NADH-dependent GlDH and ammonia produced by urease (17). An aliquot of urease solution was added to 50 mM Tris–HCl buffer (pH 7.8) containing 0.81 mM 2-oxoglutarate, 0.24 mM NADH, 10 mM urea and 3.0 U/ml GlDH. The urease activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. An aliquot of MDH solution was added to 90 mM Tris–HCl buffer (pH 7.4) containing 0.5 mM oxalacetate and 0.25 mM NADH. The MDH activity was determined by measuring the absorbance at 340 nm as a function of time at 25°C. Detailed experimental conditions are described in the figure legends. Stabilization of chaperonin co-immobilized ADH An en-
trapping method, which is one of the immobilization strategies, is routinely used for the preparation of several immobilized enzymes. Entrapment using calcium alginate gel is used for several enzymes (18, 19). In this study, ADH and T. KS-1 cpn were co-entrapped in calcium alginate gel. Sodium alginate (5%) was dissolved by heating using an autoclave (105°C, 1 min). After cooling down to room temperature, ADH and T. KS-1 cpn or BSA were added to the sodium alginate solution. This mixture was dropped into ice-cold buffer A (50 mM Tris–HCl buffer (pH 7.0) containing 1.0 M CaCl2). Four types of gel beads were prepared: entrapped ADH (ADHalone) gel beads, ADH co-entrapped with T. KS-1 α cpn (ADH-T. KS-1 α cpn) gel beads, ADH co-entrapped with T. KS-1 β cpn (ADH-T. KS-1 β cpn) gel beads and ADH co-entrapped with BSA (ADH-BSA) gel beads. These gel beads were stored at 4°C in 50 mM Tris–HCl buffer (pH 7.0) until the measurement of entrapped ADH activity. ADH activity was measured by the following method. Entrapped ADH (0.2 g wet gel) was added to 100 mM glycine– KOH buffer (pH 9.5) containing 2 mM NAD+ and 100 mM ethanol. The total reaction volume was 1.5 ml. After 5 min of stirring, the entrapped ADH activity was determined from the increase in the absorbance at 340 nm as a function of time at 25°C by measuring the absorbance at 340 nm of the reaction solution at intervals of 5 min to 15 min. The stabilization effect was evaluated as the residual activity compared to the entrapped ADH activity stored for 1 d after preparation.
RESULTS Stabilization of free enzymes at high temperatures Hyperthermophilic chaperonins are expected to increase the thermal stability of enzymes because of their high thermostability. Figure 1 shows the thermal inactivation of ADH at 50°C in the presence and absence of T. KS-1 cpn. After a 120-min incubation, ADH activity decreased in the absence of T. KS-1 cpn. A 5-fold excess concentration of T. KS-1
FIG. 1. Thermal inactivation of yeast ADH at 50°C and pH 7.8. The final concentrations of ADH, BSA and T. KS-1 cpn are 50 nM, 250 nM and 250 nM, respectively. The concentration of T. KS-1 cpn was calculated by assuming that all subunits formed a 16-mer. The residual activity was calculated as the percentage of incubated enzyme activity relative to untreated enzyme activity. Closed and open symbols indicate the incubation in the absence and presence of 5 mM ATP, respectively. Circles, Incubation in the absence of T. KS-1 cpn and BSA; diamonds, incubation in the presence of BSA; squares, incubation in the presence of T. KS-1 α cpn; triangles, incubation in the presence of T. KS-1 β cpn.
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TABLE 1. Half-life (t1/2) of free enzymes at indicated temperaturesa
FIG. 2. Thermal inactivation of jack bean urease at 65°C and pH 7.8. The final concentrations of urease, BSA and T. KS-1 cpn are 25 nM, 125 nM and 125 nM, respectively. Symbols, see Fig. 1.
FIG. 3. Thermal inactivation of T. flavus MDH at 85°C and pH 7.8. The final concentrations of MDH, BSA and T. KS-1 cpn are 75 nM, 150 nM and 150 nM, respectively. Symbols, see Fig. 1.
cpn slightly stabilized ADH in the absence of ATP, and there was no difference in the stabilization effect between T. KS-1 α and β cpns. The same concentration of BSA relative to T. KS-1 cpn did not stabilize ADH. T. KS-1 cpn plus ATP significantly increased the stability of ADH. The ADH stabilization effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. Urease, which is relatively stable, was completely inactivated after 3 h at 65°C (Fig. 2). T. KS-1 α and β cpns stabilized urease in the absence of ATP, and the urease stabilization effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. The urease stabilization effect was significantly increased in the presence of ATP, and the stabilization effect of T. KS-1 β cpn was higher than that of T. KS-1 α cpn. A thermophilic enzyme, T. flavus MDH, was also stabilized by T. KS-1 α and β cpns with or without ATP at 85°C (Fig. 3). Not only T. KS-1 cpn but also BSA prevented the inactivation of MDH in the absence of ATP. There was no significant difference in the stabilization effect between T. KS-1 α and β cpns either in the absence or presence of ATP. The stabilization effect in the presence of ATP was a
ADH urease MDH 30°C 4°C 65°C 85°C 50°C Additives t1/2 t1/2 t1/2 t1/2 t1/2 (min) (d) (d) (h) (min) 30.8 0.243 0.505 0.635 9.73 Noneb BSA 34.4 0.227 1.37 0.484 38.1 T. KS-1 α cpn 46.1 0.716 0.693 2.29 39.4 T. KS-1 α cpn+ ATP 199 3.28 1.90 4.17 95.2 T. KS-1 β cpn 42.9 0.554 0.683 1.59 54.3 T. KS-1 β cpn + ATP 123 1.51 1.88 8.19 94.0 ATP 0.504 1.99 BSA+ATP 0.665 6.07 GroEL/ES 0.729 0.821 GroEL/ES + ATP 4.89 3.46 a Half-life was determined from semilogarithmic plots shown in Figs. 1–4. b Incubation in the absence of ATP, T. KS-1 cpn, BSA and GroEL/ES.
little higher than that in the absence of ATP. From the semilogarithmic plots shown in Figs. 1–3, these inactivations can be considered first-order inactivations. Therefore, the half-life (t1/2) of ADH, urease and MDH, indicating the time when the residual activity reaches 50%, was determined from the semilogarithmic plots shown in Figs. 1–3. Table 1 shows the half-life of ADH, urease and MDH at several temperatures. In all cases, T. KS-1 cpn increased the halflife of these enzymes, and the addition of ATP in the presence of T. KS-1 cpn further increased it. These results indicate that T. KS-1 α and β cpns significantly stabilized mesophilic and thermophilic free enzymes in the presence of ATP. Long-term stabilization of free ADH at lower temperatures Considering that most enzymes are used at moderate temperature in bioreactors and stored at 4°C or −20°C, it was considered interesting to determine whether chaperonins have a long-term stabilization effect at the lower temperatures used for reaction and storage rather than the shortterm stabilization effect at high temperatures. Therefore, the ADH stabilization effect of T. KS-1 cpn at lower temperatures was examined. ADH activity was completely lost after a 4-d incubation at 30°C (Fig. 4a). T. KS-1 cpn and E. coli GroEL/ES slightly stabilized ADH in the absence of ATP. ATP also prevented the inactivation of ADH. T. KS-1 cpn significantly stabilized ADH at 30°C in the presence of ATP, and the effect of T. KS-1 α cpn was higher than that of T. KS-1 β cpn. The stabilization effects of T. KS-1 cpns were lower than that of E. coli GroEL/ES in the presence of ATP. In the case of BSA, the effect of the addition of ATP was not as significant as in the case of chaperonin. When ADH was stored at 4°C (Fig. 4b), the stabilization effect of BSA was higher than those of chaperonins in both the presence and absence of ATP. In the presence of ATP, the stabilization effects of T. KS-1 cpn and GroEL/ES at 4°C were less than those at 30°C. The half-life of ADH at 30°C and 4°C was increased by the addition of T. KS-1 cpn and GroEL/ES, and the addition of ATP in the presence of chaperonin further increased it (Table 1). These results indicate that T. KS-1 cpn has a stabilizing effect on ADH at low and high temperatures.
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FIG. 5. Storage stability of chaperonin co-immobilized ADH at 4°C and pH 7.0. The final concentrations of ADH, BSA, T. KS-1 α cpn and T. KS-1 β cpn are 0.020 µmol/g-gel, 0.040 µmol/g-gel, 0.040 µmol/g-gel and 0.040 µmol/g-gel, respectively. The residual activity is calculated as the percentage of the immobilized enzyme activity stored for the indicated period relative to that stored for 1 d after preparation. Circles, ADH-alone gel beads; diamonds, ADH-BSA gel beads; squares, ADH-T. KS-1 α cpn gel beads; triangles, ADH-T. KS-1 β cpn gel beads.
FIG. 4. Long-term stability of free yeast ADH at 30°C (a) and 4°C (b) at pH 7.8. The final concentrations of ADH, BSA, T. KS-1 cpn and GroEL/ES are 20 nM, 100 nM, 100 nM and 100 nM, respectively. Closed and open symbols indicate the incubation in the absence and presence of 5 mM ATP, respectively. Circles, Incubation in the absence of T. KS-1 cpn, GroEL/ES and BSA; diamonds, incubation in the presence of BSA; squares, incubation in the presence of T. KS-1 α cpn; triangles, incubation in the presence of T. KS-1 β cpn; reversed triangles, incubation in the presence of GroEL/ES.
Stabilization effect of chaperonin for gel-entrapped ADH In this study, four types of calcium alginate-entrapped gel beads were prepared: entrapped ADH (ADHalone) gel beads, ADH co-entrapped with T. KS-1 α cpn (ADH-T. KS-1 α cpn) gel beads, ADH co-entrapped with T. KS-1 β cpn (ADH-T. KS-1 β cpn) gel beads and ADH coentrapped with BSA (ADH-BSA) gel beads. The calcium alginate gel beads prepared in this study were 2.5–3.5 mm in diameter. The leakage of ADH or T. KS-1 cpn from these gel beads was not observed (data not shown). Then, when these gel beads were stored at 4°C, the stabilization effect of T. KS-1 cpn for entrapped ADH was investigated (Fig. 5). The residual ADH activity of ADH-alone gel beads decreased to 15% after 5 d at 4°C. One day following the gel preparation, the ADH activities of ADH-BSA, and ADH-T. KS-1 α or β cpn gel beads were 1.3-fold higher than that of ADH-alone gel beads (data not shown). BSA and T. KS-1 cpn prevented the inactivation of entrapped ADH for 5 d at 4°C. The inactivation rates of ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were lower than that of ADH-BSA gel beads even in the absence of ATP. After 5 d,
the residual ADH activities of ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were maintained at 72% and 77%, respectively. When calcium alginate entrapped ADHalone, ADH-T. KS-1 α cpn and ADH-T. KS-1 β cpn gel beads were used repeatedly for the measurement of ADH activity, the residual ADH activities decreased to 35%, 45% and 35%, respectively, after 5 d (data not shown). This result indicates that T. KS-1 cpn stabilized the entrapped ADH as well as the free ADH. DISCUSSION T. KS-1 α and β cpns expressed in E. coli cells contained a large amount of monomer after the heat treatment purification (8). In this study, the average weight percentage of the T. KS-1 α and β 16-mer were 56.4 and 23.4% (16-mer/ total chaperonin [w/w]), respectively. It has been shown that T. KS-1 cpn 16-mer facilitated the refolding of proteins in an ATP-dependent manner (5, 6, 20), and the T. KS-1 cpn monomer facilitated the refolding of proteins even in the absence of ATP (8). Because the hyperthermophilic group II chaperonin from S. solfataricus as well as the thermophilic group I chaperonin from Bacillus strain MS facilitated protein refolding and prevented the thermal inactivation of enzymes (3, 9, 13), it was expected that T. KS-1 cpn would also be effective for the prevention of the thermal inactivation of enzymes. Therefore, we used T. KS-1 cpn containing the 16-mer and the monomer for the stabilization of enzymes. T. KS-1 cpn prevented the thermal inactivation of mesophilic and thermophilic enzymes at several temperatures higher than 50°C in the presence of ATP (Figs. 1–3). These results are consistent with those of previous studies (13, 15). Although T. KS-1 α cpn stabilized ADH better than T. KS-1 β cpn at 50°C, T. KS-1 β cpn stabilized urease better than T. KS-1 α cpn at 65°C. Moreover, T. KS-1 α cpn stabilized
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MDH to the same degree as T. KS-1 β cpn at 85°C. The ATPase activities of the T. KS-1 α and β cpn 16-mers were almost the same from 40°C to 70°C, and the ATPase activity of the T. KS-1 β cpn 16-mer was higher than that of the T. KS-1 α cpn 16-mer at temperatures higher than 70°C (6, 21). Therefore, the difference in the stabilization effect between the T. KS-1 α and β cpns is probably due to the difference in not only the ATPase activity of T. KS-1 cpn but also the characteristics of target enzymes. T. KS-1 cpn also prevented the thermal inactivation of urease and MDH even in the absence of ATP (Figs. 2 and 3). It was reported that other hyperthermophilic group II chaperonins stabilized enzymes at various temperatures in the absence of ATP (13, 15). Our results are consistent with those of previous studies. Considering that the T. KS-1 cpn 16-mer exhibits ATPase activity at temperatures higher than 50°C, this stabilization effect is probably due to the T. KS-1 cpn monomer. Therefore, it is assumed that the stabilization effect at high temperatures, at which T. KS-1 cpn expresses ATPase activity, is due to both the ATP-dependent effect of the T. KS-1 cpn 16-mer and the ATP-independent effect of the T. KS-1 cpn monomer. It has been shown that some group I chaperonins enhanced the thermal stability of several enzymes at various temperatures (9–12). Our results suggest that group II chaperonins as well as group I chaperonins are effective for the stabilization of enzymes. In particular, it will be of economic advantage not to require ATP for the heat stabilization of enzymes due to the use of T. KS-1 cpn. It should be investigated whether the T. KS-1 cpn 16-mer or monomer is more suitable for the stabilization of enzymes. The stabilization properties were dependent on incubation temperature. T. KS-1 cpn had high stabilization effects at 30°C and 4°C as well as 50°C in the presence of ATP (Figs. 1 and 4). From previous results (6, 21), it is presumed that the T. KS-1 cpn 16-mer has no detectable ATPase activity at 30°C and 4°C. Furthermore, the T. KS-1 cpn monomer had no detectable ATPase activity (21). Therefore, it is assumed that these stabilization effects at 30°C and 4°C are not due to the T. KS-1 cpn 16-mer but due to the T. KS-1 cpn monomer. Although the T. KS-1 cpn 16-mer and monomer have no detectable ATPase activities at 30°C and 4°C, apparent ATP dependency was observed. This is presumed to be caused by the stabilization effect of ATP on ADH, and then ADH was synergistically stabilized by ATP and T. KS-1 cpn, probably by the T. KS-1 cpn monomer. Because GroEL/ES had ATPase activity at 30°C (22), the stabilization effect of GroEL/ES was higher than that of T. KS-1 cpn. It has been reported that the ATPase activity of GroEL at 10°C was lower than at 25°C (23). Therefore, it was assumed that the ADH stabilization effect of T. KS-1 cpn or GroEL/ES at 4°C was lower than that at 30°C. From these results, it is assumed that the stabilization effect at lower temperatures, at which T. KS-1 cpn has no detectable ATPase activity, is mainly due to the ATP-independent effect of the T. KS-1 cpn monomer. BSA or T. KS-1 cpn co-entrapped with ADH increased the ADH activity (data not shown). This is probably because BSA and T. KS-1 cpn prevent the inactivation of ADH during immobilization. Although both BSA and T.
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KS-1 cpn prevented the inactivation of ADH in the gel matrix for 5 d at 4°C, the inactivation rate of ADH-T. KS-1 cpn gel beads was lower than that of ADH-BSA gel beads (Fig. 5). Therefore, T. KS-1 cpn is more efficient for the stabilization of immobilized enzymes than BSA. Although T. KS-1 cpn prevented the inactivation of ADH in the gel matrix in the absence of ATP, this stabilization effect was not observed in free ADH (Fig. 4b). It is thought that the function of T. KS-1 cpn 16-mer is inhibited in the gel matrix at 4°C for the reasons outlined above. Therefore, it is assumed that the T. KS-1 cpn monomer, which locally concentrated in the gel matrix, interacted with ADH more strongly in the gel matrix than in the solution, and then prevented the inactivation of ADH in the gel matrix. Although T. KS-1 cpn was efficient for the stabilization of immobilized enzymes, the ADH activity of ADH-T. KS-1 cpn gel beads decreased during repeated use (data not shown). Immobilized ADH was assayed in 100 mM glycine–KOH buffer (pH 9.5). Although the highest activity of ADH was observed at around pH 9, ADH stability was not very high at around this pH. From these results, it is assumed that ADH was inactivated at this pH and T. KS-1 cpn did not stabilize ADH at this pH. Our results show that T. KS-1 cpn can be used for the stabilization of free and entrapped enzymes. In a previous study, although covalently immobilized recombinant human HSP70 onto surface modified glass with silane was able to reactivate the thermally denatured firefly luciferase, HSP70 co-immobilized luciferase did not exhibit increased thermal stability (24). Therefore, entrapment immobilization is suitable for the stabilization of immobilized enzymes by the co-immobilization of chaperonin and enzymes because the degree of interaction between enzymes and chaperonin is higher than that which occurs in other immobilization strategies and because this method does not involve chemical bonds between proteins and support materials. In particular, calcium alginate gel entrapment is a more suitable method than other entrapment methods, because other gel entrapment methods involve some factors contributing to the inactivation such as high gelation temperatures, the heat of polymerization and radicals. When an entrapped ADH gel was prepared with agar gel, ADH activity was not observed even after 1 d (data not shown). To apply hyperthermophilic chaperonins to various fields of biotechnology, the efficiency of chaperonins in the stabilization of immobilized enzymes should be investigated for other immobilization methods such as covalent immobilization and physical adsorption. ACKNOWLEDGMENTS We are grateful to Dr. T. Yoshida and Dr. T. Maruyama, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), and Prof. M. Yohda, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology for providing the chaperonin expression plasmid and helpful discussion. This work was supported by the Hiroshima City University Grant for Special Academic Research (General Studies).
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