Intracellular Lipases for Enzymatic Synthesis of Phenylalanine Butyramide in a Biphasic Reaction System

Abstract

Phenylalanine butyramide (FBA) is a novel butyric acid derivative with favorable sensory properties, which has broad prospects in medicine and feed processing. However, there is currently limited research on the enzymatic synthesis of FBA. As is well known, lipase plays a crucial role in amide bond synthesis, but it typically requires completely anhydrous conditions. The lipase from Sphingomonas sp. HXN-200 (SpL) is the only intracellular lipase identified to date, capable of catalyzing the ammonolysis of esters or acids in an aqueous phase. In this study, we developed a method for the synthesis of FBA catalyzed by SpL in a biphasic reaction system of water and n-hexane. SpL was successfully expressed in E. coli BL21, and the optimal induction conditions were 0.4 mM IPTG and 18 h. It was ascertained that the n-hexane system containing 2% water was conducive to the reaction. Under optimized reaction conditions, 0.89 mg/mL of FBA was obtained within 15 h at 30 °C. Additionally, we found that SpL also has the ability to hydrolyze amides in the reaction of SpL catalyzing the formation of amides, so we further analyzed its catalytic mechanism.

1. Introduction

The phenylalanine butyramide (FBA), an unnatural amide, possesses potential as a postbiotic derivative, attributed to its significant improvement in organoleptic and physicochemical properties compared to butyric acid and salts [1,2,3]. The augmented properties of FBA endow it with the ability to endure the harsh gastric environment and successfully reach the intestine. It can gradually release butyric acid, which has beneficial effects on gut health, such as promoting the growth of beneficial gut microbiota and maintaining the integrity of intestinal epithelium [4,5,6]. Additionally, in terms of doxorubicin-induced cardiotoxicity, FBA demonstrates the capacity to alleviate oxidative stress and enhance mitochondrial function, thereby mitigating doxorubicin toxicity [7]. Consequently, FBA holds significant promise for development and potential application value.

There is a paucity of research concerning the biosynthesis of FBA. Nevertheless, given that FBA is an amide compound, numerous investigations have been conducted on amide synthesis [8]. The traditional chemical method for the synthesis of amide involves coupling carboxylic acids or their derivatives with amines [9,10]. However, it requires the use of toxic or hazardous reagents, as well as expensive coupling agents, and faces challenges related to safety, complicated purification processes, and poor atom economy. Consequently, the enzymatic synthesis of amide is gradually gaining increasing attention [10]. Reports have indicated that the enzymes utilized in the synthesis of amide bonds can be neatly classified into two distinct categories: ATP-dependent enzymes and hydrolases [8,11,12]. ATP-dependent enzymes include carboxylate reductase, ATP-grabbing enzyme, ligase, amide bond synthetase, non-ribosomal peptide reductase, etc., of which the adenylation domain can independently facilitate the synthesis of amide bonds. In previous studies, the synthesis of FBA has been catalyzed by acetyl coenzyme A synthetase [13]. However, the catalytic reaction system of ATP-dependent enzymes is cumbersome and ATP-consuming which poses challenges in large-scale applications.

Lipases, as typical hydrolytic enzymes, have a wide range of applications in the formation of amide bonds, although these reactions must be conducted in organic solvent systems [14]. In the process of amide bond formation, the catalytic triad of lipase consists of serine, histidine, and either aspartic acid or glutamic acid. When serine was activated by deprotonation, its nucleophilicity increased, allowing it to attack the carbonyl group of the substrate to form the acyl-enzyme intermediate. Subsequently, the nucleophilic reagent attacks the acyl-enzyme intermediate, resulting in the release of product and regeneration of the catalytic site [15]. For example, Candida antarctica lipase B (CALB) [16,17] and Pseudomonas stutzeri lipase (PSL) [18] are commonly used biocatalysts for the synthesis of amide bonds. It has been reported that subtilisin Carlsberg can produce FBA through the racemic reaction of amine, although accompanied by the formation of trifluoroethanol, which needs to be reacted in anhydrous organic phases with a low conversion rate [19].

Currently, the lipases in use are exclusively extracellular enzymes. Zukic et al. had identified a novel intracellular lipase from Sphingomonas sp. HXN-200 (SpL) that is capable of promoting the amidation of esters or carboxylic acids in the presence of water [20]. In this study, SpL was employed as a catalyst for the first time to facilitate the synthesis of FBA in a hydrated organic phase. At the same time, we found that SpL has a mixed catalytic ability and analyzed its structural mechanism.

2. Results and Discussion

2.1. Expression Purification and Activity Verification of SpL

SpL was recombined with pET-28a (+) and successfully expressed in E. coli BL21. The SDS PAGE of cell-free extract before and after purification showed the histidine-tagged SpL molecular weight of 37 kDa, which was consistent with the prediction. The p-nitrophenyl butyrate (PNPB) was used as the substrate to verify the enzyme activity (Figure 1a,b). The results showed that SpL exhibited a strong ability to hydrolyze PNPB. IPTG has been demonstrated to regulate the expression of genes, thereby indirectly affecting the enzyme activity. On one hand, IPTG can facilitate the expression of target enzymes; on the other hand, excessive IPTG may lead to the synthesis of target enzymes in large quantities at a rapid rate within the cell, which may result in a proportionate reduction in the active enzyme due to its inability to fold correctly into an active conformation. An analysis was conducted to determine the effect of varying concentrations of IPTG on enzyme activity (Figure 1c). The results showed that SpL exhibited the highest enzyme activity at an IPTG concentration of 0.4 mM. Beyond this concentration, a decrease in activity was observed. This suggests that the improvement concentration of 0.4 mM not only facilitates the expression of SpL but also ensures the proper folding of the enzyme. Subsequently, the induction time was optimized in the presence of 0.4 mM IPTG (Figure 1d). It was observed that when the induction time was between 6 and 15 h, there was a gradual and significant enhancement of enzyme activity until the highest activity was reached at 18 h.

2.2. SpL-Catalyzed Ammonolysis Reaction for FBA Synthesis

Lipase-catalyzed aminolysis of esters was typically conducted in anhydrous organic solvents, which was undesirable for green catalysis. However, SpL, a newly discovered intracellular lipase, can facilitate aminolysis of both esters and acids under aqueous conditions. Consequently, we utilized butyric acid and ethyl butyrate as substrates for the aminolysis reaction with L-phenylalaninamide for the synthesis of FBA. We catalyze this reaction in n-hexane with 10% water and dimethyl sulfoxide (DMSO) solvents, respectively. Interestingly, FBA generation was only detected in the n-hexane system, but not in the DMSO system (

Table 1. The detection of FBA in different reaction systems.

	Acyl Substrates	10% Aqueous	Anhydrous
n-hexane	Butyric acid	-	-
	Ethyl butyrate	+	-
DMSO	Butyric acid	-	-
	Ethyl butyrate	-	-

+ means detected; - means not detected.

, Figure 2a). Subsequently, we measured it in a completely anhydrous n-hexane and DMSO reaction system. The results showed that there was no FBA production in either of these completely anhydrous systems. This result suggests that in the n-hexane system, the appropriate amount of water participates in and promotes the reaction process, which leads to the production of FBA, whereas in the DMSO system, the reaction cannot proceed in the direction of FBA generation. Therefore, we selected a biphasic reaction system of n-hexane with 10% water for the synthesis of FBA. These results indicate that the presence of water is essential for the catalytic activity of SpL. Therefore, we assessed the catalytic activity at different aqueous ratios (Figure 2b). Under an equal amount of substrates, the activity gradually decreased as the ratio of aqueous increased, with optimal activity observed at an aqueous content of 2%.

2.3. The Optimization of the Catalytic Conditions of SpL

The effect of temperature on the activity and stability of recombinted SpL was illustrated in Figure 3a,b. SpL exhibited mesophilic properties, with the highest catalytic capacity at 30 °C. When the reaction temperature reached 40 °C, the activity decreased significantly, with relative catalytic activity dropping to only 20%. There was little activity above 45 °C. Subsequently, we evaluated the temperature stability of SpL at 25 °C, 30 °C, 35 °C, and 40 °C. SpL showed poor stability at 40 °C, becoming completely inactivated after 2.5 h of incubation. Furthermore, during the incubation process, it was evident that the protein gradually denatured and formed a precipitate as the incubation time extended. The stability at 25 °C and 30 °C were relatively good, and there was still more than 60% residual activity after incubation for 4 h. The results of the analysis of the effect of pH on the synthesis of FBA showed that it was beneficial to the synthesis of FBA under weak alkaline conditions, and the optimal pH was 8.5 (Figure 3c). Subsequently, we evaluated the effect of reaction time on the synthesized FBA concentration under the optimal temperature and pH conditions for SpL (Figure 3d). The FBA titer gradually increased with the reaction time, reaching a maximum after 15 h. However, as the reaction continued, a gradual decrease in the concentration of FBA was noted, which may be attributed to SpL’s ability to degrade amides. We also assessed the impact of enzyme concentration on FBA synthesis. We measured the concentration of FBA produced using SpL pure enzyme solutions ranging from 0 to 10 mg/mL (Figure 3e). As the enzyme concentration gradually increased, the concentration of FBA also rose correspondingly. When the concentration of SpL was 10 mg/mL, 0.89 mg/mL FBA can be finally produced with a yield of 76%. It was showed that the appropriate ratio of ethyl butyrate to enzyme usage was 3:1 (mg/mg) within the enzyme concentration range of 0–10 mg/mL. After the reaction, the product in aqueous phase was extracted with ethyl acetate. The mass spectrometry and NMR showed that the product is FBA (Figure 4).

At present, there are few studies on the enzymatic synthesis of FBA. Kitaguchi et al. reported that FBA can be produced in the racemic reaction of amines using Subtilisin Carlsberg. The use of SpL does not require expensive ethyl trifluorobutyrate and allows the reaction to occur even in the presence of water. Moreover, compared to previous studies in our lab that used acetyl-CoA synthetase to synthesize FBA, SpL does not require an expensive ATP, which has relatively good atomic economy.

2.4. Analysis of Mechanism of SpL Catalyzed FBA Synthesis

Over the past few decades, lipases have been thoroughly studied. The amino acid sequence of SpL was submitted to the NCBI for BLASTp analysis using the non-redundant protein sequence (nr) database, which indicated that it belonged to the hormone-sensitive lipase-like protein family, exhibiting the same structural framework and catalytic mechanism in the form of α/β hydrolase folds [21]. Further BLASTp analysis with the Protein Data Bank showed that SpL had the highest sequence identity of 46.52% with the alkalophilic esterase from Erythrobacter longus (E53). Through multiple sequence alignment of SpL with carboxylesterase from Sulfolobus tokodaii (Sto-Est), thermostable esterase from Pyrobaculum calidifontis (PestE), and E53, three highly conserved motifs were identified: GDSAGG, HGGG, and DYRLAPEXXFPAA (Figure 5a). Structural identification of SpL revealed a cap above the active site, consisting of an N-terminal α-helix. The excellent catalytic activity of SpL in duplex systems may be due to the fact that the cap located above the active site of lipase is both hydrophilic and lipophilic. In the aqueous phase, although the protein molecules are well dispersed, the lid is closed, preventing the substrate molecules from approaching the active site. In the organic phase, the lid is open, but the protein molecules tend to coalesce and have a large diffusion limit. At the oil/water interface, the lid is opened by the organic phase and, at the same time, well dispersed by the aqueous phase, so the lipase has greater catalytic activity.

In the center of microbial lipase active site, serine, aspartic acid, and histidine of the GDSAGG motif constitute a catalytic triad that mediates the catalytic reaction of lipase. The HGGG motif is involved in the formation of the oxyanion hole of the substrate, which plays a crucial role in stabilizing the tetrahedral intermediate of the reaction [22,23,24]. The molecular docking of FBA with the 3D structure of SpL was carried out, and the resulting complexed model was used for structural mechanism analysis (Figure 5b,c). As depicted in the figure, the two α-helices (α1 and α2) at the N-terminus form the CAP domain, which overlays the catalytic site and functions as a substrate entry channel. The active pocket of SpL was composed of two channels: one serves as an acyl-binding tunnel, and the other functions for amine donors. Within the active pocket at a range of 4 Å, aside from the catalytic triad Ser159, His281, and Asp251, the active center of SpL was predominantly constituted by hydrophobic residues like glycine, methionine, leucine, isoleucine, and alanine (Figure 5d). When ethyl butyrate was bound to the active pocket, Asp251 first polarized His281 by electrostatic action, causing the imidazole ring of histidine to be partially negatively charged, thereby enhancing its alkalinity. The polarized histidine then seized the proton of the Ser hydroxyl group, making the oxygen atom of serine a strong nucleophile. At this point, the negatively charged serine oxygen atom initiates a nucleophilic attack on the carbonyl carbon atom of the substrate ester bond, generating acyl-enzyme intermediates. The amine donor then enters the active center and is activated by polarized His281. The activated amine donor acts as a nucleophile to attack the carbonyl carbon atom in the acyl-enzyme intermediate to form the amide product FBA.

2.5. Analysis of the Hydrolysis Mechanism of SpL

Notably, we discovered that SpL possesses a unique mixed catalytic ability, enabling it to catalyze both the formation and hydrolysis of amides. This dual functionality is particularly intriguing, as it suggests potential applications in synthetic and biodegradation processes. When investigating the correlation between the catalytic activity of SpL and reaction time, it was observed that the concentration of FBA underwent a substantial decline after 15 h of reaction commencement. Simultaneously, the reaction was accompanied by the conversion of phenylalaninamide to phenylalanine. To further verify this phenomenon, we conducted separate measurements of the degradation of phenylalaninamide and FBA by SpL, as illustrated in Figure 2a. It is well known that esterases and lipases generally lack the ability to catalyze the degradation of amide bonds. However, the p-nitrobenzyl (PNB) esterase from Bacillus subtilis (Bs2) and CALB have been reported to be members of a homofunctional family of non-homologous enzymes. Bs2 and CALB exhibit mixed catalytic activities, encompassing the hydrolysis of carboxylates as well as the hydrolysis of amides [25,26]. The hydrolysis of amides presents a significant challenge as it requires the overcoming of a substantial energy barrier, rendering it a secondary reaction. Galmes et al. conducted an in-depth analysis of the reaction mechanism underlying the amide hydrolysis catalyzed by Bs2 and CALB [27]. This mechanism was elucidated to occur in four distinct steps: substrate acylation, release of the amine donor, hydrolysis of the acyl enzyme complex, and finally, product formation along with the regeneration of the active site. The active site residues Ser159, His281, and Asp251 of SpL correspond to the equivalent residues Ser189/105, His399/224, and Glu310/Asp187 in Bs2 and CALB, respectively. We speculated that the mechanism by which SpL catalyzes the hydrolysis of amide bonds is as follows: The electrophilicity of the amide carbonyl carbon facilitates the attack of Ser159. Subsequently, the transfer of protons from His281 to the amide nitrogen atoms leads to the cleavage of the C-N bond and the formation of the phenylpropionamide leaving group. Meanwhile, the acyl moiety binds to the protein through both covalent and hydrogen bonds. The acid-base characteristics of His281 are modulated by its interaction with Asp251. During the reaction, water molecules attack the activated C1 atoms. Eventually, His281 transfers protons to Ser159, which promotes the release of butyric acid and the regeneration of the active site.

3. Materials and Methods

3.1. Materials

Standard FBA was synthesized in accordance with the patent US20230416189. Butyric acid, ethyl butyrate, L-phenylalaninamide, and p-nitrophenyl butyrate (PNPB) were purchased from Adamas (Titan^®, Shanghai, China). Kanamycin and Isopropyl-β-D-thiogalactoside (IPTG) were purchased from Sangon Biotech (Shanghai, China). Unless otherwise specified, the rest of the chemicals are purchased from reputable suppliers such as Aladdin (Shanghai, China), Macklin (Shanghai, China), or Sinopharm (Shanghai, China). The expression strains utilized in this study were E. coli BL21 (DE3), which were cultured in Luria-Bertani (LB) medium at 37 °C for 18 h and meticulously preserved at −80 °C in our laboratory.

3.2. Cloning and Expression of the SpL

The codon-optimized genes of SpL were designed and synthesized commercially, which were carried out by Azenta (Suzhou, China). The gene of SpL was subcloned into the BamH I/Hind III restriction site of the pET-28a (+), and the His-tag was located at the N-terminus of the gene. Then, the recombinant plasmid was transformed into E. coli BL21 (DE3). The transformed cells were then cultured on Luria-Bertani (LB) solid medium supplemented with 50 µg/mL kanamycin at 37 °C for 16 h. After the incubation period, a single transformant colony was selected and inoculated into a fresh LB liquid medium for expansion of the culture. The cultures were then transferred to 50 mL fresh LB medium to be incubated at 37 °C with shaking at 220 rpm until the optical density at 600 nm (OD₆₀₀) reached a range of 0.6–0.8. At this point, 0.1–0.5 mM IPTG was added to the culture to induce the expression of the target protein. The induced culture was then incubated at 20 °C for 24 h to allow for protein expression.

3.3. Purification and Lyophilization of the SpL

Cells were harvested through low-temperature centrifugation at 8000 rpm and 4 °C for 10 min and resuspended in 20 mM sodium phosphate buffer. This cell suspension was then subjected to sonication in an ice bath for 30 min with sonicators working for 2 s and stopping for 3 s. Following sonication, further centrifugation was carried out. The resulting crude enzyme was purified using nickel affinity chromatography on an AKTA avant 25 (GE Healthcare, Fairfield, CT, USA). The purification buffer consisted of 20 mM sodium phosphate, 200 mM NaCl, and a gradient of 20–300 mM imidazole, with a pH of 7.4. The 5 mL Ni-HisTrap HP column (GE Healthcare, Shanghai, China) was equilibrated and washed with 20 mM phosphate buffer containing imidazole (20 mM) and NaCl (200 mM). Continuous gradient elution buffer (20 mM phosphate buffer containing 20–300 mM imidazole and 200 mM NaCl) was used to elution. Subsequently, the purified enzyme was desalted using a dialysis tube (28 mm, 14 kDa, Biosharp, Hefei, China). The concentration of the pure enzyme was determined via the Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China). After desalting, the enzyme solution was processed in a freeze dryer (FreeZone 4.5 L, Labconco company, Kansas, MO, USA) to yield a lyophilized powder. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to further analyze the enzyme using a 10% PAGE Gel Quick preparation Kit (YEASEN, Shanghai, China) for further analysis of the enzyme. The voltage and current during electrophoresis were 140 V and 45 mA, respectively, using Tris-Gly buffer.

3.4. Activity Assay of SpL

The PNPB method was employed to verify SpL activity. The reaction system consisted of 50 mM phosphate buffer, 0.1 mM PNPB (prepared from a 10 mM stock solution in acetonitrile), and 0.1 μg of SpL. The reaction was carried out at a pH of 7.5 and a temperature of 30 °C.

For the FBA synthesis reaction system, it contained 5 mM ethyl butyrate, and 5 mM phenylalaninamide. DMSO or n-hexane was used as the solvent, respectively, along with an appropriate amount of enzyme solution. The reaction was incubated at 30 °C with 110 rpm for 12 h. After the reaction, the reaction solution was mixed with an equal volume of acetonitrile and centrifuged at 12,000 rpm for 5 min. The synthesis of FBA was analyzed using a Waters HPLC system equipped with a C18-H column (4.6 mm × 250 mm, 3 μm, Diacel, Tokyo, Japan). The mobile phase consisted of water and acetonitrile, both containing 0.1% TFA and was used for gradient elution at a flow rate of 1 mL/min and a temperature of 35 °C. The detection wavelength was set at 217 nm. The enzyme activity for the synthesis of FBA was defined as the generation of 1 μmol of FBA per minute as one enzyme activity unit (U), and the enzyme activity is calculated as follows:

$$U=c\times V÷T$$

where U is the enzyme activity (μmol·min⁻¹), c is the concentration of FBA (μmol/mL), V is the total reaction system (mL), and T is the reaction time (min).

3.5. Effect of pH and Temperature on Enzymatic Activity

The enzyme activity was measured under the following experimental conditions. Using ethyl butyrate and L-phenylalaninamide as substrates, the reaction mixture was incubated at temperatures of 20, 30, 40, 50, and 60 °C for 12 h, with 110 rpm and a pH of 7.5, to determine the enzyme activity. Subsequently, the optimal reaction temperature was derived through data analysis. To assess the optimal pH, different buffer systems were employed within a pH range of 3.0–10.0. Specifically, a (20 mM) standard disodium hydrogen phosphate-citric acid buffer was used for pH 3.0–5.0, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer for pH 6.0–8.0, and a sodium carbonate-sodium bicarbonate buffer for pH 9.0–10.0. The reaction system was maintained at 30 °C with 110 rpm for 12 h while the pH varied from 3.0 to 10.0 to measure the enzyme activity, from which the optimum reaction pH was determined.

3.6. Thermal and pH Stability

In stability assays, the reaction mixture was incubated at 30 °C for 12 h, with 110 rpm and a pH of 8.5. An appropriate volume of the pure enzyme solution was incubated at each of the aforementioned temperatures for 4 h, followed by a 10 min ice bath treatment. The residual activity of the enzyme was measured at half-hour intervals during this incubation period. The remaining enzyme activities were determined after subjecting the enzyme solution to different temperature treatments with ethyl butyrate and L-phenylalaninamide as substrates. Subsequently, an appropriate volume of the pure enzyme solution was incubated at each of the various pH values specified above for 2 h to assess the remaining enzyme activity.

3.7. The Preparation and Characterization of FBA

FBA was prepared under the optimal reaction conditions in a 500 mL reaction volume. Since FBA was present in the aqueous phase, an equal volume of ethyl acetate was added for extraction after the reaction, the organic phase was collected and washed twice with the same volume of water, and then the organic phase was evaporated at 40 °C. The precipitate product was recrystallized in hot water to obtain FBA.

3.8. Molecular Docking

Molecular docking was carried out using MOE 2019 software. The pdb file of SpL and the mol file of small molecule FBA were imported into the MOE software for protein structure pretreatment and small molecule energy minimization, respectively. The FBA was then docked to the specified active site region. After the docking is completed, it is analyzed according to criteria such as score and docking posture. The structural analysis was performed in pymol.

4. Conclusions

In this study, we successfully synthesized FBA in a biphasic reaction system of water and n-hexane using the only intracellular lipase identified to date. Our initial validation of SpL’s effectiveness through the PNPB method, along with the evaluation of IPTG concentration and induction time, revealed that optimal SpL expression and catalytic activity occur at 0.4 mM IPTG after 18 h of induction. Further investigations demonstrated that the water and n-hexane duplex system significantly enhances FBA synthesis, with the optimal water content identified as 2%. This finding highlights the critical role of water in facilitating the catalytic process. Additionally, we established that the optimal conditions for SpL-catalyzed FBA synthesis are 30 °C and pH 8.5. Our exploration of enzyme concentration revealed that an SpL concentration of 10 mg/mL yields 0.89 mg/mL of FBA, indicating a promising efficiency that could be further optimized in future studies. Notably, we discovered that SpL also was able to catalyze the hydrolysis of amides. Our predictions regarding the mechanism of amide hydrolysis catalyzed by SpL, based on comparative studies with Bs2 and CALB lipases, open new avenues for understanding the enzymatic mechanisms at play and highlight the potential of SpL as a versatile biocatalyst. In conclusion, our findings not only advance the understanding of FBA synthesis and its catalytic mechanisms but also position SpL as a promising candidate for biotechnological applications. Future research should focus on further elucidating the structural basis for SpL’s dual catalytic activity and exploring its potential in industrial applications.