Biotechnology Education How to Calculate Thermostability of Enzymes Using a Simple Approach
Abdul A. N. Saqib †* Khawar S. Siddiqui‡
From the †Green Biologics Ltd, Milton Park, Abingdon, Oxfordshire, OX14 4SD, United Kingdom, ‡Life Sciences Department, King Fahd University of Petroleum & Minerals, Dhahran, Saudi Arabia
Abstract Determination of thermostability of enzymes is of prime importance for their successful industrial applications and, yet, the published data has often been incompletely analyzed to assess the suitability of enzymes. It is possible to determine meaningful thermostability parameters from the routinely acquired data through a straightforward method that is not only more informative but also provides a means to compare thermostability of enzymes from different sources. Here, we describe a simple, effective, and economical way to determine enzyme thermostability. In our
opinion, including this method in Biochemistry and Molecular Biology curricula will encourage students to include thermostability analysis in their future work, leading to a more meaningful approach to evaluate and compare enzymes. Furthermore, as the method requires minimum specialized equipment, the analysis will be particularly suitC 2018 by able for labs that cannot afford expensive setup. V The International Union of Biochemistry and Molecular Biology, 46:398–402, 2018.
Keywords: Biochemistry; enzymology; thermostability; biotechnology education; activation thermodynamics
Introduction Many industrial processes run at elevated temperatures for long periods of times require thermostable enzymes. For example, corn-based bioprocesses require a-amylases to typically stand >808C for 1–2 hr and glucoamylases for few hours at >658C. A designed hybrid a-amylase is intended to work at even higher temperatures (95 to 1608C) for 1–2 hr [1]. Amylases are also used at high temperatures in fabric desizing as well as in oil and gas drilling. Immobilized glucose isomerases used in High Fructose Syrup production sustain 55 to 608C for days and need to be replaced once their activities drop by 10–15% of the initial levels. In addition, there is a search for thermostable isoamylases that are cost-effective for certain corn-based bioprocesses [2]. The technology in cellulosic bioprocesses requires hydrolysis to be carried out for up to 72 hr at >508C [3]. Enzymes Volume 46, Number 4, July/August 2018, Pages 398–402 *To whom correspondence should be addressed. Tel.: 144-7939-260599. E-mail:
[email protected] Received 29 September 2017; Revised 8 February 2018; Accepted 8 April 2018 DOI 10.1002/bmb.21127 Published online 2 May 2018 in Wiley Online Library (wileyonlinelibrary.com)
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can be highly active at high temperatures at the expense of reduced stability due to activity-stability trade-off [4]. Therefore, activity and stability parameters become complementary to each other when it comes to assessing an enzyme’s suitability for industrial processes, because neither of these would be very practicable on its own. This clearly demonstrates the need to accurately assess the thermostability of an enzyme along with its activity. Thermostability of an enzyme refers to its resistance to attain irreversibly denatured form. A schematic energy diagram depicting the thermal denaturation of enzymes is shown in Fig. 1, in which the direction of the reaction is determined by the free energy level of the reactants, that is, different states of enzyme structure. The denaturation process proceeds through an unstable intermediate transition (or activated) state, INT#, which is in equilibrium with the native enzyme (N) at a given temperature and, therefore, can fold back to N upon cooling [5]. Thermal denaturation of enzymes is a first order reaction, that is, its rate (kIN) depends on the concentration of only one species, which is INT# in this case. As the temperature is increased, the proportion of native enzyme (N) going into the transition state (INT#) increases, which leads to increased rate of irreversible denaturation (kIN); the depletion of INT# drives the equilibrium forward towards INT#. In other words, as the temperature is increased, the concentration of D would
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FIG 1
Schematic representation of the energy changes during thermal denaturation of enzymes: (Top). Structural depiction of the thermal unfolding process leading to enzyme denaturation (D) and showing equilibrium between the native (N) and the transition (INT# ) structures of an enzyme. (Bottom) Energy diagram showing that a minimum amount of activation energy (DG# ) must be supplied to the native enzyme (N) so that the unstable activated intermediate transition state (INT# ) is achieved that is in equilibrium with the N (N ! INT# ). Note that D has the lowest free energy as the protein unfolds spontaneously with temperature. At a given temperature, a certain proportion of INT# is then converted into the fully denatured enzyme (D). The solid curve represents the denaturation of a more thermostable enzyme, whereas the dotted curve represents a less thermostable enzyme (From Ortbauer, M. Abiotic Stress Adaptation: Protein Folding Stability and Dynamics, In: Vahdati, K. and Leslie, C., Eds., Abiotic Stress - Plant Responses and Applications in Agriculture, InTech Open Access Publisher, DOI: 10.5772/45842). [Color figure can be viewed at wileyonlinelibrary.com]
increase, leaving behind less enzyme in the N INT# equilibrium, which can revert back to N upon cooling and contribute to Amin (Fig. 2A). An enzyme can denature irreversibly to “D” if the unfolded protein undergoes some permanent change such as aggregation due to intermolecular hydrophobic interactions upon unfolding, chemical degradation, subunit dissociation, or disulfide-bridges exchange. This enzyme denaturation scheme can be represented as: kIN N INT# !D
Enzymes are proteins and a number of techniques are employed to determine the thermostability of proteins. Differential Scanning Calorimetry is an excellent method because the enthalpy of unfolding of a protein is directly
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determined, in addition to other parameters such as rate constants of thermal denaturation (kd) and melting temperature (Tm). Thermostability of proteins may also be monitored using far-UV circular dichroism (CD) for the unfolding of secondary structure whereas near-UV CD and Trp/Tyr fluorescence is used to determine the unfolding of tertiary structure [6]. A major limitation of the spectrometric and calorimetric techniques, however, is the absence of enzyme activity from the data, which does not give information regarding activity-stability relationship. Indeed, it was noticed that CD of an exo-glucanase enzyme was unchanged at both near- and far-UV wavelengths even though 90% of the enzyme activity was lost under various experimental conditions leading to a molten-globule state [7]. A MG state is an intermediate state that has lost majority of tertiary structure but retains significant amount of secondary structure [8]. Moreover, the active site of an enzyme may unfold first and, thus, loose its activity prior to unfolding of the rest of the protein structure [4, 5], which may result in significant differences between the protein’s melting temperature (Tm) and the temperature of maximum activity (Topt) – the difference being farther apart for psychrophilic and mesophilic enzymes than thermophilic ones [4]. Therefore, it is essential to base the thermostability assay of enzymes on the reaction catalyzed rather than simply on structural changes. A simpler and inexpensive method is transverse urea-gradient-PAGE (TUG-PAGE); however, it is more suitable for small single domain proteins that unfold reversibly rather than large multidomain enzymes that generally unfolds irreversibly [9]. It is possible to determine thermostability based on the unfolding of the active-site of an enzyme through activity assays performed at various temperatures. This is especially useful since high concentrations of substrates may stabilize the enzyme [10]. Mathematical models have been proposed to perform the stability analysis coupled with activity measurements [5, 11], but require either good mathematical skills or the use of specialized software and have certain limitations. An alternative method is heating the enzyme at different temperatures followed by measuring the residual activity; and due to its ease, this is the method of choice for many enzymologists and is generally reported as a part of wider studies [2]. The data on thermostability, however, is not always fully analyzed making it difficult to compare results from different studies. It is possible to extract clear-cut parameters from the routinely published thermostability data and present them in some standard format that allows meaningful comparison of thermostability between two enzymes [4]. In this article, we show a simple and easy-to-follow method for calculating various thermostability parameters and describe their suitability and significance. The easy-to-follow approach will make it possible to adopt it as a part of biochemistry and molecular biology curricula or even as a quick reference for researchers working with enzymes. The method can be
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FIG 2
Determination of thermostability parameters: The enzyme (endoglucanase in this example) was incubated at three different temperatures, 40 8C (D), 50 8C (䉱) and 60 8C (O) and samples were drawn at various time intervals for measurement of residual activity (Ares). A, Reduction in the proportion of Ares compared with native activity (A0) over time. Amin is the minimum activity at a given temperature that corresponds to the enzyme in the equilibrium state (N ! INT# ), which represents the total amount of active enzyme upon cooling. B, Estimation of melting temperature (Tm) that corresponds to the temperature when Amin drops down to 50% of the native activity (A0). C, First order plot of the data represented in figure A to obtain the rates of thermal inactivation, kIN (s21). D, Arrhenius plot of the ln kIN (figure C) vs 1/T, the slope gives the activation energy of denaturation, Ea# (J mol21). The Arrhenius plot may be curved under certain circumstances (see text for details). The error bars represent standard deviation of mean of duplicate measurements.
easily applied to convert the generally published basic stability data into key parameters and, as such, provides a powerful tool without the need of advanced mathematical background or the use of specialized equipment or software.
Method The method described below can be applied to any enzyme for which an assay is available. Therefore, the description has been kept generic rather than narrowing it down to a specific enzyme, because the intention of the article is to encourage teachers and students to apply it to any enzyme of their interest. A known amount of the enzyme is incubated, in the absence of substrate, at a range of temperatures, generally 30 to 908C (depending upon whether the enzyme is psychrophilic, mesophilic, or thermophilic), with an increment of 5–108C. If a cofactor stabilizes the enzyme structure [1, 5, 12] and is added during the actual (industrial) process, it may be necessary to add it during the thermostability analysis. The required duration of heat treatment varies depending on the temperature and the enzyme itself, but in all cases, it is carried out until a constant residual activity
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(Amin) is attained (Fig. 2A). During the heat treatment, the enzyme samples are drawn at various time intervals and immediately cooled by placing them on ice for 1–3 hr (note that the earlier samples will stay longer on ice). This step ensures that the temperature of all samples is similar and low (4–68C) for a sufficient length of time (minimum 1 hr) during which the reversibly denatured protein (INT#) is converted back to native active form (N) while the irreversibly denatured form (D) remains inactive. The interval at which enzyme samples are drawn is arbitrary, but should be designed to capture the initial drop in activity during thermal treatment. The residual activities of the drawn enzyme samples are then measured and plotted against time (Fig. 2A). As with any standard enzyme assay, the substrate and other reaction solutions, except enzyme, are equilibrated to the required temperature when enzyme activity is measured. The addition of cold enzyme (typically
