Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges

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Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges Hao Wu, Qiuming Chen, Wenli Zhang & Wanmeng Mu To cite this article: Hao Wu, Qiuming Chen, Wenli Zhang & Wanmeng Mu (2021): Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2021.1970508 To link to this article: https://doi.org/10.1080/10408398.2021.1970508

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Review

Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges Hao Wua , Qiuming Chena, Wenli Zhanga and Wanmeng Mua,b

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China; bInternational Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu, China a ABSTRACT

Biocatalysts such as enzymes are environmentally friendly and have substrate specificity, which are preferred in the production of various industrial products. However, the strict reaction conditions in industry including high temperature, organic solvents, strong acids and bases and other harsh environments often destabilize enzymes, and thus substantially compromise their catalytic functions, and greatly restrict their applications in food, pharmaceutical, textile, bio-refining and feed industries. Therefore, developing industrial enzymes with high thermostability becomes very important in industry as thermozymes have more advantages under high temperature. Discovering new thermostable enzymes using genome sequencing, metagenomics and sample isolation from extreme environments, or performing molecular modification of the existing enzymes with poor thermostability using emerging protein engineering technology have become an effective means of obtaining thermozymes. Based on the thermozymes as biocatalytic chips in industry, this review systematically analyzes the ways to discover thermostable enzymes from extreme environment, clarifies various interaction forces that will affect thermal stability of enzymes, and proposes different strategies to improve enzymes’ thermostability. Furthermore, latest development in the thermal stability modification of industrial enzymes through rational design strategies is comprehensively introduced from structure-activity relationship point of view. Challenges and future research perspectives are put forward as well.

1. Introduction As an effective alternative to traditional chemical catalysts, thermozymes have been widely applied in food, pharmaceutical, textile, bio-refining, and feed industries (Han et al. 2019). A good enzyme often meets industrial requirements like high substrate specificity, high catalytic efficiency, and high thermal stability, among which the thermal stability of enzyme is particularly important because of the resistance to the severe environment and maintenance of long-term catalysis at high temperatures (Karnaouri et al. 2019; Suresh et al. 2021). During the preparation of industrial products in industry, the chemical reaction often occurs in the system with high temperature owing to many merits, such as higher reaction rate, lower risk of microbial contamination (Wu, Zhang, and Mu 2019; Kumar et al. 2019). Enzymes exert the best biological activities in the living systems during the long-term natural evolution (Liu, Xun, and Feng et al. 2019). However, most enzymes are derived from the mesophilic bacteria, and the tolerance is poor, severely restricting the broad industrial application (Atalah et al. 2019). Therefore, new enzymes with high thermostability or modification of the available mesophilic enzymes are significant for industrial application. In addition, it is necessary to CONTACT Wenli Zhang

Thermostability; protein engineering; industrial enzymes; extreme environment; interaction force establish an efficient molecular modification method to improve the thermal stability of enzymes. The evaluation of the thermostability of enzymes from the perspective of structure and energy usually uses thermodynamic stability and kinetic stability as parameters. Thermodynamic stability is used to characterize the tendency of protein unfolding. In such a situation, the protein structure is in a relatively stable energy state determined by the entropy and enthalpy, and the unfolding trend is not spontaneous. Natural enzymes often evolve into a thermodynamically stable state to adapt to environmental temperature (Rothschild and Mancinelli 2001). Thermodynamic stability of a protein is usually assessed by free energy of unfolding (ΔGu), melting temperature (Tm), and unfolding equilibrium constant (Ku). Kinetic stability refers to the time or temperature required for a protein to maintain half activity when undergoing irreversibility (Polizzi et al. 2007). The measurement of kinetic stability requires detecting the residue activity of enzymes after incubating at specific conditions. Generally, Topt (the optimal temperature), T50 (the temperature when enzyme loses half activity), t1/2 (the time when enzyme loses half activity), and kd,obs (observed deactivation rate constant) (Bommarius and Paye 2013) should be measured.

The recent development of bioinformatics and sequencing technology has promoted the discovery of new coding sequences of enzymes (Wang, Nie, and Xu 2019). More and more thermophilic enzymes have been isolated or cloned from hyperthermophilic and thermophilic strains. It was found that the sequence similarity between thermophilic enzymes and mesophilic enzymes could reach about 40%–85% (Vieille and Zeikus 2001), while the tertiary structure of which was also highly similar, and the catalytic mechanism was the same, indicating that there were other mechanisms and factors affecting enzymes’ thermal stability. Extensive studies have revealed that there are many interaction forces being in the amino acid residues of enzymes which are including hydrophobic interactions, salt bridges, aromatic ring interactions, disulfide bonds and hydrogen bonds, and these interaction forces play a very pivotal role in maintaining the conformational stability of enzymes. Additionally, enzymes with better thermal stability tend to have a more stable conformation, such as higher rigidity, higher stacking efficiency, lower de-folding entropy, and alpha-helix stability (Vieille and Zeikus 2001). Higher thermostability makes enzymes more competitive and desirable in the industrial application. In the past few decades, continuous efforts have been made to obtain desired industrial enzymes with high thermostability through two main ways. One way is to screen hyperthermophilic and thermophilic organisms from extreme environment (Figure 1). However, this screening process is complicated and tedious, and the enzyme activity is relatively low. The other way is to improve the thermostability of existing industrial enzymes derived from mesophilic organisms through advanced protein engineering strategies like rational design, semi-rational design, directed evolution and de novo design (Liu, Xun, and

Feng et al. 2019). Additionally, the computational tools for enzyme engineering are widely used aiding this process to better understand the thermostability mechanism of industrial enzymes (Chen et al. 2020). However, rational design requires a comprehensive understanding of the relationship of the structure, function, catalytic mechanisms of enzymes. Semi-rational design involves mutations based on sequence, structure or computational models, followed by small-scale mutagenesis and screening methods (Zhang, Geary, and Simpson 2019). Directed evolution requires the construction of a large mutant library and efficient high throughput screening (HTS) methods. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding (Huang, Boyken, and Baker 2016). This review systematically summarizes some achievements in the thermal stability of industrial enzymes applied in food, pharmaceutical, textile, bio-refining and feed industries. The mechanisms and factors affecting the thermostability of enzymes are discussed in detail. Then, strategies for developing high thermostability enzymes including how to obtain enzymes and how to modify the molecular structure of existing enzymes with weak thermal stability through advanced protein engineering techniques are proposed as well. Mature methods to improve the thermal stability of enzymes through rational design are analyzed.

2. Factors affecting protein thermal stability 2.1. Hydrogen bond Hydrogen bonding is an important non-covalent interaction in protein structure, which not only exists between the

Figure 1. The flow chart of obtaining thermostable enzymes from extreme environment using different separation strategies. Critical Reviews in Food Science and Nutrition

amino acid residues in the protein but also manifests in the interaction between the protein and the surrounding water molecules (Figure 2). This is because there are many hydrogen donors and hydrogen acceptors between the water molecules inside and around the protein. Generally, the distance of hydrogen donors and hydrogen acceptors is not more than 3 Å and the angle is less than 90 degrees. The energy provided by each pair of hydrogen bonds formed in the protein is about 0.6 kcal/mol (Li, Zhou, and Lu 2005). Vogt et al., have found that the thermostability of protein is closely related to the number of hydrogen bonds after comparing different families of proteins in terms of their respective fractional polar atom surface areas (Vogt, Woell, and Argos 1997). Vieira et al., have dealt with two structurally similar family 11 xylanases, BCX (mesophilic xylanse from Bacillus circulans) and TLX (thermophilic xylanase from Thermomyces lanuginosus) by molecular dynamics simulations, and demonstrated that the intramolecular hydrogen bonds and salt bridges were key factors for the maintenance of the backbone rigidity at high temperatures (Vieira and Degrève 2009). Later research has reaffirmed the conclusion that thermophilic enzymes have more hydrogen bonds than mesophilic enzymes (Tompa, Gromiha, and Saraboji 2016). Besides, Ishak et al., have found that the melting temperature (Tm) of recombinant lipase mutants D43E and E226D from Geobacillus zalihae increased up to

76 °C and 77.4 °C, respectively, while that of the wild-type enzymes were 70.9 °C (Ishak et al. 2020). Further structural investigations about G. zalihae lipase mutants D43E and E226D have indicated that the improvement of lipase stability was attributed to the additional hydrogen bonds and ion-pair interactions. Interestingly, the introduction of interchain hydrogen bonds at the interface regions also contributed to the thermostability and structural stability of D-allulose 3-epimerase from Dorea sp. CAG317 with Tm increased by 17.54 °C compared to wild type (Zhang et al. 2018). 2.2. Salt bridge Salt bridge is formed through the electrostatic attraction of amino acid residues with opposite charges (Figure 2). As a driving force for maintaining the structure thermostability of protein at high temperatures, salt bridge frequently appears on the surface of proteins (Xu, Cen et al. 2020). Thermophilic proteins tend to possess more surface salt bridges than homologous mesophilic proteins at high temperatures (de Bakker, Hünenberger, and McCammon 1999; Szilágyi and Závodszky 2000). Also, salt bridge is considered as a more important factor than hydrogen bond in thermophilic proteins. The molecular dynamics simulation results illustrated that high temperature would lead to more intra-protein

Figure 2. Factors and characteristics affecting the thermal stability of industrial enzymes. 4 H. WU ET AL.

interaction energy due to the tightening of salt bridges, which also explained that the reason why salt bridges may stabilize hyperthermophilic proteins at elevated temperatures rather than at room temperature. Another reason was that the large desolvation penalty incurred by the association of two charged residues forming a salt bridge was not compensated by intra-protein interaction at room temperature (Szilágyi and Závodszky 2000). Therefore, introduction of a desirable salt bridge remains a very difficult task as sometimes it will result slight stabilization or destabilization of proteins. Additionally, Szilágyi and Závodszky have compiled a non-redundant data set, comprising high-quality structures of thermophilic proteins and the mesophilic homologues to draw general evolution of the heat stability of proteins (Szilágyi and Závodszky 2000). It was found that the number of salt bridges was a significant factor affecting the stability of proteins, and the number of salt bridges in thermophilic proteins was higher than that of mesophilic proteins. To increase the thermostability of 1,4-α-glucan branching enzymes from Geobacillus thermoglucosidans (GtGBE), Ban et al., have introduced additional local salt bridges into GtGBE and results showed that five separately introduced mutants, namely, Q231R-D227, Q231K-D227, T339E-K335, T339D-K335, and I571D-R569 had longer half-life of 17% to 51% than that of wild-type (Ban et al. 2020). Further circular dichroism and intrinsic fluorescence experiments indicated that the improved thermostability of GtGBE mutants may be ascribed to the enhanced rigidity in newly formed salt bridge networks. Chan et al., have investigated how salt-bridge influenced protein stability with the research on the pair-wise interaction energy and ΔCp (heat capacity change of unfolding) (Chan et al. 2011), and the results indicated that extra salt-bridges enhanced the thermostability of proteins by reducing ΔCp, which would make protein stability curves rise and widen.

2.3. Hydrophobic interaction Many studies have confirmed that hydrophobic interaction plays an important role in maintaining protein stability and forming protein tertiary structure, where the hydrophobic environment is the major factor affecting the stability of thermophilic proteins when compared with other interactions, such as salt bridges and hydrogen bonds. The hydrophobicity content has also been proposed to be informative for discriminating between mesophilic and thermophilic proteins (Modarres, Mofrad, and Sanati-Nezhad 2016). Gromiha et al., have found that 80% of thermophilic proteins show higher hydrophobicity than mesophilic ones based on a dataset of 373 protein families (Gromiha et al. 2013). The interaction force is generated mainly due to the cluster and hydrophobic amino acid side chains which instinctively repels the contact with water (Folch, Rooman, and Dehouck 2008). Therefore, the hydrophobic amino acids (nonpolar amino acids) in aqueous solutions are usually buried inside proteins, forming a hydrophobic core, whereas polar amino acids are distributed in the hydrophilic environment on the protein surface. It was reported that the

stable protein could obtain 1.3 ± 0.5 kcal/mol when every additional −CH2− group was buried in the protein (Figure 2) (Pace 1992). By introducing a methyl group into the hydrophobic core of E. coli ribonuclease H1, Ishikawa et al., have successfully achieved the thermostability of the enzyme (Ishikawa et al. 1993). This can be explained by the later research findings that more hydrophobic interaction would make the unfolding rate slower in thermostable proteins (Okada et al. 2010). Relevant literature had reported that the thermostability of D-allulose 3-epimerase from Rhodopirellula baltica was enhanced to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C after the site-directed mutation of L144F (Mao et al. 2020). Further detailed analysis of the structure indicated that a new hydrophobic interaction was produced in the mutation protein. A similar result was also observed in the double point mutant T130M/ E133F of (R)-selective amine transaminase from Aspergillus terreus, with 3.3-fold in t1/2 at 40 °C and 5 °C higher in T1/210 min thermal stability than wild type enzyme, which was due to the newly formed hydrophobic interactions and hydrogen bonds (Huang, Xie, and Feng 2017). Furthermore, when error-prone PCR and rational design based on B-factors were used, the thermostability of optimal variant Xyn376 from GH 11 family showed a half-life 820-fold higher than that of the wild-type enzyme at 70 °C (Xing et al. 2021). The hydrogen bonds and hydrophobic interactions were the major forces for improved thermostability by the structural analysis.

2.4. Aromatic ring interaction Aromatic ring interaction is also an important driving force for protein thermostability, mainly including the interaction of cation (positively charged amino acids, such as Lys, Arg and protonated His) and aromatic ring (cation-π), or aromatic-aromatic interaction (π-π). The cation-π interaction was a general, strong and non-covalent binding force, providing the energy twice as strong as that of salt bridges (Chakravarty and Varadarajan 2002; Dougherty 2007). The interaction of aromatic ring amino acids (Try, Tyr and Phe) mainly occurs when the distance between the phenyl ring centroids is less than 7.0 Å (Figure 2). A π-π interaction usually stabilizes a protein molecule by 0.6–1.3 kcal/mol when the preferential distance of the centroids of the two aromatic rings are 5.5 Å (Ohmura et al. 2001). However, the aromatic interactions happen when the orientation of aromatic rings is in offset stacked or edge-face, which results in some challenges to design aromatic interactions. To systematically clarify the contribution of aromatic interactions to the thermostability of proteins, Kannan et al., have investigated a dataset of 24 protein families with known crystal structures from the thermophilic and the mesophilic homologues, and found that 17 thermophilic protein families possessed additional aromatic clusters or enlarged aromatic networks located on the protein surface than the corresponding mesophilic homologues (Kannan and Vishveshwara 2000). Yoneda et al., have revealed that the higher thermostability of B. smithii indio reductase was due to the

intersubunit aromatic interactions (F105-F172′ and F172 and F105′), and F105 played a governing role in these aromatic interactions (Yoneda et al. 2020). The additional aromatic interaction of Y11-Y16 could increase the stability of the N-terminal part of mesophilic family 11 xylanase from Streptomyces sp. S38, which increased Tm by 9 °C, indicating that adding π-π interaction at the appropriate place could improve the thermostability of the protein (Georis et al. 2000). A recent study has declared that half-lives of the nitrilase mutants AcN-T201F and AcN-T201W constructed by error-prone PCR were 13.5- and 10.8-fold longer than that of wild-type AcN, respectively (Xu et al. 2018). The molecular modeling suggested that W201 or F201 residue of nitrilase mutant not only improved the stability of the dimer interface but also formed π-π interaction with W165 residue to stabilize the substrate binding pocket, indirectly enhancing the thermostability.

2.5. Disulfide bond Disulfide bonds are formed between the cysteine residues (Cys) with the distance of the alpha carbon atoms in space ranging from 4 to 9 Å (Figure 2). This process is one of the posttranslational modifications dependent on the conformation of proteins and requires a specific redox environment and chaperones proteins (Creighton 1984). Disulfide bonds, as covalent interactions, which stabilize the structure of proteins by reducing conformational entropy, play a vital role in the folding and activity of proteins. Compared with the non-covalent interactions forces, such as hydrogen bonds, salt bridges, hydrophobic interactions and aromatic interactions, disulfide bonds provide the greatest energy for protein stability. It has been reported that the average separation between Cys forming a disulfide cross-link is 15 residues for natural protein, which can provide 3.0 kcal/mol energy if assuming negligible effect on the folded form (Kazlauskas 2018). However, many factors should be taken into account to construct disulfide bonds. To figure out the factors that may affect the successful construction of disulfide bonds, Dani et al., have reevaluated and refined an automated procedure for modeling the disulfide bonds in proteins through a computational MODIP program (Dani, Ramakrishnan, and Varadarajan 2003), and found that the stabilized disulfide bonds were associated with the proper stereochemistry, lower depth regions, relatively higher mobility (higher B-factors) and longer loop lengths (25–75 residues). To effectively improve the protein stability and modify functional properties, a software named Disulfide by Design (DbD) and an accessible web server hosting site (http:// cptweb.cpt.wayne.edu/DbD2/) have been developed by Craig and Dombkowski to auxiliary predict pairs of residues that would likely form a disulfide bond if mutated to Cys (Craig and Dombkowski 2013). Compared with other non-covalent interaction forces including hydrogen bonds and hydrophobic forces, it seems to be a more convenient and simple method to design disulfide bonds assisting by computational calculation programs and software. However, the formation and correct folding of disulfide bonds are also full of

uncertainty due to the redox instability which is not fit for enzymes that function in reducing environment. Extensive studies have been carried out to enhance the thermostability of enzymes by introducing disulfide bonds (Vasudevan et al. 2019). A recent study had successfully enhanced the thermostability of phytase from B. licheniformis WHU by DbD v2.05 program coupled with homology models analysis, whose mutant G197C/A358C was 3.8-fold than wild type enzyme in half-life at 60 °C (Zhang et al. 2020). Molecular dynamics results indicated that the thermostability mechanism of this G197C/A358C mutant was that the newly formed disulfide bonds might anchor the C-terminus of phytase and reinforce the local packing rigidity. In the study of Tang et al., two disulfide bonds had been separately constructed, namely Xyn2C14–52 and Xyn2C59– 149 , between the N-terminal and α-helix to the β-sheet core of Xyn2 using site-directed mutagenesis at the corresponding residues, respectively, and resulted 2.5- and 1.8-fold longer half-lives than the wild type Xyn2 at 60 °C (Tang et al. 2017). The newly formed disulfide bonds effectively stabilized the Xyn2 structure by preventing the structure from unfolding, which may be the mechanism for the improved thermostability. Li et al., have engineered different regional multiple disulfide bonds to improve the thermostability of lipase from Yarrowia lipolytica (Li, Zhang et al. 2019). The sextuple mutant 6 s showed that Tm and T5015 (Temperature that the activity loss half at 15 min) increased by 22.53 °C and 31.23 °C, respectively, due to more rigidified enzyme structures and longer unfolding times after introducing additional disulfide bonds. 2.6. Protein packaging efficiency Protein packaging efficiency, which is related to the thermal stability of a protein, is defined as the ratio of the hydrophobic surface area to the total area of the protein surface (Vieille and Zeikus 2001) (Figure 2). Karshikoff et al., had analyzed the structures of 80 non-homologous mesophilic proteins, 20 thermophilic proteins and 4 hyperthermophilic proteins, and found that thermophilic proteins often had higher protein packaging efficiency (Karshikoff and Ladenstein 1998). An improvement in the inner packing effectively enhances protein stability since an increase in buried cavities destabilizes protein structure (Eriksson et al. 1992; Xu et al. 1998). Abraham et al., had chosen 43 residues of Lipase A from B. subtilis with exposure ratio below 5% and packing value below 0.55 as first-round mutant candidates and found that less-packed residues with no water-contact were good target sites for enhancing thermostability (Abraham et al. 2005). Based on the strategies of Gly mutation to Ala, and Ala mutation to Val, six mutants, namely, A38V, A75V, G80A, A105V, A146V and G172A were obtained with higher thermostability than that of wild-type. Among them, half-lives of A38V, G80A, and G172A mutant increased 64- to 70-fold than wild type lipase A. One study has also shown that reducing the hydrophobic surface area of flavodoxin by changing the hydrophobic amino acids on the surface into hydrophilic amino acids

could significantly improve the thermal stability of the protein (Ayuso-Tejedor, Abián, and Sancho 2011).

3. Discovery natural enzymes with high thermostability It is believed that the properties of enzymes are closely related to the environment in which the host lives owing to the long-term natural evolution (Ueno, Ibarra, and Gojobori 2016). Accordingly, it is reasonable to think that microorganisms that grow in a high-temperature environment may secrete enzymes with good thermal stability (Table 1). Previous studies have revealed that extremophiles survived in extreme conditions could secrete novel enzymes with various properties, which could be applied in chemical, food and pharmaceutical industries. Therefore, extremophiles have gained much research attention (Chettri et al. 2021; Herbert 1992; van den Burg 2003; Verma, Meghwanshi, and Kumar 2021), and thermostable enzymes can be obtained by novel proteins from extremophiles such as hyperthermophiles or thermophiles (Figure 1). At present, only a minor fraction of microorganisms that can resist extreme heat have been reported, and most of which belong to archaea. Many of these available microorganisms that grow in extreme conditions, such as deep-sea, hot-spring environment, are difficult to be cultivated in the laboratory environment, which makes the production of enzymes from extremophiles infeasible through fermentation technology. Nonetheless, the development of genome sequencing, molecular biology and metagenomics has accelerated the gene mining, discovery and identification of enzymes, such as xylanase (Chadha et al. 2019; Ferrer et al. 2016; Lorenz and Eck 2005; Mhiri et al. 2020; Patel et al. 2019). Besides, many plasmid vectors and mature expression systems allow the extremophiles-derived enzymes to

be cloned and expressed in the mesophilic hosts that are easy to be cultivated in the laboratory with mature molecular biotechnology. Furthermore, many prokaryote systems such as Escherichia coli, Bacillus, Lactobacillus, and eukaryotic systems such as Pichia, Saccharomyces cerevisiae, and Candida have been developed as selectable expressing hosts for producing different source enzymes (Peng et al. 2021). For example, both the D-lyxose isomerase from Thermoprotei archaeon and L-aminoacylase from Thermococcus litoralis have been successfully cloned, expressed and characterized in E. coli hosts (Toogood et al. 2002; Wu et al. 2020). The β-glycosidase from extreme thermoacidophilic archaeon Sulfolobus solfataricus has been expressed in yeast systems (D’Auria et al. 1996). Fortunately, the majority of hyperthermophilic enzymes expressed in these expressing systems retain all of the native enzymes’ biochemical properties, including proper folding and thermostability (Ebaid et al. 2019; Grättinger et al. 1998; Shi et al. 2019; Vieille and Zeikus 2001).

4. Protein engineering strategies for developing thermostable enzyme 4.1. Rational design In recent years, with the rapid development of bioinformatics technology and structural biology, rational design strategies have become important means to modify the properties of proteins (Table 2). A practical rational design is based on the comprehensive understanding of the protein structure, function and structure-activity relationship, and then purposeful modification of the specific sites of the protein through selective substitution, insertion or truncation, and further experimental analysis of the property changes of the designed protein. However, the application range of rational

Table 1. Different strategies used to discovery natural enzymes with high thermostability. Microorganisms Sources Microorganism Topt (°C) Methods Production Enzymes Topt (°C) Thermostable ability