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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
Published online: 26 Aug 2021.
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Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges
Hao Wua , Qiuming Chena, Wenli Zhanga and Wanmeng Mua,b aState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China; bInternational Joint Laboratory on Food
Safety, Jiangnan University, Wuxi, Jiangsu, China 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, pharma- ceutical, 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 meso- philic bacteria, and the tolerance is poor, severely restricting the broad industrial application (Atalah et al. 2019).
Therefore, new enzymes with high thermostability or mod- ification of the available mesophilic enzymes are significant for industrial application. In addition, it is necessary to 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 ther- modynamic stability and kinetic stability as parameters.
Thermodynamic stability is used to characterize the ten- dency 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 thermo- dynamically stable state to adapt to environmental tempera- ture (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 activ- ity when undergoing irreversibility (Polizzi et al. 2007). The measurement of kinetic stability requires detecting the res- idue activity of enzymes after incubating at specific condi- tions. 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 deac- tivation rate constant) (Bommarius and Paye 2013) should be measured.
© 2021 Taylor & Francis Group, LLC CONTACT Wenli Zhang wenlizhang@jiangnan.edu.cn https://doi.org/10.1080/10408398.2021.1970508
KEYWORDS Thermostability; protein engineering; industrial enzymes; extreme environment; interaction force
2 H. WU ET AL.
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 con- formation, 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 hyperthermo- philic 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 screen- ing (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 indus- tries. The mechanisms and factors affecting the thermosta- bility of enzymes are discussed in detail. Then, strategies for developing high thermostability enzymes including how to obtain enzymes and how to modify the molecular struc- ture 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 3 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 struc- turally 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 hydro- gen 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 sta- bility was attributed to the additional hydrogen bonds and ion-pair interactions. Interestingly, the introduction of inter- chain hydrogen bonds at the interface regions also contrib- uted 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 pro- teins. 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 com- pensated by intra-protein interaction at room temperature (Szilágyi and Závodszky 2000). Therefore, introduction of a desirable salt bridge remains a very difficult task as some- times 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 interac- tions, such as salt bridges and hydrogen bonds. The hydro- phobicity 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 pro- teins 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 envi- ronment 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 pro- teins (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 ΔT50 60 of 22 °C after the site-directed mutation of L144F (Mao et al. 2020). Further detailed anal- ysis of the structure indicated that a new hydrophobic inter- action 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
T1/2 10
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, pro- viding 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 aro- matic rings is in offset stacked or edge-face, which results in some challenges to design aromatic interactions. To sys- tematically clarify the contribution of aromatic interactions to the thermostability of proteins, Kannan et al., have inves- tigated a dataset of 24 protein families with known crystal structures from the thermophilic and the mesophilic homo- logues, and found that 17 thermophilic protein families possessed additional aromatic clusters or enlarged aromatic networks located on the protein surface than the corre- sponding mesophilic homologues (Kannan and Vishveshwara
2000). Yoneda et al., have revealed that the higher thermo- stability of B. smithii indio reductase was due to the
Critical Reviews in Food Science and Nutrition 5 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, indi- cating 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 con- formation of proteins and requires a specific redox envi- ronment 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 sep- aration 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 disul- fide 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 mobil- ity (higher B-factors) and longer loop lengths (25–75 resi- dues). 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 hydropho- bic 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. licheni- formis 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 ther- mostability 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 rigid- ity. 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 stabi- lized 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 T50
15 (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 addi- tional 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 hydro- phobic 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 resi- dues 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 thermo- stability (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
6 H. WU ET AL. could significantly improve the thermal stability of the pro- tein (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 environ- ment may secrete enzymes with good thermal stability (Table
1). Previous studies have revealed that extremophiles sur- vived 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 hyperthermo- philes 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 microor- ganisms 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 accel- erated 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 expres- sion 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 molec- ular biotechnology. Furthermore, many prokaryote systems such as Escherichia coli, Bacillus, Lactobacillus, and eukary- otic 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 hyperther- mophilic 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 pur- poseful 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 References
Pyrococcus furiosus DSM 3638 Shallow thermal waters near
Vulcano Island, Italy 100 Sample isolation Native α-amylase
106 Retain approximately 30% of initial activity after 8 h at 98 °C (Brown,
Costantino, and Kelly 1990) Thermococcus sp.
Hot-spring environment 85 Sample isolation Native cyclomaltodextrin glucanotransferase
110 40 min half-life at 110 °C (Tachibana et al. 1999)
T. aggregans Deep-sea from the Guaymas Basin 85 Cloning and expression in
Escherichia coli Recombinant pullulan hydrolase III
100 2.5 h half-life at 100 °C (Niehaus et al.
2000) P. furiosus DSM 3638 Shallow thermal waters near
Vulcano Island, Italy 100 Cloning and expression in E. coli
Recombinant esterase 100 50 min half-life at 126 °C (Ikeda and
Clark 1998) NS Offshore oil reservoir on the Norwegian
Continental Shelf NR Metagenome isolation Recombinant esterase
NR 90 °C for 1 h without any notable decrease in activity (Lewin et al.
2016) NS Compost NR Metagenome isolation Recombinant endoglucanase
85 Retain full activity after 24 h at 60 °C (Jensen et al.
2018) NR, not reported; NS, not specified.
Critical Reviews in Food Science and Nutrition 7 design is restricted due to a limited understanding of the catalytic mechanism and thermostability mechanism.
Currently, the rational design strategies that have been suc- cessfully applied mainly include homologous sequence align- ment, protein surface charge engineering, disulfide bonds design, proline and glycine design, free energy of protein unfolding design, temperature factor design and anchoring of unstable regions (Figure 3).
4.1.1. Based on homologous sequence alignment The primary structure of a protein is defined as the sequence of amino acid residues in the polypeptide chain of the protein, determining other high-level structures of the protein.
Therefore, the sequence alignment of proteins with various thermal stabilities would provide useful amino acid residues affecting the proteins’ functions. Previous studies have reported that the sequence similarity of thermophilic enzymes and mesophilic enzymes could reach 40%–85% (Vieille and Zeikus
2001). Accordingly, the amino acid sites related to thermal stability could be found by sequence alignment between pro- teins with high or poor thermal stability, and then possible mutation sites could be selected or designed to improve the thermal stability of proteins (Table 3). Based on homologous sequence alignment, Xiao et al., have successfully improved the thermostability of pectase lyase from Xanthomonas camp- estris, with Tm increasing by 6 °C and half-life being 23-fold longer at 45 °C (Xiao et al. 2008). Similarly, G312 and K436 have been selected as the site-directed mutagenesis targets to improve the thermostability of maltogenic amylase from
Bacillus sp. US149 by sequence alignments and homology modeling (Mabrouk et al. 2011). The maltogenic amylase dou- ble point mutant G312A/K436R displayed Topt and half-life time increased by 5 °C and 10 min at 55 °C, respectively. Based on the multiple sequence alignment of D-psicose 3-epimerase from various organisms, t1/2 value of mutant G109P from
Clostridium bolteae ATCC BAA-613 increased by 2.1-fold to that of wild-type enzyme (Zhang et al. 2016).
4.1.2. Based on protein surface charge engineering
Protein surface charge engineering is a powerful tool to change some enzyme properties (Zhou et al. 2019). Almost all natural proteins contain charged amino acids which are distributing on the surface of the protein to increase the stability of the protein through the interaction between charges. Optimizing the charged amino acids on the protein surface is a promising method to improve the thermal stability of protein (Strickler et al. 2006; Zouari Ayadi et al. 2015). As expected, this engineering successfully improved the activity, thermostability and ionic liquid tol- erance of B. subtilis lipase A, further convincing it a pow- erful approach in surface charge engineering to enhance thermostability (Zhou et al. 2019). It has also been reported the remarkable significance of protein surface charge engi- neering in strengthening the thermostability of endogluca- nase II from Penicillium verruculosum and stability in
1-butyl-3-methylimidazolium chloride (Dotsenko et al.
2020). Through analysis of protein surface topography, mul- tiple sequence alignment and ΔG calculations, it was found that the half-life time of endoglucanase mutant E70S and
V150L increased by 1.2 and 1.4-fold at 80 °C, respectively (Table 3). In addition, the thermal activation and T50 of laccase mutant E188K from Bacillus HR03 were 3-fold and
5 °C higher than wild type enzyme, respectively, by intro- ducing positive charge on its surface loop (Mollania et al.
2011; Liu et al. 2020).
4.1.3. Based on disulfide bonds design Disulfide bond is a kind of interaction force formed by the covalent bonding of two Cysteines (Figure 3). The DbD
Table 2. Some of the popular softwares applied in the protein engineering for improving thermostability.
Software Input Function Output Access websites References
FoldX Structure A force filed for energy calculations and protein design (ΔΔG) between the final state (the mutant) and the reference state (the wild-type protein) http://foldx.embl.de/ (Schymkowitz et al.
2005) PoPMuSiC Structure Predicting stability changes (ΔΔG) between the final state (the mutant) and the reference state (the wild-type protein) http://dezyme.com/. (Dehouck et al. 2009)
B-FITTER Structure Predicting the flexibility of protein structure based on B-factor
A ranked classification of all the residues according to the B-factor http://www.kofo.mpg.de/ en/research/ biocatalysis. (Reetz and Carballeira
2007) Disulfide by Design Structure Predicting pairs of residues that will likely form a disulfide bond
A series of potential disulfide bonds pairs, including bond energy, angle and B factor values http://cptweb.cpt.wayne. edu/DbD2/. (Craig and
Dombkowski 2013) Molecular dynamic simulation Structure
Performing dynamic properties of a protein at atomic level during a period of time.
Root mean square fluctuation (RMSF), Root mean square deviation (RMSD), Radius of rotation (Rg)
Gromacs, Amber, NAMD or Charmm (Zeiske, Stafford, and
Palmer 2016) Swiss-model Sequence Building a three-dimensional structure of protein
Generating a three-dimensional structure of target protein based on the available homologous crystal protein structure https://swissmodel.expasy. org/interactive. (Waterhouse et al.
2018) HotSpot Wizard 3.0 Sequence/ Structure Automated prediction of hotspots and the design of smart libraries in semi-rational protein design
Single-point mutant or multiple-point mutant or smart library https://loschmidt.chemi. muni.cz/ hotspotwizard/. (Sumbalova et al.
2018) 8 H. WU ET AL. software provides a convenient pathway to identify potential disulfide bonds to improve the thermal stability of the pro- tein based on the integration analysis of bonding energy, bonding dihedral angles, bonding distances, bonding geom- etry and values of temperature factor (B-factor) (Craig and
Dombkowski 2013). Currently, extensive studies have paid much attention on disulfide bond strategy to alter the ther- mostability of proteins (Table 3). For example, the thermo- stability of diisopropyl-fluorophosphatase mutant V24C/C76 at 60 °C was successfully enhanced after using the disulfide bond engineering, because the designed V24C/C76 displayed the lowest energy (0.33 kcal/mol) and a lower B-factor (15.07) which were predicted by DbD software (Mohammadi et al. 2018). Samson et al. have found that the thermosta- bility of catalase from B. pumilus was strengthened by intro- ducing a disulfide bond between S286C and D289C with the half-life showing 48 min longer than that of the wild type at 60 °C (Samson et al. 2018). Furthermore, the con- structed glucose dehydrogenase mutant, V149C/G190C, showed a 110 min half-life of thermal inactivation at 45 °C, which was 13-fold greater than that of the wild-type enzyme, and that the mutant’s catalytic activity and kinetic param- eters were not affected (Sakai et al. 2015).
4.1.4. Based on proline and glycine design It was reported that the mutation of Xaa to Proline or
Glycine to Xaa could increase the stability of protein because these substitutions could decrease the unfolding entropy of proteins (Matthews, Nicholson, and Becktel 1987). Proline has a pyrrolidine ring on the side chain, and the rotation of N-Cα is restricted, contributing to a smaller degree of conformational freedom and conformational rigidity increase (Figure 3) (Dotsenko et al. 2019). Nonetheless, only the replacement of proline at the appropriate site could improve the thermal stability of the protein (Tian et al. 2010; Zhou,
Xue, and Ma 2010). The decreasing content of glycine is also beneficial for the stability of proteins because glycine has high conformational entropy (Xu, Cen et al. 2020). For example, the mutation of Gly to Ala within the α-helix could improve the protein thermostability because of high helical propensity and low conformational entropy (Scott et al. 2007). Interestingly, Yi et al., have reported that intro- ducing glycine to the protein surface would improve the thermostability of endoglucanase CelA from Clostridium thermocellum (Yi, Pei, and Wu 2011). Previous study has indicated that the substitutions of Xaa to proline located in regions of the β-turn and the N-cap of the α-helix were
Figure 3. Different strategies to improve thermal stability of enzymes.
Critical Reviews in Food Science and Nutrition 9 Table 3. Different protein engineering strategies to improve the thermostability of various industrial enzymes.
Enzymes Microorganisms or Plasmids Strategies Mutants
Results (than wild type) Thermostable mechanisms Applications
References Pectase lyase Xanthomonas campestris Sequence alignment strategy of mesophilic and thermophilic proteins
R236F Half-life: 23-fold at 45 °C Tm: 6 °C higher Hydrophobic desolvation analyzed by structure analysis and computational methods
Hemp fiber-processing, fruit ripening (Xiao et al. 2008)
Maltogenic amylase Bacillus sp. US149 Sequence alignments and homology modeling
G312A/K436R Half-life: 10 min higher at 55 °C Topm: 5 °C higher
Hydrophobic interactions, salt bridges, hydrogen bonds
Bread, baking industry (Mabrouk et al.
2011) Endoglucanase II Penicillium verruculosum Protein surface topography, multiple sequence alignment and ΔΔG calculations
A52K, E70S, and V150L Half-life: 1.3–1.6 fold at 70 °C
Half-life: 1.2–1.4 fold at 80 °C NR Wood industry (Dotsenko et al.
2020) Laccase Bacillus HR03 Introducing positive charge on the surface loop
E188K T50: 5 °C higher Higher tightness of the enzyme structure
Beverages, wastewater treatment, drug analysis, bioremediation (Mollania et al.
2011) Diisopropyl- fluorohosphatase (DFPase) Loligo vulgaris
Introducing disulfide bridges V24C/C76 Thermostability is higher at 60 °C
More stable secondary structures, Rigidifying flexible regions
Medicine (Mohammadi et al.
2018) Catalase Bacillus pumilus ML413 Introducing disulfide bridges
S286C/D289C Half-life: 48 min higher at 60 °C Generating long inter-residues interactions
Food, textile, healthcare (Samson et al.
2018) Luciferase NR Proline substitutions within flexible regions
H489P Half-life: 1.4-fold at 35 °C Rigidifying flexible regions
487–495 Diagnostic, therapeutic application (Yu et al. 2015)
Feruloyl esterase Aspergillus niger Calculating the folding free energy change
D93G/S187F Half-life: 9.6-fold at 50 °C Lower conformational energy
Agrifood, pharmaceutical industries (Zhang and Wu 2011)
Transglutaminase Streptomyces hygroscopicus Calculating the folding free energy change
P132I Half-life: 31% higher at 50 °C Increased hydrogen bonds,
Stronger internal hydrophobicity Food, textile (Tong et al. 2018)
Tyrosinase Streptomyces kathirae SC-1 Calculating the folding free energy change
R95Y/G123W Half-life: 1.5-fold at 60 °C Additional hydrogen bonds
Medicine, cosmetic, food, environmental protection (Guo et al. 2017)
Lipase Rhizopus chinensis Calculating the folding free energy change
S142A/D217V/ Q239F/S250Y Half-life: 41.7-fold at 60 °C,
Topm: 5 °C higher T50 30: 15.8 °C higher Decreased solvent accessible surface area, newly formed salt bridges, increased folding free energy
Food, oil, pharmaceuticals, paper, leather, detergent, cosmetics (Wang et al. 2020) α-L-Rhamnosidase
Aspergillus terreus B-factor-saturation mutagenesis strategy
G827K/D594Q Half-life: 2.3-fold at 70 °C Newly formed hydrogen bonds and salt bridges
Food, pharmaceutical industries (Ge et al. 2018) Feruloyl esterase
Aspergillus usamii B-factor, folding free energy, iterative saturation mutagenesis
S33E/N92R Half-life: 3.96-fold at 50 °C Tm: 4.7 °C higher
Additional hydrogen bonds Food, textile, pharmaceutical industries (Yin et al. 2015)
Lipase B Candida antarctica B-factor, RosettaDesign, packing analysis tool “Voronoia”
R249L Tm: 2.3 °C higher Decreasing number of cavities
Dairy, nutrition, cosmetics, pharmaceutical (Kim, Le, and Kim
2010) Xylanase 2 Trichoderma reesei Introducing disulfide bonds between
N-terminus and β-sheet F14C/Q52C Half-life: 2.5-fold at 70 °C
Decreasing the entropy of unfolded state Food, feed, pulp, biofuel (Tang et al. 2017) (Continued)
10 H. WU ET AL.
Xylanase Thermomyces lanuginosus Introducing disulfide bond into
N-terminal region Q1C-Q24C Half-life: 20-fold at 70 °C
Tm: 8 °C higher Rigidifying flexible regions Animal feeding, pulp bleaching, baking (Wang et al. 2012)
Transglutaminase Streptomyces mobaraensis Directed evolution based on error-prone PCR
E164L Half-life: 1.66-fold at 50 °C Weakened the interactions between the two loop regions adjacent
Food, textile (Liu, Huang et al.
2019) Phospholipase D Streptomyces halstedii Directed evolution based on error-prone PCR
S163F Half-life: 3.04-fold at 50 °C Topt: 10 °C higher
Newly formed salt bridges, stronger hydrophobic interactions
Pharmaceutical, food, cosmetic industries (Huang et al. 2020)
Xylanase pET28a vector Directed evolution based on error-prone PCR and rational design guided by B-factor analysis
L28V/N30S/ K133E/ G172D/ Q14H/A29S/ A203V/N30P/ T31H/A52L
Half-life: 820-fold at 70 °C Newly formed hydrogen bonds
Animal feeding, pulp bleaching, baking (Xing et al. 2021)
Chitinase Paenibacillus pasadenensis Sequence- and structure-based semi-rational design in combination with introduction of disulfide bond
S244C-I139C/ T259P Half-life: 26.3-fold at 50 °C Topt: 7.5 °C higher
Stabilized folding form Agrifood, biological control, medical rehabilitation (Xu, Ni et al. 2020)
Lipase Lip2 Yarrowia lipolytica Directed evolution based on error-prone PCR, semi-rational design based on B-factor iterative test and site-directed mutagenesis
A103S/T117G and T117G/ F237C Half-life: 7-fold at 50 °C
Anchoring the flexible loop region Food, oil, pharmaceuticals, paper, leather, detergent, cosmetics (Wen, Tan, and
Zhao 2012) NR, not reported.
T50 30: Temperature at which the enzyme remained 50% of its activity after 30 min of heat-treatment; Topt: Optimal temperature; Tm: Melting temperature; T50: Temperature at which 50% of the initial activity is retained after 30 min incubation.
Enzymes Microorganisms or Plasmids Strategies Mutants
Results (than wild type) Thermostable mechanisms Applications
References Table 3. (Continued) Critical Reviews in Food Science and Nutrition
11 highly effective for increasing the stability of proteins (Wang et al. 2014). Besides, the comparative analysis of mesophilic and thermophilic alcohol dehydrogenases had revealed that a higher number of conserved proline residues existed in the two most stable enzymes, horse liver alcohol dehydro- genase (HLADH) and Thermoanaerobacter brockii alcohol dehydrogenase (TBADH), and these prolines residues were responsible for the increased thermostability of HLADH and
TBADH by rigidifying the loop regions (Barzegar et al.
2009). Similar result was also obtained to enhance the ther- mostability of luciferase and α-glucosidase by introducing proline to rigidify flexible regions (Zhou, Xue, and Ma 2010;
Yu et al. 2015). The half-life time of the improved luciferase
H489P mutant was 1.4-fold longer than that of wild type, further proving that proline strengthened the overall rigidity of proteins (Yu et al. 2015).
4.1.5. Based on the folding free energy of protein design
Unfolding free energy is the difference in free energy between the folded state and the unfolded state of the protein. The higher the unfolding free energy, the more energy needed to destroy the higher-level structure of the protein, indicating the more stable the protein. Protein folding free energy is consid- ered as an important characteristic related to the thermal stability of protein (Zhang et al. 2012). Some bioinformatic softwares like FoldX (Guerois, Nielsen, and Serrano 2002;
Schymkowitz et al. 2005) and PoPMuSiC were adopted to predict the effect of protein mutants on the folding free energy based on the protein sequence or three-dimensional structure (Table 2) (Zhang and Wu 2011). By predicting the folding free energy change of amino acid substitutions using PoPMuSiC algorithm, four positive mutants of feruloyl esterase A from
Aspergillus niger, namely S92A, D93G, D174A and S187F, were found to have the most stabilizing effect (Zhang and Wu
2011). Especially, half-life of the double point mutant D93G/
S187F increased by 9.6-fold at 50 °C when compared with that of the wild type (Table 3). By employing the same algorithm, the half-life of the transglutaminase mutant P132I from S. hygroscopicus reached 5.0 min at 50 °C, which was 31% higher than that of the wild type (Tong et al. 2018). The tyrosinase double point mutant R95Y/G123W from S. kathirae increased by 5 °C higher Topt and 1.5-fold half-life at 60 °C when com- pared with that of the wild type (Guo et al. 2017). Moreover, the combining use of FoldX and molecular dynamics simula- tions approaches also significantly enhanced the thermostability of Rhizopus chinensis lipase (Wang et al. 2020). The quadru- plemutation m31 (S142A/D217V/Q239F/S250Y) mutant exhib- ited a half-life at 60 °C 41.7-fold longer, a Topt 5 °C higher and a T50
30 of 15.8 °C higher than that of the wild type r27RCL.
Further analysis revealed that the thermostability mechanism was improved due to the decreased solvent accessible surface area, newly formed salt bridge and increased folding free energy of m31.
4.1.6. Based on temperature factor design The temperature factor (B-factor) is often used to reflect the conformational state of amino acid residues in the protein (Figure 3). The higher the B-factor value means the more flexibility in the conformation of the corresponding part (Yu and Huang 2014). Sun et al., have comprehensively reviewed the exploitation of B-factors in protein science in terms of the application in interpreting rigidity, flexibility, and internal motion and engineering thermostability (Sun et al. 2019), and declared that B-factor analyses could provide structural and mechanistic insights when combined with other exper- imental and computational techniques. The B-FITTER pro- gram was considered to be effective in predicting the flexible residues and calculating the amino acid B-factor of proteins, but available crystal structure information was required since
B-factor was obtained from X-ray data (Reetz and Carballeira
2007). Using the B-factor-saturation mutagenesis strategy, the half-life of D594Q and G827K/D594Q mutants of α-L-rham- nosidase from A. terreus increased by 2.1- and 2.3-fold, respectively, compared to that of the wild type at 70 °C, which was due to the newly formed hydrogen bonds and salt bridges (Ge et al. 2018). The Tm and half-life at 35 °C of levansucrase mutant E404L increased by 2.8 °C and
12.5-fold to that of wild type, respectively (Xu et al. 2019).
Similarly, according to B-factor analyzed by B-FITTER and folding free energy predicted by PoPMuSiC algorithm, S33 and N92 of type A feruloyl esterase from A. usamii were chosen for further iterative saturation mutagenesis (Yin et al. 2015). The S33E/N92R mutant displayed a Tm 4.7 °C higher and a half-life 3.96-fold longer at 50 °C compared with that of the wild type (Table 3). Moreover, Tm of mutant
R249L lipase B from Candida antarctica increased to 56.8 °C through in silico design after employing B-factor and
RosettaDesign approach (Kim, Le, and Kim 2010).
4.1.7. Based on the anchoring of unstable regions
Loops and N- and C-termini, the regions with the highest thermal factors in a protein crystal structure (Figure 3), are considered to be closely related to protein stability. Many studies have found that anchoring these regions can enhance the thermal stability of proteins (Ben Ali et al. 2011; Sun et al. 2005; Tang et al. 2017). To improve the thermostability of a mesophilic family 11 xylanase A from A. niger (AnxA), researchers have tried to replace AnxA’s N-terminus with the corresponding region of thermostable xylanase A from
Thermomonospora fusca (TfxA) (Sun et al. 2005). The newly hybrid xylanase ATx was more thermostable than AnxA, and it was similar to that of TfxA, demonstrating the important role of the N-terminus of TfxA in the thermo- stability of ATx. This work has also proved the feasibility of N-terminus replacement to improve the thermal stability of the protein. Thermostability of Trichoderma reesei xylanase
2 (Xyn2) is also reported to be enhanced by introducing disulfide bonds between N-terminus and β-sheet, with
2.5-fold higher half-life at 60 °C (Tang et al. 2017). Likewise,
Tm and half-life time of GH11 xylanase from Thermomyces lanuginosus increased about 8 °C and 20-fold, respectively, after introducing disulfide bond Q1C-Q24C into the
N-terminal region of the enzyme (Wang et al. 2012).
However, most studies have declared that the modification of the N-terminus sometimes decreased the activity of the
12 H. WU ET AL. enzyme (Liu et al. 2015; Sun et al. 2005). Accordingly, the
C-terminal modification might be an alternative to alter the enzyme properties. Coincidently, Li et al., have reported that the thermostability of A. fumigatus Z5 xylanase could be enhanced by introducing a poly-threonine region located in a linker region of the C-terminus between catalytic domain and carbohydrate-binding module domain (Li, Chen et al. 2019).
4.2. Directed evolution strategy Directed evolution is a very important means of non-rational design to change the properties of proteins, such as thermal stability, substrate specificity and catalytic reaction efficiency (Figure 3). Since proposed in the 1990s, this technology has been significant in protein engineering and has been broadly adopted in the enzyme fields (Chen et al. 1991; Zhang,
Geary, and Simpson 2019). Directed evolution is an accel- erated process that mimics the evolution of enzymes in nature by artificially creating a large number of mutants in vitro. Although directed evolution does not need to a prior understanding of the structure, catalytic mechanism and structure-activity relationship of the enzyme, the successful implementation of this technique requires, firstly to con- struct highly efficient mutant library by random mutagenesis such as error-prone PCR or DNA-shuffling techniques, and secondly to create high-throughput screening (HTS) method based on the chromogenic substrate or fluorescent color or physical and chemical properties of the products (Ladevèze et al. 2013; Liu, Xun, and Feng et al. 2019).
Transglutaminase has been widely used in the food and textile industry due to the cross-linking modification of proteins. To meet industrial requirements, three mutants with enhanced activity and thermostability were obtained from 5700 mutant libraries via directed evolution using two round error-prone PCR, and the E164L mutant of
Streptomyces mobaraensis transglutaminase showed a half-life
1.66-fold longer than that of the wild type at 50 °C (Liu,
Huang et al. 2019). The thermal stability of the mutant lipase (ep-231-51) from B. licheniformis was 13.5-fold higher than that of the wild type (Madan and Mishra 2014).
Besides, an enhancement thermostability S163F mutant of
Streptomyces halstedii phospholipase D was produced from
7700 random mutagenesis clones generated by error-prone
PCR strategy, showing a Topt 10 °C higher and a half-life at
50 °C 3.04-fold longer than that of wild type enzyme (Huang et al. 2020). Although directed evolution can achieve the modification of the thermal stability of protein, this process is labor-intensive and time-consuming. The blind charac- teristic of random mutations will generate a large number of unexpected and useless mutants, which require to be further screened.
4.3. Semi-rational design strategy Rational design requires an in-depth understanding of
protein structure and function relationship, whereas directed evolution needs to establish a huge mutant library and HTS methods (Figure 3). The semi-rational design provides a new direction for mutation according to the structural/ sequence-based information and the feasible HTS system because this strategy integrates the advantages of the other two methods (Kapoor, Rafiq, and Sharma 2017). With the aid of bioinformatics and computational predictive algo- rithms, the mutation sites of semi-rational design are con- centrated to one or a few sites, which will be further mutated using random mutation, site-directed mutation or site-directed saturation mutation technology, thereby creating a high-quality and small-scale mutagenesis library (Liu, Xun, and Feng et al. 2019). There are several feasible tools, such as FoldX (Schymkowitz et al. 2005), PoPMuSiC (Dehouck et al. 2009) and MultiMutate (Deutsch and Krishnamoorthy
2007), for predicting changes in protein stability following point mutation.
Xing et al. have employed the directed evolution based on error-prone PCR and rational design guided by B-factor analysis to improve the thermostability of GH11 xylanase (Table 3) (Xing et al. 2021). After conducting two rounds of error-prone PCR, site-directed mutagenesis, site-saturation mutagenesis and B-factor analysis, the mutant Xyn376 (L28V/N30S/K133E/G172D/Q14H/A29S/A203V/N30P/T31H/
A52L) was screened from 30,000 clones in the random mutagenesis library with a half-life of 410 min at 70 °C, which was 820-fold higher than that of the wild type enzyme (Table 3). The chitinase from Paenibacillus pasadenensis was also engineered to improve its thermostability and activity through semi-rational design from sequence- and structure-based strategy analysis (Xu, Ni et al. 2020). The obtained mutant (S244C-I139C/T259P) displayed 26.3-fold longer half-life at 50 °C and a Topt 7.5 °C higher than that of the wild type. Furthermore, the thermostability of lipase
Lip2 from Yarrowia lipolytica was significantly enhanced with a half-life 7-fold longer than that of the wild type using error-prone PCR based on directed evolution and
B-factor iterative test based on semi-rational design and site-directed mutagenesis (Wen, Tan, and Zhao 2012).
5. Challenges and future perspectives Comparing with chemical catalysts, biocatalysts such as enzymes have unparalleled advantages in terms of catalytic efficiency, substrate specificity and environmental friendli- ness, and are widely used in food, pharmaceutical, textile, bio-refining and feed industries. The high thermostability of enzymes is the prerequisite for industrial application. The ways to obtain highly thermostable enzymes include metag- enomic screening, genome sequencing of extremophiles from extreme environments and employing protein engineering methods to modify available enzymes with poor function.
It is the goal for all biologists and enzyme engineers to optimize and modify proteins’ properties to meet industrial requirements. The developed protein engineering technolo- gies, such as rational design, directed evolution and semi-rational design, have made great progress in protein function modification, and have been widely adopted in the
Critical Reviews in Food Science and Nutrition 13 industrial enzyme fields. There are still many challenges in engineering the properties of enzymes. The rational design based on sequence or structure information of protein requires an in-depth understanding of the structure, function and structure-activity relationship. In most cases, the obtained three-dimensional structure of a target protein is usually provided by homology modeling construction according to primary sequences. Accordingly, obtaining the crystal structure of the target enzyme is an urgent challenge to be overcome. Directed evolution does not rely on the three-dimensional structure of protein, but it requires the construction of a large number of mutant libraries and HTS methods. However, it is relatively difficult to establish a corresponding index correlation between the enzyme’s ther- mal stability and activity. During the screening process, the mutants generally need to be heat-treated in advance and then subject to high-throughput detection of enzyme activity.
Actually, the whole screening process is complex and time-consuming. When performing protein engineering for a specific enzyme, the aforementioned factors should be fully considered, and appropriate molecular modification strategies should be flexibly selected. Fortunately, the devel- opment of hadoop-driven artificial intelligence and compu- tational biology has greatly accelerated protein engineering design. The intelligent computational design of proteins will be a new trend in future development.
Disclosure statement The authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relation- ships with other people or organizations within five years of beginning the submitted work that could inappropriately influence, or be per- ceived to influence, their work.
Funding This work was funded by National Natural Science Foundation of
China (No. 31801583 and 31922073), the Natural Science Foundation of Jiangsu Province (No. BK20180607), the Key Technology R&D
Program of Jiangsu Province (BE2019629), Tianjin Synthetic
Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-003), and the Key-Area Research and Development
Program of Guangdong Province (2020B020226007).
ORCID Hao Wu http://orcid.org/0000-0002-5532-6564 Wenli Zhang http://orcid.org/0000-0002-7861-0170
Wanmeng Mu http://orcid.org/0000-0001-6597-527X References
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