Letter Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Stabilization of Multimeric Enzymes against Heat Inactivation by Chitosan-graf t-poly(N‑isopropylacrylamide) in Confined Spaces Qian Tao,† Ang Li,‡ Zhenkun Zhang,‡ Rujiang Ma,‡ and Linqi Shi*,†,§ †
School of Chemistry and Materials Science, Ludong University, Yantai 264025, China State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, and §Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China
‡
S Supporting Information *
ABSTRACT: The inactivation of multimeric enzymes is a more complicated process compared with that of monomeric enzymes. Stabilization of multimeric enzymes is regarded as a challenge with practical values in enzyme technology. Temperature-sensitive copolymer chitosan-graf t- poly(Nisopropylacrylamide) was synthesized and encapsulated with multimeric enzymes in the confined spaces constructed by the W/O microemulsion. In this way, the quaternary structures of multimeric enzymes are stabilized and the thermal stabilities of them are enhanced. The whole process was studied and discussed. This method, which works well for both glucose oxidase and catalase, can be developed as a general protection strategy for multimeric enzymes. KEYWORDS: thermal stability, multimeric enzyme, reversible thermoresponse, confined space
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there are some limitations of these methods. The medium engineering method usually only works for some specific enzymes. The stability of genetically engineered enzymes under normal conditions is unpredictable. The reaction conditions and agents of the cross-linking between subunits need to be strictly selected, in order to avoid irreversible damage for the enzyme. Therefore, a more convenient, mild, and general system for stabilizing multimeric enzymes is highly desired. We think that an effective multimeric enzyme stabilizing system can be simplified by isolating the enzyme molecules from each other. In this way, it only needs to consider the subunit dissociation of each enzyme molecule. Even though the dissociation occurs, it is difficult for the detached subunits to meet the subunits of other enzyme molecules, which may result in more complex structure changes and irreversible inactivation. The water in oil (W/O) microemulsions have been used as confined spaces in studies of the protein folding.22,23 Proteins can be easily encapsulated in the inner aqueous phases and properties of the confined spaces are highly tunable. Only one enzyme molecule in one droplet can be achieved by adjusting the concentration of enzyme to make the number of enzyme molecules is less than that of droplets. However, microemulsions alone are not efficient to completely isolate multimeric enzymes because the detached
long with the rapid developments of enzyme technology, inactivation becomes a main obstacle that hinders enzymes to be fully exploited as biocatalysts. Lots of methods have been devised to stabilize enzymes against inactivation and proved their effectiveness for certain specific enzymes, especially the monomeric ones. However, many enzymes with practical applications are the multimeric enzymes (such as catalase,1 oxidase,2,3 dehydrogenase,4,5 aldolase,6,7 etc.), which consist of several peptide subunits and cofactors in some cases. The inactivation procedure of multimeric enzymes is much more complicated and difficult to be prevented through the methods aimed at monomeric enzymes. The current means to stabilize monomeric enzymes focused on maintaining the native tertiary structure or avoiding the wrong folding of secondary structure.8,9 The quaternary structures of multimeric enzymes were not concern in these methods. Therefore, stabilization of multimeric enzymes against inactivation is considered as a particular challenge. Inactivation of multimeric enzymes under severe conditions is mainly related to the changes of their quaternary structures. In the inactivation of most multimeric enzymes, the first step is the dissociation of subunits, followed by distortion of the subunits.10,11 Hence, preventing the dissociation of subunits has been recognized as an important strategy to stabilize multimeric enzymes.12 Some methods based on this idea have been reported. The subunit dissociation of some multimeric enzymes can be avoided through medium engineering,13−16 protein engineering,17,18 chemical cross-linking,19−21 etc. Nevertheless, © XXXX American Chemical Society
Received: October 10, 2017 Accepted: November 28, 2017 Published: November 28, 2017 A
DOI: 10.1021/acsbiomaterials.7b00764 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering subunits may contact with others during the collision among microemulsion droplets. Hence, a “lock” is required to prevent the confined space against the content exchanges, and even lock the quaternary structure of the enzyme to avoid the subunit dissociation. This locking effect is best to be taken only under the denaturation conditions (e.g., high temperature), so as not to interfere the normal enzyme activity. Therefore, it is required locked at high temperature and unlocked at room temperature. Thermoresponsive networks are ideal candidates to fulfill this goal. If it is introduced in the aqueous phase of microemulsions, we think that an effective strategy for stabilizing multimeric enzymes against heat inactivation can be devised. The network with reversible thermoresponse was implemented by chitosan grafted with the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). By employing the graft copolymer chitosan-g-PNIPAAm as the “lock”, we developed a method for protecting multimeric enzymes against heat inactivation (Scheme 1). Mixed solutions of
Figure 1. Evolutions of the storage modulus (G′) and loss modulus (G′’) of chitosan-g-PNIPAAm in MES solution with temperature increases. The inset shows chitosan-g-PNIPAAm in (a, b) MES and (c, d) PBS solutions with the change of temperature. The photographs were taken (a, c) at room temperature or (b, d) immediately after heating at 60 °C. The concentrations of chitosan-g-PNIPAAm were 100 mg mL−1.
Scheme 1. Schematic Illustration of (a) the Subunit Dissociation and Inactivation of Multimeric Enzyme and (b) Stabilization of Multimeric Enzyme by Chitosan-gPNIPAAm Confined in W/O Microemulsion
solutions show that it is a flowing liquid at room temperature, but a soft solid after heating. And this transition is reversible and nearly the same between MES and PBS solution. These changes of chitosan-g-PNIPAAm were in agreement with those in refs 24−26, which is qualified for constructing the thermoresponsive and reversible network. Glucose oxidase (GOD) and catalase are typical multimeric enzymes and are composed of 2 and 4 subunits, respectively. Both of them were used to study the efficiency of the above method to enhance the stability of multimeric enzymes. Microemulsions containing only enzymes and those containing both enzymes and chitosan-g-PNIPAAm were incubated at the denaturation temperature for 30 min and then kept at 4 °C to renature. The heat treatments were carried out at different temperatures in consideration of both enzymatic species and application conditions. The activities of renatured enzymes were assayed and compared with the activities of native enzymes under the identical conditions (Figure 2). The results show that the residual activities of the enzymes in the presence of chitosan-g-PNIPAAm are higher than that of the enzymes alone. In the case of GOD, 62.4 and 48.9% of enzyme can be renatured after heated at 65 and 70 °C for 30 min, respectively, by only encapsulated in the microemulsions. However, in the presence of chitosan-g-PNIPAAm, 90.0 and 78.0% of GOD can be renatured after heated at 65 and 70 °C for 30 min, respectively. It is affirmed that this method is efficient for stabilizing GOD on heating. Even for catalase with four subunits, 21.1 and 45.1% of enzyme can be renatured without and with chitosan-g-PNIPAAm, respectively, after heating at 50 °C for 30 min. These results demonstrate that the thermal stabilities of both GOD and catalase can be improved through encapsulating them in confined spaces with chitosan-gPNIPAAm. In the same experiments performed in MES solutions, it is found that the residual enzymatic activities in solutions are much less than those in microemulsions (Figure S3). Without the confinement effect of the microemulsion, only 18.9 and 7.36% of GOD can be renatured after heated at 65 and 70 °C, respectively. Even adding chitosan-g-PNIPAAm into the MES solutions, the renatured rates have no significant improvement. Obviously, the confined space constructed by microemulsion is a key to the success of this method, which is consistent with our
multimeric enzymes and chitosan-g-PNIPAAm were used as the water phase to prepare W/O microemulsions, making multimeric enzymes coexist with the copolymers in the confined droplets. Upon heating, polymeric networks were formed because of the junction of hydrophobic PNIPAAm, which would play the “lock” role. With cooling, PNIPAAm became hydrophilic and the network was dissolved in the water droplet. In this way, chitosan-g-PNIPAAm spontaneously carries out the enzyme stabilization function according to the change of the environmental temperature. The thermoresponse of chitosan-g-PNIPAAm was studied first. The transmittances of chitosan-g-PNIPAAm in MES (20 mM, pH = 5.5) and PBS (10 mM, pH = 7.5) are almost 100% at 25 °C, and decrease sharply when heated to 31.5 °C. The lower critical solution temperature (LCST) of the chitosan-gPNIPAAm solution nearly the same at these two different pH values and has a value of 32.5 °C approximately (Figure S2). The phase transition was characterized by rheological measurements. At the temperature less than LCST, the loss modulus (G′’) is larger than the storage modulus (G′), both of which are independent of the temperature, indicating a liquid system. With the temperature increasing, both G′ and G′’ increase. However, the increase of G′ is faster than that of G′’ and eventually larger than the latter, suggesting that the structure was affected by the hydrophobicity of PNIPAAm above the LCST (Figure 1). The photographs of chitosan-g-PNIPAAm B
DOI: 10.1021/acsbiomaterials.7b00764 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering
Figure 2. (a) Residual enzymatic activity of free GOD and GOD with chitosan-g-PNIPAAm (C-g-PN) in microemulsion after heat treatments. The concentrations of GOD and chitosan-g-PNIPAAm were 0.012 and 3.0 mg mL−1, respectively. The heat treatment was carried out at 65 or 70 °C for 30 min. (b) Residual enzymatic activity of free catalase and catalase with chitosan-g-PNIPAAm in microemulsion after heat treatments. The concentrations of catalase and chitosan-g-PNIPAAm were 0.0014 and 0.75 mg mL−1, respectively. The heat treatment was carried out at 50 or 55 °C for 30 min. Error bars denote standard deviation (SD) values over three different experiments.
Figure 3. FAD fluorescence spectra of free GOD in microemulsion (a) and GOD with chitosan-g-PNIPAAm in microemulsion (b) in different conditions (N, native; D, denatured at 70 °C for 30 min and measured immediately at 70 °C; R, after being renatured for 12 h). The concentrations of GOD and chitosan-g-PNIPAAm were 0.012 and 3.0 mg mL−1, respectively.
previous results. 27,28 Without the confined space, the dissociated subunits that could have been assembled into a correct quaternary structure may be far away from each other and cannot gather together on renaturaton. Similarly, the polymeric network is difficult to lock the enzyme structure, which would greatly reduce the efficiency of stabilization. Homopolymer PNIPAAm was proved effective on stabilizing monomeric enzymes through the hydrophobic interaction with unfolded enzymes upon heating.27,28 The side chain of chitosan-g-PNIPAAm may also interact with the unfolded multimeric enzymes, but have to do that after the enzyme dissociation. It is less efficient than preventing the subunits dissociation, which is discussed in the beginning and confirmed by the enzymatic activity assays (Figure S4). The interaction between PNIPAAm and enzyme enhances the stability, but is not a complete explanation herein. To fully account for the mechanism of this method, we make two assumptions for explaining the stabilization effects on multimeric enzymes. First, the confinement effect of microemulsion is strengthened by the chitosan-g-PNIPAAm network at high temperature, which blocks the interactions among the detached subunits of different droplets. At the same time, the subunits of the same multimeric enzyme are fixed in one droplet and can selfreassemble into the correct quaternary structure with cooling. Second, the quaternary structure of enzyme is “locked” by the
polymeric network at high temperature. With temperature increase, the PNIPAAm part of the graft copolymer becomes hydrophobic and interacts with that of other copolymer molecule. The formed network coats on the enzyme, making the subunits hard to dissociate even though the interactions among subunits are weak on heating. The significant difference between the two assumptions is whether the quaternary structure of enzyme is changed. Therefore, the quaternary structure changes were studied to confirm which assumption is correct or plays the dominate role. The quaternary structure changes of GOD can be conveniently detected by taking advantage of the FAD (flavin adenine dinucleotide) fluorescence, because there is one FAD as the cofactor in each subunit of GOD. The FAD fluorescence is sensitive to variation in the surrounding environment and quenched under the native state while being enhanced on denaturation because the FAD molecules leave each other during the subunits dissociation.29,30 First, the FAD fluorescence of GOD in aqueous solution was measured under three different states (native, denatured and renatured) (Figure S5). The fluorescence increases significantly after heating and remains unchanged after renaturation for 12 h at 4 °C, which demonstrates that the subunits of GOD dissociate at high temperature and the change of quaternary structure is irreversible in aqueous solution. In the next, the microC
DOI: 10.1021/acsbiomaterials.7b00764 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
ACS Biomaterials Science & Engineering
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emulsions (GOD@E and GOD/chitosan-g-PNIPAAm@E) were studied by the same way (Figure 3). In the absence of chitosan-g-PNIPAAm, the FAD fluorescence increases on heating but falls back to certain degree after renaturation (Figure 3a). These results well explain the differences of renatured rates between GOD in MES solution and that in microemulsion. The confined space cannot prevent the subunit dissociation, but help the subunit to regain correct quaternary structure, at least partially. Some of the subunits that dissociate at high temperature reassemble during cooling. It is similar to the first assumption above, and microemulsion can play the role by itself. The FAD fluorescence intensity of GOD/chitosan-gPNIPAAm@E also increases on heating, but the increment is significantly less than that of GOD@E. This means quite a few GOD molecules keep the original quaternary structure during heating when chitosan-g-PNIPAAm exists. Thus, the second assumption, “locking” the quaternary structure, is verified. Moreover, the fluorescence intensity also decreases on renaturation. This structure recovery is probably caused by the confined space, based on the previous discussion. Therefore, the mechanism of this method for stabilizing multimeric enzymes can be summarized that chitosan-gPNIPAAm at high temperature “locks” the quaternary structure to prevent the subunit dissociation, and the confinement effect of the microemulsion helps the denatured enzyme recover the correct structure. The confined space is essential for taking effect, while chitosan-g-PNIPAAm ensures high efficiency. These two ways work synergistically, efficiently improving the thermal stabilities of the multimeric enzymes. For monomeric enzymes, this method is also effective. A similar experiment performed on lipase shows that chitosan-gPNIPAAm can protect lipase against heat inactivation and the protecting efficiency is higher than that using homopolymer PNIPAAm (Figure S6). PNIPAAm forms a complex with lipase via hydrophobic interaction to avoid the aggregation of lipase molecules, as discussed in our previous work.27 Besides the same function as PNIPAAm, chitosan-g-PNIPAAm also forms a network coating on lipase to protect the whole structure. Thus, the efficiency for stabilizing monomeric enzymes is enhanced. Moreover, the effect of chitosan was also evaluated. The residual enzymatic activity of lipase/chitosan@E is higher than that of lipase@E, which can be explained by the increased viscosity of the water droplets because of the high molecular weight of chitosan (Figure S6). However, it is much lower than that of lipase/PNIPAAm@E and lipase/C-g-PN@E. It is believed that chitosan alone has less effect on the improved enzymatic stability than PNIPAAm or chitosan-g-PNIPAAm. In conclusion, a novel method is devised to stabilize multimeric enzymes against heat inactivation, utilizing the reversible thermoresponse of copolymer chitosan-g-PNIPAAm and the confined space of microemulsions. It is proved to be effective and general. The mechanism is also discussed. With heating, chitosan-g-PNIPAAm forms network and coats on the enzymes to prevent the subunit dissociation. With cooling, the copolymer becomes hydrophilic and the enzymes regain their activities. Besides extending the applications of multimeric enzymes, this study may provide some new perspectives for stabilizing the multilevel structures of enzymes.
Letter
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00764. Detailed materials, methods, and additional experimental results (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhenkun Zhang: 0000-0002-3480-2381 Rujiang Ma: 0000-0002-2021-3850 Linqi Shi: 0000-0002-9534-795X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51403096, 91527306, and 51503104) and the Natural Science Foundation of Tianjin, China (16JCQNJC03000).
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REFERENCES
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DOI: 10.1021/acsbiomaterials.7b00764 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
