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Microbiology and Biotechnology Letters

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Microbial Biotechnology (MB)  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2024; 52(2): 122-134

https://doi.org/10.48022/mbl.2403.03003

Received: March 5, 2024; Revised: April 16, 2024; Accepted: April 17, 2024

Enhancement of Pseudomonas savastanoi pv. glycinea Levansucrase Thermostability by Site-Directed Mutagenesis

Jun-Soo Kim1, Nack-Sick Choi2, and Woo-Yiel Lee1*

1Department of Biomedical Materials, Konyang University, Daejeon 32992, Republic of Korea
2Kprotec Co., Ltd., Daejeon 34141, Republic of Korea

Correspondence to :
Woo-Yiel Lee, 대전광역시 서구 관저동로 158 [35365]
Tel : 042-600-8534, Fax : 0642-600-8477, E-mail : lee0519@konyang.ac.kr

Abstract

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Levan is a functional fructooligosaccharide that belongs to the class of fructans and displays a range of physiological and dietary benefits. Levansucrase, classified as a fructosyltransferase, catalyzes the synthesis of levan fructans through the transfer of d-fructosyl residues from sucrose. Recombinant levansucrase exhibits a significantly high level of expression and activity in E. coli, however, its industrial applicability is limited due to its relatively poor thermal stability. In this study, we generated six single mutants by targeting either the high flexibility regions (242-250) or the 382Glu residue within the -TEAP- motif, and further developed two double mutants featuring concurrent mutations encompassing both regions. We expressed multiple levansucrase mutant proteins in E. coli DH5α using site-directed mutagenesis and measured the enzymatic activity and thermal stability of each mutant by levan formation reactions. The E246Y mutant displayed half-time 5.02-fold and 1.89-fold higher at 55℃ and 56℃, respectively, when compared to the WT, whereas the E382L mutant exhibited a 1.06-fold enhancement in half-time at 55℃. Furthermore, E246Y/E382L exhibited 1.95-fold and 1.86-fold higher thermal stability at 55℃ and 56℃, respectively. These findings suggest the potential for the developed thermally stable levansucrases to be applied in industrial settings, highlighting the effectiveness of our targeted mutagenesis approach.

Keywords: Levansucrase, levan, site-directed mutagenesis, protein expression, enzyme thermostabiliy

Introduction

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Levan is a fructan biopolymer consisting of β-D-fructofuranose residues linked by β-(2-6) glycosidic bonds with a terminal glucose molecule [1]. Levan is synthesized in nature by a few plant species, and a diverse range of microorganisms, including Bacillus subtilis, Zymomonas mobilis, Brenneria goodwini, and Pseudomonas savastanoi [2−5]. Levan possesses several functional properties, including low viscosity, high solubility, antioxidant activity, and moisturizing effects, which make it highly suitable for a wide range of industrial applications, particularly in the food, cosmetic, and pharmaceutical industries [6−8]. Because of this characteristics, levan exhibits promising potential as an emulsifying agent, stabilizer, thickener, and nanocarrier for drug delivery across various industrial sectors [9−11].

Levansucrase (EC 2.4.1.10), beta-2,6-fructosyltransferase, is classified as the glycoside hydrolase 68 (GH 68) family and catalyzes the synthesis of levan oligosaccharide from the substrate sucrose [12, 13]. Considering the high levels of protein expression and enzymatic activity exhibited by levansucrase in microorganisms, particularly E. coli., the primary approach for levan synthesis involves utilizing recombinant levansucrase as a catalyst [14, 15]. While the majority of bacterial levansucrases are efficiently overexpressed in E. coli, the enzymatic reactions of levan synthesis exhibit a limitation resulted from the short half-time of the enzyme at high temperatures. To overcome these limitations, previous studies have explored methods such as enzyme immobilization and creating mutants of levansucrase with improved thermal stability through site-directed mutagenesis [19, 20].

The thermal stability of enzymes is closely related to their protein structure, and specifically, the chemical stability can vary depending on the flexibility of the polypeptide chain. The B-factor, also known as temperature factor or atomic displacement parameter, reflects the flexibility of a protein and describes the probability distribution of atomic positions within a crystal structure. High B-factor values indicate a higher probability of electrons being located in incorrect positions, which in turn signifies a decrease in thermal stability of protein [21, 22]. It is a crucial parameter for understanding the dynamics and stability of proteins, as well as for rational design and optimization of enzymes for various biotechnological applications [23].

RMSF (root mean square fluctuation) is a measure of the average deviation of atomic positions in a protein structure from their average positions over time, reflecting the protein's flexibility and mobility, and is commonly employed in conjunction with B-factor analysis to investigate protein dynamics and flexibility [24−26]. Either introducing point mutations to increase the rigidity of a flexible polypeptide chain or substituting amino acid residues with high B-factors can be effective methods for enhancing the thermal stability of mutant proteins [27−29]. The B-factor values conventionally measured from X-ray crystallography experiments, but in recent years, it has become possible to predict these values using large sets of deep learning data [30−32]. This approach can be used to efficiently identify amino acid substitutions for point mutations that result in improved thermal stability of proteins.

In the present study, we conducted site-directed mutagenesis on the levansucrase gene from Pseudomonas savastanoi pv. glycinea and expressed the resulting mutants in E. coli. By analyzing half-time values at a specific temperature, we identified a levansucrase variant with improved thermal stability.

Materials and Methods

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Plasmid onstruction

The levansucrase gene from Pseudomonas avastanoi pv. glycinea (accession number: AAC36056.1) was subjected to codon optimization for E. coli. and the synthetic gene (Bioneer, Korea) was utilized for PCR amplification. The nucleotide sequence of the optimized levansucrase gene is presented in Fig. 1. The forward primer was 5’-CGCGGATCCATGTCTAACATCAACTATGCAC CTAC-3’, which included a BamHI recognition site and an additional CGC site. The reverse primer was 5’-CCCAAGCTTTTAGCTCAAAACGACATTCTTCATCGC-3, incorporates a HindIII recognition site and a stop codon (TTA).

Figure 1.The optimized nucleotide sequence of levansucrase from Pseudomonas savastanoi pv. Glycinea.

The PCR products and pMAL-p4e vector (NEB, England), which utilizes the tac promoter for inducible expression, were both digested with BamHI and HindIII, and ligated to produce the pMAL-p4e-levansucrase (pMPLV) recombinant plasmid. The pMPLV plasmid was transformed into E. coli DH5α and plated on SOB agar plates supplemented with ampicillin.

Expression and purification

The transformed E. coli DH5α harboring recombinant pMPLV was grown in SOB broth containing 0.2% glucose and 100 mg/l ampicillin at 37℃. When the optical density value at 600 nm reached 0.6, the recombinant levansucrase was induced with 0.3 mM isopropyl-β-Dthiogalactopyranoside (IPTG) at 30℃ for 3 h. The cell pellet was suspended in cell lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, pH 7.4) and lysed by ultrasonication. Subsequently, cell lysates were centrifuged at 16,000 rpm for 20 min to separate soluble and inclusion body. Amylose-based chromatography was performed to purify soluble levansucrase. The amylose resin was equilibrated with cell lysis buffer, and the lysate was loaded onto the column. After washing the column with the cell lysis buffer, recombinant levansucrase was eluted using an elution buffer (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 10 mM Maltose, pH 7.4). Following the elution, the MBP-levansucrase fusion protein was cleaved by enterokinase to separate the MBP tag from levansucrase.

Modelling of levansucrase mutants

In this study, multiple sequence alignment was performed to compare amino acid sequences of levansucrases from Pseudomonas savastanoi pv. Glycinea, Bacillus subtilis, Zymomonas mobilis subsp. mobilis and Rahnella aquatilis (Fig. 2). This analysis contributes to understanding the structural and functional relationships within the glycoside hydrolase (GH) family. It lays a foundation for deeper insights into the structural flexibility of amino acid residues and their B-factor values, crucial for protein stability. The conformational flexibility of amino acid residues and their corresponding B-factor values are important factors that influence the structural stability of proteins. The flexibility of amino acid residues can induce structural fluctuations, while high B-factor values may indicate unstable regions of the protein structure (Fig. 3). For this reason, a deep learning-based protein flexibility prediction tool, Predy-Flexy(https://www.dsimb.inserm.fr/dsimb_tools/predyflexy/) and MEDUSA (https://www.dsimb.inserm.fr/MEDUSA), were utilized to estimate the flexibility of levansucrase from Pseudomonas savastanoi pv. glycinea [33, 34].

Figure 2.Multiple sequence alignment of levansucrase from Pseudomonas savastanoi pv. Glycinea (GeneBank accession No: AAC36056.1), Bacillus subtilis (AAA22725.1), Zymomonas mobilis subsp. mobilis (BAA04475.1) and Rahnella aquatilis (AAK14794.1).

Figure 3.Color-coded B-factor representation of Pseudomonas savastanoi pv. glycinea-derived levansucrase structure visualized in PyMOL. The lower B-factor values are represented in blue, while the higher B-factor values are shown in red.

The structure of the levansucrase protein was visualized using PyMOL, where the average B-factors were indicated by a color gradient, with red and blue representing higher and lower values, respectively. In the levansucrase protein derived from Pseudomonas savastanoi pv. glycinea, amino acid residues from 242 to 250 display high B-factor values and conformational flexibility, making this region an ideal candidate for sitedirected mutagenesis to enhance the protein's thermal stability.

In previous studies, it has been identified that the Glu404 residue of levansucrase, located within the conserved “-TEAP-” motif, serves as a key determinant of the protein's thermostability [35]. In particular, substitution of Glu404 with hydrophobic or large side chain amino acids has been shown to increase thermal stability.

Site-directed mutagenesis

Point mutations were introduced into the levansucrase protein in two distinct regions: G243W, E246Y, and G248Y within the 242−250 segment, as well as E382W, E382L, and E382F within the conserved -TEAP- motif.

Site-directed mutagenesis of the levansucrase was conducted using in-fusion cloning. The nucleotide sequences of the primers used for the mutagenesis are shown in Table 1. The conditions for site-directed mutagenesis were as follows: 30 sec at 94℃, 30 sec at 62℃, and 7 min at 72℃. The amplified linear vector was ligated with T5 exonuclease and transformed into E. coli. DH5α. Plasmids were isolated and purified from each colony by the alkaline lysis method, and DNA sequencing was performed to select the strains with the correct mutant insertions.

Table 1 . Primers of the site-directed mutagenesis for levansucrase from Pseudomonas savastanoi pv. glycinea.



Levansucrase mutants were expressed and purified using the same method as the wild-type.

Effects of temperature and pH on enzyme activity

The optimization of reaction temperatures for both WT and mutants was carried out in a reaction mixture containing 50 mM acetate buffer, 5% sucrose, and 1 unit of levansucrase. The reactions were conducted for 1 h at a series of temperature points, specifically 40, 45, 50, 55, and 60℃, with 5℃ intervals between measurements.

(1 Unit is defined as the amount capable of hydrolyzing One mg of glucose in 1 h at 40℃.)

The optimal pH of the expressed WT and mutant was evaluated by determining their relative activity in three different buffer systems: sodium acetate (50 mM, pH 4.5−5.5), sodium phosphate (50 mM, pH 6.0−7.0), and Tris-HCl (50 mM, pH 7.5−9.0). The enzyme activity was measured by the DNS method [36, 37], and all experiments were independently performed in triplicate.

Thermostability measurement

To assess the thermostability of levansucrase, the enzyme was subjected to incubation at temperatures of 55℃, 56℃, and 57℃ for specific time intervals using a PCR thermocycler. These temperatures were selected based on the significant decrease in the activity of the wild-type levansucrase from 55℃. After incubation, 1 unit of levansucrase was added to a solution consisting of 50 mM sodium acetate buffer and 5% sucrose, and this mixture was then incubated at 40℃ for 1 h. The obtained data was fit to a first-order logarithmic function, and the corresponding curve was used to calculate the half-time of the enzyme.

TLC analysis

Thin-layer chromatography (TLC) was employed to examine the thermal stability of levansucrase mutants following a 1-h incubation. The enzyme was added to a solution prepared with 50 mM sodium acetate buffer and 5% sucrose and incubated at 40℃ for this duration. The products obtained were then analyzed with TLC to better understand the enzymés stability under these reaction conditions.

The reaction mixture was loaded onto TLC silica gel 60 F254 plates (Merck) using a 1:2:1:1 (v/v/v/v) mixture of acetic acid:acetonitrile:1-butanol:water as the mobile phase. Visualization of the reaction products was achieved by spraying the plate, loaded with samples, with a 5% sulfuric acid in methanol solution and subsequently heating it at 121℃ for 20 min.

Results and Discussion

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Protein expression and purification of recombinant levansucrase

The codon-optimized gene of Pseudomonas savastanoi pv. glycinea was amplified using PCR and subsequently cloned into the pMAL-p4e vector to construct an expression vector for levansucrase production. The recombinant vector was transformed into E. coli DH5α after which the harvested cells were sonicated and the resulting cell lysate was fractionated into soluble protein and inclusion bodies by centrifugation.

Utilizing the fusion with the MBP tag, the soluble protein was purified through affinity chromatography using amylose resin. The purified enzyme displayed a single protein band at approximately 87.8 kDa on SDS-PAGE (Fig. 4), being consistent with their predicted values, which confirmed the successful expression and purification of the recombinant enzyme in E. coli.

Figure 4.SDS-PAGE analysis of recombinant Pseudomonas savastanoi pv. glycinea-derived levansucrase expression and purification using amylose affinity chromatography. Lane 1: protein marker, Lane 2,3: purified levansucrase, Lane 4: the cell lysate from the recombinant E. coli DH5α after IPTG induction.

Construction of levansucrase mutants

B-factors and RMSFs represent the thermal motion of atoms or residues in proteins. A higher value suggests that the protein structure is more flexible, while a lower value indicates that the protein structure is more rigid. From this perspective, it can be anticipated that as the B-factor decreases, the protein becomes more rigid and its thermal stability increases. Levansucrase sourced from Pseudomonas savastanoi pv. glycinea demonstrates notably high B-factor and RMSF values within the residue range of 242−250. This region is therefore highly conducive for selecting mutants with the goal of enhancing thermal stability.

As evidenced in Table 2 and Fig. 5, the selected mutants, G243W, E246Y, and G248Y, exhibited an increase in the S2 values and thus, increased rigidity compared to the wild type. Furthermore, as shown in Fig. 6, residue E245 in the wild type enzyme exhibited a remarkably high RMSF value of 1.254, indicating significant flexibility. However, the introduction of mutations G243W, E246Y, and G248Y led to a significant reduction in the RMSF values to 0.764, 0.646, and 0.884, respectively, suggesting that these mutations enhance the rigidity of the enzyme, potentially improving its thermal stability.

Table 2 . Comparison of total S2 prediction differences for wild-type levansucrase and its mutants.

G243WE246YG248YE382WE382LE382F
Region242+0.1088+0.0305+0.0175---
243+0.1263+0.0362+0.0256---
244+0.121+0.0399+0.0339---
245+0.1002+0.0447+0.0493---
246+0.0766+0.049+0.0697---
247+0.0579+0.0504+0.0927---
248+0.0421+0.0455+0.1079---
249+0.0333+0.0374+0.0995---
…------
378---+0.0376+0.04+0.0369
379---+0.0461+0.0461+0.0443
380---+0.0526+0.0437+0.0476
381---+0.0608+0.0398+0.0527
382---+0.0674+0.0379+0.0559
383---+0.0719+0.0389+0.0549
384---+0.0668+0.0368+0.0488
385---+0.0583+0.0299+0.0374
ΔΣ S2 Prediction+1.2564+0.8981+0.9626+1.1919+0.741+0.9631


Figure 5.(A, B) Backbone dynamics of wild-type and mutant levansucrase from Pseudomonas savastanoi pv. glycinea.

Figure 6.(A, B) RMSF value of wild-type and mutant levansucrase from Pseudomonas savastanoi pv. glycinea.

According to a previous study, thermal stability was enhanced through mutation of the Glu404 residue within the “-TEAP-” motif. In this study, we strategically selected and analyzed three specific mutations: E382W, E382L, and E382F. These specific mutations were chosen based on their lower B-factor values as depicted in Fig. 7, indicating that they may confer increased rigidity and thus potentially enhance the thermal stability of the enzyme. Moreover, two double mutants, G243W/E382W and E246Y/E382L, were engineered by concurrently introducing mutations into two distinct regions: the 242−250 sequence and the “-TEAP-” motif. The expression vectors of mutated Levauscrase were constructed by PCR amplification using pMPLv as a template DNA for site-directed mutagenesis. Each of the mutants was expressed and purified in E. coli DH5α under conditions matching those used for the WT.

Figure 7.(A, B) B-factor value of wild-type and mutant levansucrase from Pseudomonas savastanoi pv. glycinea.

Effects of reaction temperature and pH

To determine the optimized temperature conditions for both the wild-type and the prepared mutant levansucrases, enzymatic assays were conducted within a 35−60℃ range at 5-min intervals.

WT and the mutant enzymes G243W, E246Y, G248Y, E382L and E246Y/E382L showed the highest activity at 55℃, while E382W, E382F and G243W/E382W exhibited the highest activity at 50℃ (Fig. 8).

Figure 8.(A, B) Effect of temperature (35℃ to 60℃) and pH (4.5 to 8.0) on the activity of wild-type and mutant levansucrase from Pseudomonas savastanoi pv. glycinea.

The optimization of pH for enzymatic activity involved testing a range of pH values (4.5−9.0) using sodium acetate, phosphate, and tris buffers. The optimal pH condition for all the tested enzymes, including WT and the mutants, was found to be pH 5.5 (Fig. 8B), which results indicated that, unlike temperature-dependent properties, the B-factor and RMSF parameters do not exhibit a direct correlation with optimal pH conditions. Moreover, this finding implies that the mutation did not significantly alter key aspects of the enzyme such as its active site or charge distribution.

Comparison of thermal stability and half-time between wild-type and mutant variants

The thermal stability of mutated levansucrase enzymes was investigated by incubating them at temperatures of 55℃, 56℃, and 57℃ for various time periods, then measuring the changes in enzymatic activity post-incubation.

The E246Y mutant exhibited a higher half-time compared to the wild-type (wt), approximately 1.89-fold at 55℃ and 1.89-fold at 56℃. At 57℃, the enzymatic activity of wt was no longer detectable after 45 min, while E246Y maintained approximately 40% of its activity even after 60 min.

The E382L mutant did not show significant differences in thermal stability compared to wt at 55℃ and 56℃. However, at 57℃, it exhibited considerable enzymatic activity even after 45 min.

In contrast, mutants G243W, G248Y, E382W and E382F showed a trend of decreased thermal stability at all temperature conditions. The double point mutant, G243W/E382W, displayed a significant reduction in thermal stability at all temperature conditions, whereas mutant E246Y/E382L demonstrated enhanced thermal stability specifically at 57℃ (Fig. 9).

Figure 9.(A-C) Thermal stability of wild-type and mutant levansucrase from Pseudomonas savastanoi pv. glycinea at 55℃ (A), 56℃ (B), at 57℃ (C).

The E382W, E382F and G243W/E382W mutants demonstrated not only a decrease in the optimal temperature to 50℃, but also a significant reduction in their thermal stability (Table 3). To further assess the thermal stability of mutant levansucrase, TLC was performed to verify the hydrolysis of the sucrose substrate (Fig. 10).

Table 3 . Comparison of total unit and half-time for wild-type levansucrase and its mutants.

MutantsTotal activity (U/mg)t1/2 (min)Km (Mm)
40℃55℃56℃55℃56℃
WT242.089.281.269.2032.1413.84
G243W329.262.855.829.7717.7125.81
E246Y292.0134.7113.0347.5660.9648.98
G248Y258.544.939.125.0512.7728.74
E382W273.632.721.227.2611.6245.8
E382L482.770.668.573.5231.7417.48
E382F357.833.632.114.459.5211.8
G243W/E382W385.330.522.27--21.89
E246Y/E382L459.1121.995.6135.0359.9431.79


Figure 10.Thin layer chromatography (TLC) analysis of enzyme reactions for wild-type and mutants of levansucrase. The reaction was performed for 1 h at 40℃ using a reaction mixture consisting of 50 mM sodium acetate buffer and 5% sucrose. The positive control group underwent pre-incubation with the enzyme at 55℃ for 1 h in order to evaluate its thermal stability.

Conclusion

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In this study, a deep learning program was utilized to design thermally stable mutant variants of levansucrase derived from Pseudomonas savastanoi pv. glycinea by assessing protein flexibility through the measurement of B-factor and RMSF values.

Notably, the E246Y mutant exhibited a 5.02-fold increase in half-time at 55℃, a 1.06-fold increase in halftime at 56℃ compared to the wild-type levansucrase, and also demonstrated significant thermal stability enhancements at 57℃. Incorporating the TEAP-motif, the E382L mutant showed modest thermal stability enhancements, with the double mutant E246Y/E382L demonstrating a similar level of improvement. Contrastingly, the thermal stability of G243W, G248Y, E382W, and E382F mutants was moderately diminished across all temperature conditions, with the double-point mutant G243W/E382W exhibiting the lowest thermal stability among all variants. The mutations G243W and G248Y involve the replacement of small, flexible glycine residues with larger amino acid side chains. These substitutions could potentially interfere or clash with adjacent secondary structures, thereby reducing the thermal stability of the protein. On the other hand, the E246Y mutation, which involves replacing glutamic acid that has a negatively charged side chain capable of forming ionic bonds with other amino acids with tyrosine possessing a neutral polar side chain, could potentially impact the internal electrostatic balance within the protein.

It may facilitate the formation of new hydrogen bonds or van der Waals interactions leading to an enhanced network of intermolecular interactions that increases thermal stability. These findings indicate that considering factors such as B-factor, RMSF, and protein flexibility can be beneficial in designing enzymes with enhanced thermal stability. However, alterations to these parameters do not appear to consistently enhance thermal stability across the entire spectrum of proteins, thereby suggesting the potential influence of additional factors such as spatial orientation, hydrogen bonding, hydrophobic interactions or Gibbs free energy.

In conclusion, this study illustrates the development of levansucrase mutants with enhanced thermal stability, which could potentially facilitate more efficient levan production in industrial applications. By utilizing innovative techniques in protein engineering, this research serves as a guideline for designing and producing practical and efficient enzyme mutants, contributing to advancements in the field of protein engineering.

Acknowledgment

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This paper was supported by the Konyang University Research Fund in 2022.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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