CRITICAL REVIEWS IN BIOTECHNOLOGY, 2018 VOL. 38, NO. 3, 335–350 https://doi.org/10.1080/07388551.2017.1355294 REVIEW ARTICLE Practical insights on enzyme stabilization aà aà b c a,b Carla Silva , Madalena Martins , Su Jing , Jiajia Fu and Artur Cavaco-Paulo aCentre of Biological Engineering (CEB), University of Minho, Braga, Portugal; bInternational Joint Research Laboratory for Textile and Fiber Bioprocesses, Jiangnan University, Wuxi, China; cKey Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China ABSTRACT ARTICLE HISTORY Enzymes are efficient catalysts designed by nature to work in physiological environments of living Received 9 November 2016 systems. The best operational conditions to access and convert substrates at the industrial level Revised 4 January 2017 are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles Accepted 17 May 2017 can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in indus- KEYWORDS trial processes which require the use of extreme environments that can undergo enzyme instabil- Stabilization; enzymes; ity. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a formulation; molecular practical approach. This review intends to compile and describe the most used approaches for interactions; industrial enzyme stabilization highlighting case studies in a practical point of view. catalysis Industrial demands of enzymes Enzyme properties and the need of stabilization strategies Major applications of enzymes are in industries of deter- gents, food processing, animal nutrition, juice and fla- Enzymes are protein molecules consisting of folded vorings, cosmetics, medication, pharmaceuticals, polypeptide chains of amino acids that are essential to leather, silk, chemical, and for research and develop- perform an array of biological functions. The order of ment [1,2]. The industrial applications are only feasible these amino acids in a protein determines its tertiary if catalysts are stabilized against temperature, extreme structure through molecular geometry and intramolecu- pH, and in the presence of alkalis, acids, salts, and sur- lar chemical interactions [7,8]. Depending on the amino factants [3,4]. Major applications of enzymes are at high acid composition, these proteins can incorporate both temperature (e.g. washing at 60–70 C, starch gelatiniza- acidic and basic functional groups, which play an tion at 100 C, textile desizing at 80–90 C), or may vary important role in their structure. Therefore, their expres- depending on the substrate and product solubility and sion can be related to resistance of unfolding forces stability. They can occur under high salt concentration because of protein’s conformation results in a less sol- (food industry) and alkaline conditions and/or in the uble state due to the occurrence of structural changes, presence of surfactants (detergents). Reactions can be aggregation, and/or precipitation [9]. Enzyme’s solubil- realized at gas–liquid interface (for reactions consuming ity can change as their structure undergoes modifica- (e.g. O2) or producing (e.g. CO2) gas), liquid–liquid inter- tions resulting from the exposure of different residues face for aqueous-organic two-liquid phase reactions to the surrounding environment. A few of the remark- where organic phase is used as a carrier for the sub- able features of enzymes are their functional diversity strate and/or product and in the presence of organic and versatility derived from each constituent amino solvents [3,5]. Hence, the need to stabilize enzymes acid having a different side chain with a specific chemis- against thermodeactivation, deactivation in the pres- try and polarity, and from flexible and numerous ways ence of surfactants and alkaline pH is imperative [6]. in which polypeptide chains can fold. Enzyme stability can be enhanced following different Enzymes undergo a variety of denaturation reactions routes which include strategies for stabilization in aque- during production, storage, and their application in ous and non-aqueous environments. industry. Denaturation is the unfolding of the tertiary CONTACT Artur Cavaco-Paulo [email protected] Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal à These authors contributed equally to this work. ß 2017 Informa UK Limited, trading as Taylor & Francis Group 336 C. SILVA ET AL. Figure 1. Illustration of (a) electron density profiles perpendicular to the air/water interface for lysozyme injected into buffer solu- tions with pH 3 and pH 11.5 (pI) (adapted from Yano et al. [19]); (b) net charge enzyme as a function of pH (adapted from Pihlasalo et al. [142]); topdown image shows the effect of several physical and biological agents responsible for denaturation with the respective target sites and the corresponding effects on enzymes. structure of the enzyme to a disordered polypeptide in contrast, the adsorption efficiency decreases as pH which key residues are no longer aligned closely increases, since the particles and protein repel each enough to continue the participation in functional or other due to repulsive charges, both having a negative structure stabilizing interactions [3]. If the denaturing charge [20,21]. influence is removed this tendency can be reversed. The achievement of stable and active enzymes is Enzymes must be formulated in order to maintain often a challenging effort because they have not proper folding to perform their biological functions evolved naturally to be used in industrial environments. [10–12]. Their integrity under extreme conditions might Their biological activity depends on the three-dimen- involve high excipient concentrations [11] whose inter- sional native structure, hence catalytically active, and actions are aided by several forces, such as hydrogen any significant conformational change can lead to their bonds, solvation forces, electrostatic, Van der Waal inactivation [12]. Manifestations of enzyme instability forces, amongst others [13–15]. These interactions can arise from aggregation, loss of biological functionality, be controlled through screening of the charge by and exposure to extreme conditions or even slight var- increasing the salt concentration of the solution or by iations of temperature or pH, that can induce abrupt changing the charge density on the surface [16,17]. conformational changes and subsequent loss of their Generally, at low ionic strength, the observed adsorp- biological activity [22]. A number of enzyme-based tion is to be maximumal at the isoelectric point [18], processes have been commercialized for producing whereas the unfolding of the tertiary structure of pro- valuable products, however despite their great potential tein reaches a maximum because of the large intramo- they have been hampered by undesirable stability, cata- lecular electrostatic repulsion, as observed in Figure 1(a) lytic efficiency, and low specificity. For this reason, the [19,20]. The unfolding of the tertiary structure is greater exploitation of methodologies for enzyme stabilization at pH 11.5 which is its isoelectric point (pI). Proteins is imperative for the progress in biotechnology and for have low solubility at pI, because the effective charge potential protein-templates discovery (Figure 2). of the molecule is zero (Figure 1(b)) and with decreased Along this review, practical insights related with repulsion and electrostatic interaction between the mol- enzyme stabilization, highlighting experimental aspects ecules, they form clumps that tend to precipitate. In for each methodology developed will be provide. This CRITICAL REVIEWS IN BIOTECHNOLOGY 337 Figure 2. Schematic representation of different enzyme stabilization methodologies. will be described along the text and practical examples interesting enzyme classes that are active and stable will be specified in Table 1. under extreme conditions have been used to maximize reactions in the food and paper industry, detergents, drugs, toxic waste removal, and drilling for oil. These Enzyme-stabilization strategies enzymes can be produced from the thermophiles Screening and isolation of enzymes from through either optimized fermentation of the microor- extremophiles ganisms or cloning of fast-growing mesophiles by recombinant DNA technology. It has been found that The interest in extremophiles stems from their surpris- psychrophilic enzymes can assist enhance yield of heat- ing properties being superior to traditional catalysts sensitive products, halophilic enzymes that are stable in and allowing the performance of industrial processes high salt concentrations serve as models for biocatalysis under harsh conditions in which conventional proteins in low-water media and thermophilic enzymes that are are denaturated. There has been extensive research on highly resistant to proteases, detergents, and chaotropic the structural proteins and key metabolic enzymes that agents, which may also afford resistance to the effects are responsible for the organisms’ unusual properties. of organic solvents. Within these enzyme classes are Recent research has focused on the identification of included: esterases/lipases, glycosidases, aldolases, nitri- extremozymes relevant for industrial biocatalysis lases/amidases, phosphatases, and racemases [24,25]. [23,24]. The most evident examples