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Article

Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate

by
Fengying Dong
1,
Xudong Tang
1,
Xiaohui Yang
1,
Lin Lin
2,3,
Dannong He
3,
Wei Wei
1,* and
Dongzhi Wei
1,*
1
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China
2
Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China
3
Research Laboratory for Functional Nanomaterial, National Engineering Research Center for Nanotechnology, Shanghai 200241, China
*
Authors to whom correspondence should be addressed.
Catalysts 2019, 9(7), 620; https://doi.org/10.3390/catal9070620
Submission received: 19 May 2019 / Revised: 15 July 2019 / Accepted: 16 July 2019 / Published: 21 July 2019
(This article belongs to the Special Issue Biocatalysts: Design and Application)
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Abstract

:
Novel gene estBAS from Bacillus altitudinis, encoding a 216-amino acid esterase (EstBAS) with a signal peptide (SP), was expressed in Escherichia coli. EstBASΔSP showed the highest activity toward p-nitrophenyl hexanoate at 50 °C and pH 8.0 and had a half-life (T1/2) of 6 h at 50 °C. EstBASΔSP was immobilized onto a novel epoxy resin (Lx-105s) with a high loading of 96 mg/g. Fourier transform infrared (FTIR) spectroscopy showed that EstBASΔSP was successfully immobilized onto Lx-105s. In addition, immobilization improved its enzymatic performance by widening the tolerable ranges of pH and temperature. The optimum temperature of immobilized EstBASΔSP (Lx-EstBASΔSP) was higher, 60 °C, and overall thermostability improved. T1/2 of Lx-EstBASΔSP and free EstBASΔSP at 60 °C was 105 and 28 min, respectively. Lx-EstBASΔSP was used as a biocatalyst to synthesize chloramphenicol palmitate by regioselective modification at the primary hydroxyl group. Conversion efficiency reached 94.7% at 0.15 M substrate concentration after 24 h. Lx-EstBASΔSP was stable and could be reused for seven cycles, after which it retained over 80% of the original activity.

Graphical Abstract

1. Introduction

Chloramphenicol was first isolated and purified from Streptomyces venezuelae by Burkholder in 1947 [1]. Following its discovery, chloramphenicol was synthesized via chemical methods and produced on an industrial scale, and then introduced into clinical practice as a therapeutic agent two years later [2]. Chloramphenicol was notably efficient against 95% of gram-negative bacteria and against a variety of gram-positive aerobic strains (including the dreaded strains of methicillin-resistant Staphylococcus aureus). However, the extensive use of chloramphenicol in the treatment of infectious diseases in humans and animals has caused increasing concerns about its effect on human health. It was subsequently proved to have severe adverse effects on humans; these effects could lead to aplastic anemia, childhood leukemia, grey baby syndrome, and bone marrow suppression [3]. Additionally, its bitter taste has led to the development of chloramphenicol alternatives. Many studies have addressed the derivatization of chloramphenicol, including selective esterification of the hydroxyl groups at positions 1 and 3, with many studies demonstrating selective esterification of hydroxyl group 1 as the best derivatization method, which yields products such as chloramphenicol palmitate or chloramphenicol succinate esters. Chloramphenicol palmitate is an important chloramphenicol derivative and can be quickly hydrolyzed in vivo into the biologically active drug [4]. Nonetheless, the selective modification at one specific hydroxyl group in a polyhydroxy compound is a difficulty in the field of organic chemistry due to time-consuming protection and deprotection procedures to discriminate between the available hydroxyl groups. Furthermore, the separation process to remove the unwanted compounds increases costs and decreases the final yield. For this reason, biocatalytic transformations with high regioselectivity have become the standard procedure for the selective acylation of polyfunctionalized derivatives.
Among all biocatalytic processes, lipase reactions have many advantages owing to their high regioselectivity and mild incubation conditions, and maximize the efficiency of trivial processes in comparison to chemical methods [5,6,7]. Recently, it was reported that lipase can serve as a catalyst to regioselectively produce chloramphenicol derivatives. Ottolina et al. reported the regioselective synthesis of several derivatives using Chromobacterium viscosum lipase and lipase G in acetone, thus obtaining excellent yields (98%) after 24 h at 45 °C [4]. Later, Lv et al. reported the synthesis of chloramphenicol vinyl esters using Lipozyme from Bacillus subtilis, thereby demonstrating the versatility of hydrolases [8]. Among chloramphenicol derivatives, chloramphenicol palmitate has been studied only in a few works. Bizerra et al. obtained a yield of 98% using Candida antarctica lipase B at 0.25 M chloramphenicol, and Wang et al. demonstrated that an imprinted lipase nanogel (Lipozyme TL 100L, Novozymes UK Ltd, Nottingham, UK) can have a yield of 99% within 12 h at 20 °C [5,9]. However, the lipases discussed above are commercially available enzymes. To date, there have been no reports of novel lipases with high regioselectivity and high activity used to effectively synthesize chloramphenicol palmitate [10,11]. Therefore, there is an increasing demand for a kind of novel lipase from different species and with industrial applicability.
Free lipase is usually obtained in an aqueous form, giving it low stability and making its recycling difficult. Immobilization of lipase is a good way to improve its enzymatic performance. Immobilization of enzymes can increase their recyclability and improve the economic value and efficiency of their use in nonaqueous conditions [12,13]. Many immobilization methods have been reported, including encapsulation, entrapment, covalent binding, and cross-linking [14,15]. Among these methods, encapsulation and entrapment can lead to multiple interactions between the enzyme and the immobilization surface; these interactions can block the active site. However, covalent immobilization methods result in strong interactions between the enzyme and some functional groups of the immobilization surface, such as epoxy groups, amide and aldehyde, which can make enzyme leaching difficult and ensure high retention of a catalytic activity [16,17,18]. Among these groups, epoxy moieties are the most extensively applied for covalent binding of lipases because epoxy moieties carry simple groups for covalent binding and contain abundant active amino, phenolic, and thiol groups at a specific pH condition. Epoxy moieties have short spacer arms that are highly stable at pH 7.0 and that can support the enzyme on the surface of particles, thereby reducing steric hindrance of some immobilization enzymes and improving the whole catalytic activity [17,19,20,21,22].
In this research, we obtained a novel esterase gene (estBAS) from Bacillus altitudinis and immobilized the recombinant protein without the signal peptide (SP) (EstBASΔSP) on a novel epoxy resin (Lx-105s). Furthermore, for the first time, we report Lx-EstBASΔSP (EstBASΔSP immobilized on Lx-105s) and free EstBASΔSP to synthesize chloramphenicol palmitate with high regioselectivity and high conversion efficiency (94.7%, at 0.15 M substrate). In short, this novel esterase shows a potential for the industrial application of chloramphenicol palmitate synthesis.

2. Results and Discussion

2.1. Cloning of the EstBAS Gene and Sequence Analysis of EstBAS

estBAS and estBASΔSP from B. altitudinis were amplified with primer pairs F1/R1 and F2/R1, respectively. The open reading frame of estBAS was found to consist of 648 bp, encoding a protein of 216 amino acid residues with a molecular mass of 22.96 kDa and a theoretical pI of 9.85, as calculated by the ExPASy compute pI/Mw tool. The nucleotide sequence was deposited in GenBank under the accession number MH898984. Homology analysis of the amino acid sequence revealed that EstBAS of B. altitudinis shares the highest identity (99%) with the triacylglycerol lipase of Bacillus sp. and B. pumilus (GI: 639684939 and 408690817) and is 94% identical to the triacylglycerol lipase of Bacillus sp. LLTC93 (GI: 1360561895) (92% to B. pumilus AVI41987, GI: 1351330082; 89% to B. pumilus AFU81785 and B. safensis, GI: 408690815 and GI:1130455194; 77% to B. atrophaeus 010787545 and B. atrophaeus 063638677, GI:498485857 and 1027703361; and 75% identical to B. velezensis, GI: 818920927; Figure 1). Visual inspection of the sequence alignment uncovered conservation of amino acid residues in the regions related with catalysis and stabilization of the protein, e.g., the catalytic triad Ser111, Asp167, and His190 (Figure 1a). The cleavage site for the SP turned out to be located at residue 25, as determined by SignalP 5.0 Server (http://www.cbs.dtu.dk/services/SignalP/) [23]. Because this esterase has not been previously reported, the secondary and three-dimensional structures of EstBAS were predicted by means of the SWISS-MODEL server, and the structure of EstBAS was generated with a specific template (Protein Data Bank (PDB) code: 1T4M (54% similar and 79.01% identical); Figure 1b,c), which was viewed in PDB Viewer.

2.2. Expression and Purification of Recombinant EstBASΔSP

Genes estBAS and estBASΔSP were subcloned into vector pET-22b. The recombinant plasmid was transfected into BL21 (DE3), and the engineered strains (BL21-EstBAS, BL21-EstBASΔSP) were grown in the Luria–Bertani (LB) medium with the ampicillin antibiotic. Optimal expression was obtained using isopropyl-β-d-thiogalactopyranoside (IPTG) at 0.1 mM and 20 °C for 20 h. Crude and purified protein are depicted in Figure 2 (purified fractions of EstBAS are not shown in the image of sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE). SDS-PAGE revealed that the recombinant proteins, EstBAS and EstBASΔSP, have similar soluble expression profiles when induced at low temperatures (20 °C; Figure 2). EstBAS purification was unsuccessful: only a small amount of the purified protein was obtained (data not shown) when compared to EstBASΔSP purified via the same Ni metal affinity chromatography (Ni-NTA) procedure. This may be because the His6 tag and SP sequences were cleaved after translation, preventing the binding of EstBAS to the Ni-NTA agarose affinity chromatography column. The above results are consistent with those of Harris et al. [24].

2.3. Optimization of EstBASΔSP Immobilization

Purified EstBASΔSP was immobilized onto novel epoxy resin Lx-105s at 30 °C. The immobilization process is outlined in Scheme 1a. The effects of the initial amount of esterase, immobilization time on the loading capacity, and the relative esterase activity were investigated. The relative activity and EstBASΔSP loading gradually increased to a peak (78.5 mg/g) when 90 mg of esterase was initially added (Figure 3a). After that, the relative activity decreased even if EstBASΔSP loading was slightly augmented. Considering both esterification activity and esterase loading, 90 mg esterase was selected for immobilization to the epoxy resin Lx-105s. The above results may be explained in that unsaturated carriers provide abundant sites for esterase to immobilize to, hence improving the relative activity; whereas when more esterase was added, it caused the overcrowded accumulation of esterase onto the epoxy resin, leading to some active sites to be blocked [25]. Silva et al. obtained the same results when immobilizing lipases derived from Candida rugosa onto dialdehyde polyethylene glycol (PEG)-modified particles with physical adsorption style. They found that the open conformation of the enzyme was necessary to keep lipase activity high and to keep the enzyme stable [26]. As shown in Figure 3b, the loading capacity was time-dependent and the optimum time for esterase immobilization was 7 h with the loading efficiency reaching approximately 96 mg/g. After an increase in duration from 7 to 14 h, the relative activity decreased gradually and loading tended to be stable. This may be because the esterase was denatured with long-term shaking, causing a further decrease in activity at 30 °C [27].

2.4. Characterization of Lx-EstBASΔSP by FTIR

FTIR was used to investigate the potential interactions between the amino groups of the enzyme and the epoxy groups of the Lx-105s resin (Figure 4). Figure 4 shows FTIR spectra of the Lx-105s resin (a), Lx-EstBASΔSP (b), and free EstBASΔSP (c). The free Lx-105s resin had characteristic peaks centered at 3063 and 908 cm−1, which are commonly assumed to be the epoxy groups stretching bands. Lx-EstBASΔSP had corresponding characteristic peaks at 3060 and 908 cm−1, but these were not present in the spectra of the free EstBASΔSP indicating the presence of epoxy groups in Lx-EstBASΔSP. Furthermore, absorbance bands at 3189 and 1631 cm−1 for the amide groups of the free lipase were present, while for Lx-EstBASΔSP the amide group bands shifted from 1631 to 1636 cm−1. Additionally, the broad peak appeared in a range of 3100–3600 cm−1 corresponding to hydroxyl and amino groups stretching. These results indicated that EstBASΔSP was successfully immobilized onto Lx-105s resin [28,29].

2.5. Substrate Specificity of EstBASΔSP

The substrate specificity of purified EstBASΔSP was determined using p-NP esters with variable chain length: p-NP acetate (pNPA), p-NP butyrate (pNPB), p-NP hexanoate (pNPH), p-NP caprylate (pNPC), p-NP decanoate (pNPD), p-NP laurate (pNPL), p-NP myristate (pNPM), and p-NP palmitate (pNPP) at 60 °C and pH 8.0. The substrate specificity of EstBASΔSP towards the p-NP esters of various fatty acids is presented in Figure 5. The results showed that EstBASΔSP hydrolyzed pNPP (acyl chains of up to 16 carbons). EstBASΔSP manifested higher activity for short-chain fatty acids (C < 10) compared with long-chain fatty acids (C ≥ 10; Figure 5). The highest specific activity toward pNPH was 1414 U mg−1. Taken together, these results indicated that EstBASΔSP is an esterase that preferentially hydrolyzes short acyl chain (C < 10) substrates [30] and has a broad spectrum of substrates: from pNPA to pNPP.

2.6. Effects of pH and Temperature on the Activity of Free EstBASΔSP and Lx-EstBASΔSP

Data on pH of a reaction help to understand the structure–function relationship of an enzyme [31]. Therefore, the impact of pH on the enzymatic properties of Lx-EstBASΔSP and free EstBASΔSP was investigated at various pH levels ranging from 4.0 to 10.0 with pNPH as a test substrate at 60 °C (Figure 6a). This figure shows that free EstBASΔSP retained a maximal activity above 60% at pH from 6.0 to 9.0 with an optimal pH of 8.0. The activity significantly decreased at pH below 6.0, with approximately 30% of the maximal activity observed at pH 4.0, meaning that free EstBASΔSP is an alkaline esterase. Nevertheless, after immobilization, EstBASΔSP retained approximately 80% of activity at pH from 6.0 to 9.0, and >50% of the maximal activity was observed at values below pH 8.0. This finding suggested that Lx-EstBASΔSP could endure a wider pH range. The possible reason is that immobilization maintains the enzyme in a more stable conformation [32,33].
The effects of temperature on the activity of Lx-EstBASΔSP and free EstBASΔSP were investigated at pH 8.0 with pNPH as the test substrate (Figure 6b). Free EstBASΔSP was found to have a relatively narrow range (40–60 °C) with an optimal temperature of 50 °C and retained only 60% of its maximal activity at 20 °C. Thermal stability of Lx-EstBASΔSP was much higher as compared with free EstBASΔSP. Lx-EstBASΔSP manifested broad temperature stability, ranging from 30 to 70 °C, and the optimal temperature of Lx-EstBASΔSP was found to be 60 °C. Additionally, Lx-EstBASΔSP is capable of retaining 75% of its maximal activity at 20 °C and 35% at 90 °C. In contrast, the activity of free EstBASΔSP decreased to 2% at 90 °C. Similar results were obtained by Li et al., who demonstrated that the activity of an immobilized lipase on an epoxy resin (ECR8285) increases by 40% when compared with free lipase at 50 °C [29]. The increased temperature stability of Lx-EstBASΔSP may be attributed to the rigid structure of EstBASΔSP; this rigidity resulted in a wider temperature endurance range.

2.7. Effects of Metal Ions and Detergents on Lx-EstBASΔSP and Free EstBASΔSP

The influence of some chemicals on Lx-EstBASΔSP and free EstBASΔSP was investigated (Table 1). Table 1 suggests that the activity of Lx-EstBASΔSP and free EstBASΔSP is inhibited by most of the heavy metal ions. Lx-EstBASΔSP and free EstBASΔSP were highly active (>90%) at low metal ion concentrations (1 mM). In contrast, Mg2+ slightly activated EstBASΔSP (105%, 108%) at low concentrations (1 mM). Nevertheless, the activity of Lx-EstBASΔSP and free EstBASΔSP decreased quickly at the concentrations ranging from 5 to 10 mM. Of note, Lx-EstBASΔSP showed a higher activity than free EstBASΔSP at concentrations ranging from 1 to 10 mM. Similarly, Mishra et al. also observed this activation function by Mg2+. He observed that Lecitase® Ultra phospholipase activity had a 1.5-fold activation in the presence of 10 mM Mg2+. He explained that salts are well known to cause changes in enzyme activities via different mechanisms, such as Mg2+, which can activate the enzyme to a lesser extent, and Mg2+ also can function without other metal ions [34].
The detergents had various effects on the activity of esterase (Table 2). As presented in Table 2, a slight decrease in the activity of esterase (94%) was observed upon the addition of 1% of Tween 20, Tween 60 (94.5%), or Tween 80 (94.8%) compared with the control. In contrast, Lx-EstBASΔSP had a higher activity (95%, 96%, and 95%, respectively). On the contrary, the activity of Lx-EstBASΔSP and free EstBASΔSP was significantly inhibited by 1% Triton X-100 (to 38% and 36.1%, respectively) and SDS (to 20% and 18.9%). Furthermore, the activity of free EstBASΔSP disappeared at SDS and Triton X-100 concentrations up to 10%, accordingly, activity of Lx-EstBASΔSP was 0% and 8%. As a result, Lx-EstBASΔSP manifested enhanced resistance to high concentrations of metal ions and detergents. The reason was that the formed covalent bond with the support limited the conformational changes of EstBASΔSP, resulting in a more rigid structure of Lx-EstBASΔSP. However, the loss of enzymatic activity was due to some specific interaction between detergents and the enzyme surface [34].

2.8. Stability of Lx-EstBASΔSP and Free EstBASΔSP

Thermal stability of Lx-EstBASΔSP and free EstBASΔSP is described in Figure 6c. The activity of Lx-EstBASΔSP decreased gradually with incubation time, whereas the activity of free EstBASΔSP decreased almost linearly during 90 min of incubation. Furthermore, Lx-EstBASΔSP retained approximately 60% of its activity at 60 °C after 90 min of incubation, whereas free EstBASΔSP lost almost all activity under the same conditions. This was likely due to the stability of the protein three-dimensional structure of the immobilized enzyme. Xie et al. reported that an immobilized enzyme has lower sensitivity to temperature and that the conformational integrity of the enzyme structure changes due to its covalent binding to the carrier when compared with the free enzyme [35,36].
Storage stability is another important factor for industrial applications of immobilized enzymes. A comparison of storage stability between free and immobilized enzymes is illustrated in Figure 6d. Free and immobilized enzymes followed a similar trend, but the decrease in the Lx-EstBASΔSP activity was lesser and slower than that of the free EstBASΔSP. Lx-EstBASΔSP retained over 85% of its initial catalytic activity after storage for six weeks (4 °C), whereas the free enzyme lost ~80% of its activity after only two weeks and lost almost all its activity in less than three weeks. These results clearly indicated that enzyme immobilization significantly increased storage stability, in line with another report [37].
In organic reaction systems, the organic-solvent tolerance of enzymes is especially important. Here, four organic solvents (1, 4-dioxane, ethanol, acetone, and acetonitrile) were tested (Table 3). The activity of both Lx-EstBASΔSP and free EstBASΔSP decreased after 70 h of incubation. For free EstBASΔSP, incubation with 90% (v/v) 1, 4-dioxane, ethanol, and acetone reduced the activity to 12.6%, 52.5%, and 25.0%, respectively, with undetectable activity in the presence of acetonitrile. Lx-EstBASΔSP retained 80.2%, 92.5%, 93.8%, and 92% of its activity in the presence of 1, 4-dioxane, ethanol, acetone, and acetonitrile, respectively, revealing that Lx-EstBASΔSP has excellent organic-solvent tolerance. These results are consistent with other research [38]. Li et al. have reported that organic solvents strongly interact with enzymatic active sites; therefore, their presence could quickly render the enzyme inactive. However, the presence of the carrier may change the enzyme structure in terms of protein folding or prevent the direct interaction of the organic solvent with the enzyme [38].

2.9. Regioselective Synthesis of Chloramphenicol Esters by Lx-EstBASΔSP and Free EstBASΔSP

To assess the catalytic activity of the immobilized enzyme and free enzyme, we tested them as catalysts in the regioselective synthesis of chloramphenicol esters in acetone at 60 °C (Scheme 1b). As illustrated in Figure 7a, free enzyme had a slightly faster conversion rate than immobilized enzyme in the first three hours, then the immobilized enzyme maintained a similar conversion trend with the free enzyme from 6 to 24 h. From 21 to 24 h, the conversion rate was stable, reaching 94.7% and 90% for Lx-EstBASΔSP and free EstBASΔSP, respectively, at 0.15 M chloramphenicol (Figure 7a). The free enzyme featured faster conversion velocity, especially in the initial three hours; this property may be attributed to improved mass transfer in the procedure of esterification. On the other hand, Lx-EstBASΔSP manifested the highest catalytic efficiency, which could be due to its good catalytic activity, organic-solvent tolerance, and thermal stability. Therefore, the enzyme could be in contact with the substrate for a sufficiently long time [29].
Reusability of the catalyst plays a significant role in potential large-scale industrial applications. Lx-EstBASΔSP retained excellent residual activity (80%) after seven cycles (Figure 7b); Li et al. also observed that free lipase immobilized on epoxy resin retains 98% of its initial activity after seven cycles [29]. The above results proved that the enzyme bound to the epoxy resin via a covalent linkage has the advantage of functional groups of the support matrix and strong bonds, which ensure the high retention of catalytic activities. However, a decrease in the enzymatic activity was caused by stripping of the bound water molecules from the enzyme by hydrophilic organic solvents in the reaction system, and the increased hydrophilic-solvent content resulted in a reduction in enzymatic activity [37].

2.10. Product Identification as Chloramphenicol Palmitate

The synthesis of chloramphenicol palmitate from vinyl palmitate and chloramphenicol was catalyzed by Lx-EstBASΔSP or free EstBASΔSP in acetone. The products were purified by thin-layer chromatography (TLC) and silica gel chromatography, and characterized by FTIR analysis (Figure 8). FTIR analysis is depicted in Figure 8. FTIR spectra featured peaks for the C=O stretching of the ester at 1742 cm−1 and for the amide group, N–H, at 3323 cm−1. The peak at 3504 cm−1 was assigned to the hydroxyl group, and the peaks at 2918 and 2850 cm−1 were assigned to stretching vibrations of CH, respectively. These findings confirmed the synthesis of the chloramphenicol esters [39].

3. Materials and Methods

3.1. Materials

Epoxy resin Lx-105s was provided by Xian Lanxiao Technology New Materials Co., Ltd. (Xi-an, China). B. altitudinis was maintained in our laboratory. PrimeSTAR HS DNA Polymerase, DNA marker, and restriction enzymes were purchased from TaKaRa Biotechnology Corporation (Otsu, Japan), the ClonExpress® II One Step Cloning Kit from Vazyme (Vazyme Biotech, Nanjing, China), and the BSA standard solution from TIANGEN (TIANGEN Biotech, Beijing, China). IPTG and ampicillin were acquired from Amresco (Shanghai Genebase Co., Ltd., Shanghai, China), whereas the DNA Mini Kit and Plasmid Mini Prepare Kit were from Axygen Biosciences (Union City, CA, USA). E. coli DH5α (Invitrogen, USA) and plasmid pMD19-T (TaKaRa, Shiga, Japan) were employed for gene cloning and sequencing. Plasmid pET-22b (Novagen, Madison, WI, USA) served as a vector to construct the protein expression plasmid for E. coli BL21 (DE3). The p-NP ester substrates were bought from Sigma-Aldrich (St. Louis, MO, USA), whereas the yeast extract and tryptone were from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). All other analytical chemicals were purchased at local markets. The substrates, p-NP esters, chloramphenicol, and vinyl palmitate were bought from Sigma-Aldrich (St. Louis, MO, USA). Acetone and other chemicals were of analytical reagent grade and were used without further purification.

3.2. Triacylglycerol Esterase and Gene Analysis

The nucleotide and predicted amino acid sequences were analyzed by means of the Basic Local Alignment Search Tool (BLAST) software (National Center for Biotechnology Information: NCBI, Bethesda, MD, USA). The primers for triacylglycerol esterase gene cloning with and without the SP were designed based on the information obtained from http://www.cbs.dtu.dk/services/SignalP/. The three-dimensional protein model (http://swissmodel.expasy.org/SWISS-MODEL.html) was visualized and analyzed in PDB viewer, and the final figure was constructed in PyMol.

3.3. Cloning of the Esterase Gene from B. altitudinis

To determine the role of the SP in EstBAS purification, two plasmids were constructed. The full-length estBAS gene and estBAS without the SP (estBASΔSP) were cloned using primer pairs F1/R1 and F2/R1, respectively (forward primer F1, 5′-TAAGAAGGAGATATACATATGATGAAAGTGATTCGATTTAAGAAA-3′; reverse primer R1, 5′-GTGGTGGTGGTGGTGCTCGAGATTCGTATTCTGTCCTCCGCCATTC-3′; forward primer F2, 5′-TAAGAAGGAGATATACATATGTTCATCCAGCCGAAAGAAGCCA-3′; the underlined nucleotides and restriction sites). The amplification was carried out as follows: initial denaturation at 94 °C for 4 min; then 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; and a final extension of 10 min at 72 °C. The polymerase chain reaction (PCR) products were cloned into the pMD-19T (simple) vector after purification with the DNA Gel Extraction Kit (Axygen, Union City, CA, USA) and then transferred into E. coli DH5α. The target gene was obtained and sequenced. After digestion with Nde I and Xho I, the PCR products were recovered and ligated into the pET-22b vector. Recombinant plasmids were transfected into E. coli BL21 (DE3) cells. The recombinant triacylglycerol esterase strains (named BL21-EstBAS, BL21-EstBASΔSP) were maintained for further analysis.

3.4. Effects of the SP on Overexpression and Purification of Triacylglycerol Esterase

Cells containing the recombinant plasmids were incubated at 37 °C in 50 mL of the LB medium containing ampicillin (50 μg mL−1). Cultures were induced at optical density at 600 nm (OD600) of 0.6–0.8 by the addition of 0.1 mM IPTG and were incubated for additional 20 h at 20 °C. The effects of the SP on overexpression and purification were investigated by SDS-PAGE. The strains were harvested by centrifugation, resuspended in lysis buffer (NPI 10: 50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 8.0) and sonicated, and the crude enzyme was collected by centrifugation (12,000× g, 10 min). The crude enzyme was passed through a 0.22 μm filter to remove any large impurities. This solution was then loaded onto a Ni-NTA Superflow Column (1 mL, Qiagen, Hilden, Germany) equilibrated with lysis buffer. The enzyme was eluted with a linear gradient of imidazole (from NPI 50 to NPI 250). The crude and purified proteins were examined by SDS-PAGE. All purification steps were carried out at 4 °C.

3.5. Immobilization of Purified EstBASΔSP

The EstBASΔSP suspension was desalted and concentrated by ultrafiltration in a 50 mL Amicon Ultra Centrifugal Filter Device with a molecular weight cutoff of 10 kDa (Millipore, Billerica, MA, USA). The hydrolytic activity of EstBASΔSP was 396 ± 10.5 U/mL, as determined by the method of Wang et al. [40]. The immobilization procedure was as follows: 20 g of the Lx-105s epoxy resin was equilibrated with 100 mM phosphate buffer (pH 8.0) in a 200 mL conical flask and stirred at 30 °C for 1 h (200 rpm). The mixture was then dried by vacuum filtration. After that, 1 g of a wet carrier and different amounts of purified EstBASΔSP mixed with 10 mL of a phosphate buffer were added into a conical flask (200 mL) and stirred in a thermostatic air bath shaker for 7 h (30 °C, 200 rpm). After that, the suspension was filtered, and the Lx-EstBASΔSP was washed with the buffer until no protein was detected in the buffer. At last, Lx-EstBASΔSP was dried (25 °C, 10 h) in a vacuum desiccator and stored (4 °C) until experiments.

3.6. Biochemical Characterization of Lx-EstBASΔSP and Free EstBASΔSP

The enzymatic activity of purified EstBASΔSP in solution was assayed by measuring the absorbance of liberated p-NP at 405 nm. One unit of enzymatic activity was defined as the amount of enzyme needed to release 1 μmol of p-NP per minute [37].
Reactions were monitored at the isosbestic point (405 nm) of p-nitrophenol and p-nitrophenolate and kinetic constants calculated on the basis that the molar absorption coefficient (△ε) was 2104 mol−1·cm−1. The reaction mixture (1.0 mL) contained 20 μL of a pNPH solution (final concentration 25 mM in a mixture of isopropanol and dimethyl sulfoxide at a volume ratio of 3:1) as the substrate, 960 μL of esterase assay buffer (50 mM Tris-HCl, pH 8.0), and 20 μL of the appropriately diluted enzyme. The reaction mixture was incubated at a specific temperature for 5 min, and the reactions were terminated by the addition of 1 mL of ethanol. We evaluated substrate specificity by means of the following substrates: pNPA, pNPB, pNPH, pNPC, pNPD, pNPL, pNPM, and pNPP. Enzymatic assays were carried out under standard assay conditions.
The effects of pH on the hydrolytic activity of Lx-EstBASΔSP and free EstBASΔSP were examined over a pH range from 4 to 10 for 5 min (60 °C). The following buffer systems were utilized: 100 mM citric acid-sodium citrate (pH 4.0–5.0), 200 mM sodium phosphate (pH 6.0–7.0), 50 mM Tris-HCl (pH 8.0), and 50 mM glycine-NaOH (pH 9.0–10.0) [41]. To identify the optimum temperature for enzymatic activity, assays were performed over a temperature range of 20 to 90 °C (pH 8.0). The thermal stability of EstBAS was evaluated by assaying its residual activity after incubation of the enzyme at 60 °C for a specific period. Aliquots were removed at various time points, and residual esterase activity was determined. A control with nonincubated enzyme was used to determine 100% activity. Enzymatic-activity experiments were conducted three times independently.

3.7. Effects of Metal Ions, Detergents, and Organic Solvents on EstBASΔSP Activity

The effects of metal ions (Ca2+, Mg2+, Cu2+, Co2+, Ni2+, Mn2+, Zn2+, and Fe2+) on the activity of Lx-EstBASΔSP and free EstBASΔSP were determined at final concentrations of 1, 5, and 10 mM.
The influence of detergents on the activity of Lx-EstBASΔSP and of free EstBASΔSP was analyzed by incubation of the enzyme with a detergent (Tween 20, Tween 60, Tween 80, SDS, or Triton X-100) for 30 min at 4 °C at a final concentration of 1% and 10%.
To estimate the organic-solvent tolerance of EstBASΔSP, Lx-EstBASΔSP or free EstBASΔSP was mixed with various organic solvents at the final concentration of 90% (v/v), as described by Li et al. [38]. Lx-EstBASΔSP or free EstBASΔSP was mixed with an organic solvent (1, 4-dioxane, ethanol, acetone, or acetonitrile) in sealed vessels and incubated for 70 h (4 °C, 200 rpm). The remaining solvent was removed by centrifugation for 10 min (8000× g), and the residual solvent was removed by evaporation. Lx-EstBASΔSP was resuspended in Tris-HCl buffer (50 mM, pH 8.0), and residual activities were determined under standard conditions.

3.8. Characterization of Lx-EstBASΔSP by Scanning Electron Microscopy (SEM) and FTIR

The surface morphology of EstBASΔSP before and after immobilization was characterized by SEM and FTIR. SEM images (Supplementary Figure S1) were acquired using an S-3400N scanning electron microscope (Hitachi, Japan). FTIR spectra were obtained on a Nicolet 6700 (Thermo Fisher Scientific, USA) using KBr pellets. FTIR (KBr): ṽ = 3189, 3063, 3060, 1636, 1631, 908 cm−1.

3.9. Regioselective Synthesis of Chloramphenicol Esters by Lx-EstBASΔSP and Free EstBASΔSP

Chloramphenicol palmitate was synthesized using Lx-EstBASΔSP or free EstBASΔSP as the catalyst. The reaction was carried out, and the physical and chemical conditions were varied. A 10-mL round-bottom flask containing a mechanical stirrer bar was filled with acetone, chloramphenicol (0.15 M), vinyl palmitate (0.75 M), and Lx-EstBASΔSP or free EstBASΔSP. The reaction temperature was set to 50 °C (24 h, 200 rpm). Subsequently, samples were taken out at equal intervals, and the mixture was centrifuged for 1 min (10,000× g). Next, ~0.5 mL of the supernatant was withdrawn for high-performance liquid chromatography analysis.
The conversion ratio of chloramphenicol is represented by the reduction rate of chloramphenicol in the resultant chromatograms:
conversion ratio = 1 − At/A0
where A0 is the area of the chloramphenicol peak at time 0, and At is the area of the chloramphenicol peak at time t.

3.10. Reusability of Lx-EstBASΔSP

After esterification of chloramphenicol with vinyl palmitate, Lx-EstBASΔSP was recovered and washed 3 times with n-hexane (20 mL). The recovered Lx-EstBASΔSP was dried for 24 h in a desiccator before the next cycle. The activity of the first-cycle reaction was set to 100%, and the activity of the subsequent reactions was calculated based on the first-cycle reaction.

3.11. Chloramphenicol Palmitate Characterization

TLC was used to analyze the lipid fractions of the reaction. Lipid fractions (1 μL) in hexane prepared were spotted on the line of origin and run for 30 min in a solvent tank comprising 2 solvents of different ratios i.e., hexane/ ethyl acetate (70:30, v/v%) at room temperature. The ZF-1 UV analyzer visualized the spots at 254 nm [42].
The conversion was calculated by high pressure liquid chromatography (Waters 2690 Separations Module; Waters 2487 UV vis Dual Absorbance Detector; readings were made at 273 nm) using a silica gel column (Agilent C18, 4.6 × 150 mm) and eluted with 0.01 M phosphoric acid/acetonitrile (85:15, v/v%) with a flow rate of 0.6 mL/min [8]. The products were identified by FTIR, 1H-NMR (400 MHz, CDCl3), and 13C-NMR (101 MHz, CDCl3) on a JEOL RESONANCE ECZ 400S spectrometer. CDCl3 was used as a solvent and trimethylsilane was used as an internal reference. Multiple signals of chloramphenicol palmitate are shown in the spectrum of 1H-NMR and 13C-NMR (Supplementary Figure S2). FTIR (KBr): ṽ = 3504, 3323, 2918, 2850, 1742 cm−1. 1H-NMR (CDCl3, 400 MHz): δ 0.87 (t, 3H, CH3), 1.26 (s, 24H, CH2), 1.62 (t, 2H, CH2), 2.37 (t, 2H, CH2), 4.17–4.19 (m, 1H, CH), 4.38–4.48 (m, 2H, CH2), 5.04 (d, 1H, CH), 5.74 (s, 1H, CH), 6.84 (d, 1H, NH), 7.55 (d, 2H, CH), 8.21 (d, 2H, CH). 13C-NMR (CDCl3, 101 MHz): δ 14.2 (CH3), 22.8 (CH2), 24.8 (CH2), 24.9 (CH2), 29.2–29.8 (8CH2), 32.0 (CH2), 33.8 (CH2), 34.2 (CH2), 54.2 (CH), 62.4(CH2), 66.1(CH), 70.8 (CH), 123.8 (2CH), 126.8 (2CH), 147.1 (C), 147.8 (C), 164.5 (C), 174.6 (C).

4. Conclusions

In this research, a novel esterase gene, estBASΔSP, was cloned from B. altitudinis. EstBASΔSP exhibited maximum activity at 50 °C and at pH 8.0. The recombinant protein was successfully immobilized on a novel epoxy resin Lx-105s with a high loading of 96 mg/g, exhibiting enzyme activity over a wider range of pH and temperature. Compared with free EstBASΔSP, the optimum temperature of Lx-EstBASΔSP is 60 °C, and the enzyme retained more than 35% activity at 90 °C. In addition, Lx-EstBASΔSP showed improvements in thermostability and organic-solvent tolerance when compared to free EstBASΔSP. Moreover, Lx-EstBASΔSP has excellent catalytic efficiency in the synthesis of chloramphenicol palmitate with regioselectivity. Conversion efficiency reached 94.7% at 0.15 M of substrate after 24 h. Lx-EstBASΔSP was reused for seven cycles, retaining over 80% of its original activity. In short, this novel esterase holds promise for industrial applications of chloramphenicol palmitate synthesis.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4344/9/7/620/s1, Figure S1: SEM of the surface of epoxy resin Lx-105s before (a–c) and after (d–f) immobilization of EstBASΔSP. Magnification: a,d × 100; b,e × 500; c,f × 15 k, Figure S2. (a) 1H-NMR spectrum of chloramphenicol palmitate in CDCl3; (b) 13C-NMR spectrum of chloramphenicol palmitate in CDCl3.

Author Contributions

F.D., D.W. and W.W. conceived and designed the experiments; F.D. and X.T. performed the experiments; F.D. and X.Y. analyzed the data; F.D. wrote the paper; L.L. and D.H. contributed reagents/materials/analysis tools; F.D. and W.W. revised the paper.

Funding

This research was financially supported by the National Natural Science Foundation of China (No. C31570795), Shanghai Outstanding Technical Leaders Plan 19XD1431800, National Natural Science Foundation of China (Grant No. 81830052, 81530053), and Shanghai Key Laboratory of Molecular Imaging (18DZ2260400).

Acknowledgments

Authors are thankful to Xiaoyu Huang and Guolin Lu for providing the precious help of the analysis of NMR spectrum in the present study.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

FTIRFourier transform infrared spectroscopy
IPTGisopropyl-β-d-thiogalactopyranoside
p-NPp-nitrophenyl
pNPAp-NP acetate
pNPBp-NP butyrate
pNPCp-NP caprylate
pNPDp-NP decanoate
pNPHp-NP hexanoate
pNPLp-NP laurate
pNPMp-NP myristate
pNPPp-NP palmitate
SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresis
SEMscanning electron microscopy;
SPsignal peptide;
TLCthin-layer chromatography

References

  1. Yang, G.; Zhao, F. Electrochemical sensor for chloramphenicol based on novel multiwalled carbon nanotubes@molecularly imprinted polymer. Biosens. Bioelectron. 2015, 64, 416–422. [Google Scholar] [CrossRef] [PubMed]
  2. Pilehvar, S.; Mehta, J.; Dardenne, F.; Robbens, J.; Blust, R.; De, W.K. Aptasensing of chloramphenicol in the presence of its analogues: Reaching the maximum residue limit. Anal. Chem. 2012, 84, 6753–6758. [Google Scholar] [CrossRef] [PubMed]
  3. Abnous, K.; Danesh, N.M.; Ramezani, M.; Emrani, A.S.; Taghdisi, S.M. A novel colorimetric sandwich aptasensor based on an indirect competitive enzyme-free method for ultrasensitive detection of chloramphenicol. Biosens. Bioelectron. 2015, 78, 80–86. [Google Scholar] [CrossRef]
  4. Ottolina, G.; Carrea, G.; Riva, S. Synthesis of ester derivatives of chloramphenicol by lipase-catalyzed transesterification in organic solvents. J. Org. Chem. 1990, 55, 2366–2369. [Google Scholar] [CrossRef]
  5. Bizerra, A.M.C.; Montenegro, T.G.C.; Lemos, T.L.G.; Oliveira, M.C.F.D.; Mattos, M.C.D.; Lavandera, I.; Gotor-Fernández, V.; Gonzalo, G.D.; Gotor, V. Enzymatic regioselective production of chloramphenicol esters. Tetrahedron 2011, 67, 2858–2862. [Google Scholar] [CrossRef]
  6. Kumar, R.; Mahajan, S.; Kumar, A.; Singh, D. Identification of variables and value optimization for optimum lipase production by Bacillus pumilus RK31 using statistical methodology. New Biotechnol. 2011, 28, 65–71. [Google Scholar] [CrossRef] [PubMed]
  7. Ghanem, E.H.; Alsayed, H.A.; Saleh, K.M. An alkalophilic thermostable lipase produced by a new isolate of Bacillus alcalophilus. World J. Microbiol. Biotechnol. 2000, 16, 459–464. [Google Scholar] [CrossRef]
  8. Lv, D.S.; Xue, X.T.; Wang, N.; Wu, Q.; Lin, X.F. Enzyme catalyzed synthesis of some vinyl drug esters in organic medium. Prep. Biochem. 2004, 34, 97–107. [Google Scholar] [CrossRef]
  9. Wang, R.; Zhang, Y.; Huang, J.; Lu, D.; Ge, J.; Zheng, L. Substrate imprinted lipase nanogel for one-step synthesis of chloramphenicol palmitate. Green Chem. 2013, 15, 1155–1158. [Google Scholar] [CrossRef]
  10. Esakkiraj, P.; Usha, R.; Palavesam, A.; Immanuel, G. Solid-state production of esterase using fish processing wastes by Bacillus altitudinis AP-MSU. Food Bioprod. Process. 2012, 90, 370–376. [Google Scholar] [CrossRef]
  11. Schmidt, M.; Henke, E.; Heinze, B.; Kourist, R.; Hidalgo, A.; Bornscheuer, U.T. A versatile esterase from Bacillus subtilis: Cloning, expression, characterization, and its application in biocatalysis. Biotechnol. J. 2010, 2, 249–253. [Google Scholar] [CrossRef] [PubMed]
  12. Hung, T.C.; Giridhar, R.; Chiou, S.H.; Wu, W.T. Binary immobilization of Candida rugosa lipase on chitosan. J. Mol. Catal. B Enzym. 2003, 26, 69–78. [Google Scholar] [CrossRef]
  13. Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzym. Microb. Technol. 2007, 40, 1451–1463. [Google Scholar] [CrossRef]
  14. Sheldon, R.A.; Van, P.S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. [Google Scholar] [CrossRef] [PubMed]
  15. Wang, W.; Zhou, W.; Li, J.; Hao, D.; Su, Z.; Ma, G. Comparison of covalent and physical immobilization of lipase in gigaporous polymeric microspheres. Bioprocess Biosyst. Eng. 2015, 38, 2107–2115. [Google Scholar] [CrossRef] [PubMed]
  16. Lei, L.; Bai, Y.; Li, Y.; Yi, L.; Yang, Y.; Xia, C. Study on immobilization of lipase onto magnetic microspheres with epoxy groups. J. Magn. Magn. Mater. 2009, 321, 252–258. [Google Scholar] [CrossRef]
  17. Bayramoglu, G.; Karagoz, B.; Altintas, B.; Arica, M.Y.; Bicak, N. Poly(styrene–divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: Performance of reversibly immobilized lipase in ester synthesis. Bioprocess Biosyst. Eng. 2011, 34, 735–746. [Google Scholar] [CrossRef] [PubMed]
  18. Gomes, F.M.; Pereira, E.B.; de Castro, H.F. Immobilization of lipase on chitin and its use in nonconventional biocatalysis. Biomacromolecules 2004, 5, 17–23. [Google Scholar] [CrossRef]
  19. Matte, C.R.; Bordinhão, C.; Poppe, J.K.; Rodrigues, R.C.; Hertz, P.F.; Ayub, M.A.Z. Synthesis of butyl butyrate in batch and continuous enzymatic reactors using Thermomyces lanuginosus lipase immobilized in Immobead 150. J. Mol. Catal. B Enzym. 2016, 127, 67–75. [Google Scholar] [CrossRef]
  20. Zhang, D.H.; Peng, L.J.; Wang, Y.; Li, Y.Q. Lipase immobilization on epoxy-activated poly(vinyl acetate-acrylamide) microspheres. Colloids Surf. B Biointerfaces 2015, 129, 206–210. [Google Scholar] [CrossRef]
  21. Ma, H.Z.; Yu, X.W.; Song, C.; Xue, Q.L.; Jiang, B. Immobilization of Candida Antarctica lipase B on epoxy modified silica by sol-gel process. J. Mol. Catal. B Enzym. 2016, 127, 76–81. [Google Scholar] [CrossRef]
  22. Ashjari, M.; Mohammadi, M.; Badri, R. Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: Modulation of stability and selectivity. J. Mol. Catal. B Enzym. 2015, 115, 128–134. [Google Scholar] [CrossRef]
  23. Petersen, T.N.; Brunak, S.; Von, H.G.; Nielsen, H. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat. Methods 2011, 8, 785–786. [Google Scholar] [CrossRef]
  24. Harris, D.A. Cellular Biology of Prion Diseases. Clin. Microbiol. Rev. 1999, 12, 429–444. [Google Scholar] [CrossRef] [Green Version]
  25. Li, Y.; Cong, Q.; Chao, C.; Fu, Y.; Ming, M.; Xie, Q. Highly sensitive phenolic biosensor based on magnetic polydopamine-laccase-Fe3O4 bionanocomposite. Sens. Actuators B 2012, 168, 46–53. [Google Scholar] [CrossRef]
  26. Silva, R.; Carmona-Ribeiro, A.; Petri, D. Catalytic Behavior of Lipase Immobilized onto Congo Red and PEG-Decorated Particles. Molecules 2014, 19, 8610–8628. [Google Scholar] [CrossRef] [Green Version]
  27. Xavier, A.; Tavares, A.; Daniel-Da-Silva, A.; Fortes, C. Optimisation of laccase immobilization on modified magnetic nanoparticles for biocatalytic reactions. New Biotechnol. 2016, 33, S15. [Google Scholar] [CrossRef]
  28. Jenjob, S.; Sunintaboon, P.; Inprakhon, P.; Anantachoke, N.; Reutrakul, V. Chitosan-functionalized poly(methyl methacrylate) particles by spinning disk processing for lipase immobilization. Carbohydr. Polym. 2012, 89, 842–848. [Google Scholar] [CrossRef]
  29. Li, X.; Li, D.; Wang, W.; Durrani, R.; Bo, Y.; Wang, Y. Immobilization of SMG1-F278N lipase onto a novel epoxy resin: Characterization and its application in synthesis of partial glycerides. J. Mol. Catal. B Enzym. 2016, 133, 154–160. [Google Scholar] [CrossRef]
  30. Dong, H.; Secundo, F.; Xue, C.; Mao, X. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus. J. Agric. Food Chem. 2017, 65, 2120–2128. [Google Scholar] [CrossRef]
  31. Oliveranappa, A.; Andrews, B.A.; Asenjo, J.A. A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity. Biotechnol. Bioeng. 2010, 86, 573–586. [Google Scholar] [CrossRef]
  32. Lin, J.; Liu, Y.; Chen, S.; Le, X.; Zhou, X.; Zhao, Z.; Ou, Y.; Yang, J. Reversible immobilization of laccase onto metal-ion-chelated magnetic microspheres for bisphenol A removal. Int. J. Biol. Macromol. 2016, 84, 189–199. [Google Scholar] [CrossRef]
  33. Hou, C.; Qi, Z.; Zhu, H. Preparation of core-shell magnetic polydopamine/alginate biocomposite for Candida rugosa lipase immobilization. Colloids Surf. B Biointerfaces 2015, 128, 544–551. [Google Scholar] [CrossRef]
  34. Mishra, M.K.; Kumaraguru, T.; Sheelu, G. Lipase activity of Lecitase® Ultra: Characterization and applications in enantioselective reactions. Tetrahedron Asymmetry 2009, 20, 2854–2860. [Google Scholar] [CrossRef]
  35. Xie, W.; Ning, M. Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles. Biomass Bioenergy 2010, 34, 890–896. [Google Scholar] [CrossRef]
  36. Freitas, L.; Paula, A.V.; Santos, J.C.D.; Zanin, G.M.; Castro, H.F.D. Enzymatic synthesis of monoglycerides by esterification reaction using Penicillium camembertii lipase immobilized on epoxy SiO 2 -PVA composite. J. Mol. Catal. B Enzym. 2010, 65, 87–90. [Google Scholar] [CrossRef]
  37. Gao, W.; Wu, K.; Chen, L.; Fan, H.; Zhao, Z.; Gao, B.; Wang, H.; Wei, D. A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters. Microb. Cell Factories 2016, 15, 41. [Google Scholar] [CrossRef] [Green Version]
  38. Li, C.; Tan, T.; Zhang, H.; Feng, W. Analysis of the conformational stability and activity of Candida antarctica lipase B in organic solvents: Insight from molecular dynamics and quantum mechanics/simulations. J. Biol. Chem. 2010, 285, 28434–28441. [Google Scholar] [CrossRef]
  39. Alemdar, A.; Sain, M. Isolation and characterization of nanofibers from agricultural residues: Wheat straw and soy hulls. Bioresour. Technol. 2008, 99, 1664–1671. [Google Scholar] [CrossRef]
  40. Wang, W.F.; Li, T.; Qin, X.L.; Ning, Z.X.; Yang, B.; Wang, Y.H. Production of lipase SMG1 and its application in synthesizing diacylglyecrol. J. Mol. Catal. B Enzym. 2012, 77, 87–91. [Google Scholar] [CrossRef]
  41. Cai, X.H.; Jing, M.; Wei, D.Z.; Lin, J.P.; Wei, W. Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production. Antonie Van Leeuwenhoek 2014, 106, 1049–1060. [Google Scholar] [CrossRef]
  42. Dong, F.; Li, L.; Lin, L.; He, D.; Chen, J.; Wei, W.; Wei, D. Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens. Molecules 2017, 22, 1523. [Google Scholar] [CrossRef]
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Figure 1. Conserved sequence alignment and structural analysis of EstBAS. (a) Conserved sequence alignment of EstBAS and other esterase from different bacterial sources. The sequences of the following strains were obtained from GenBank: Bacillus sp.; B. pumilus; Bacillus sp. LLTC93; B. pumilus AVI41987; B. safensis; B. atrophaeus 010787545; B. atrophaeus 063638677; B. velezensis; The structures are denoted as follows: ▲, the catalytic site (Ser111, Asp167, and His190). □, the signal peptide. (b) The three-dimensional structure of the entire protein (Template PDB code: 1T4M, 79.01% identical). The three-dimensional structure of EstBAS was predicted by SWISS-MODEL server. (c) The catalytic site of EstBAS structure. The catalytic triad: Ser111, Asp167, and His190 residues are marked in gray.
Figure 1. Conserved sequence alignment and structural analysis of EstBAS. (a) Conserved sequence alignment of EstBAS and other esterase from different bacterial sources. The sequences of the following strains were obtained from GenBank: Bacillus sp.; B. pumilus; Bacillus sp. LLTC93; B. pumilus AVI41987; B. safensis; B. atrophaeus 010787545; B. atrophaeus 063638677; B. velezensis; The structures are denoted as follows: ▲, the catalytic site (Ser111, Asp167, and His190). □, the signal peptide. (b) The three-dimensional structure of the entire protein (Template PDB code: 1T4M, 79.01% identical). The three-dimensional structure of EstBAS was predicted by SWISS-MODEL server. (c) The catalytic site of EstBAS structure. The catalytic triad: Ser111, Asp167, and His190 residues are marked in gray.
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Figure 2. SDS-PAGE analysis of EstBAS and EstBASΔSP. Lane M: standard molecular-weight marker proteins; lanes 3, 4: crude extract of EstBAS and EstBASΔSP; lanes 2, 5: cell lysate supernatants (EstBAS and EstBASΔSP); lanes 1, 6: precipitates of EstBAS and EstBASΔSP cell lysates; lane 7: purified EstBASΔSP.
Figure 2. SDS-PAGE analysis of EstBAS and EstBASΔSP. Lane M: standard molecular-weight marker proteins; lanes 3, 4: crude extract of EstBAS and EstBASΔSP; lanes 2, 5: cell lysate supernatants (EstBAS and EstBASΔSP); lanes 1, 6: precipitates of EstBAS and EstBASΔSP cell lysates; lane 7: purified EstBASΔSP.
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Catalysts 09 00620 sch001 550
Scheme 1. (a) EstBASΔSP immobilization on epoxy resin Lx-105s and (b) the application of immobilized EstBASΔSP in the regioselective synthesis of chloramphenicol palmitate.
Scheme 1. (a) EstBASΔSP immobilization on epoxy resin Lx-105s and (b) the application of immobilized EstBASΔSP in the regioselective synthesis of chloramphenicol palmitate.
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Figure 3. (a) Effects of the initial esterase concentration on esterase loading and relative activity. (b) The influence of the immobilization time on esterase loading and relative activity.
Figure 3. (a) Effects of the initial esterase concentration on esterase loading and relative activity. (b) The influence of the immobilization time on esterase loading and relative activity.
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Figure 4. FTIR spectra of free EstBASΔSP, Lx-EstBASΔSP, and free epoxy resin Lx-105s. a, resin Lx-105s; b, Lx-EstBASΔSP; c, free EstBASΔSP.
Figure 4. FTIR spectra of free EstBASΔSP, Lx-EstBASΔSP, and free epoxy resin Lx-105s. a, resin Lx-105s; b, Lx-EstBASΔSP; c, free EstBASΔSP.
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Figure 5. Determination of the optimum substrate for recombinant EstBASΔSP. The relative activity (%) was calculated by comparison of the data between substrates and p-NP hexanoate (pNPH).
Figure 5. Determination of the optimum substrate for recombinant EstBASΔSP. The relative activity (%) was calculated by comparison of the data between substrates and p-NP hexanoate (pNPH).
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Figure 6. Effects of (a) pH and (b) temperature on the activity of Lx-EstBASΔSP and free EstBASΔSP, and the influence of (c) temperature at 60 °C and (d) storage duration (at 4 °C) on the stability of Lx-EstBASΔSP and free EstBASΔSP.
Figure 6. Effects of (a) pH and (b) temperature on the activity of Lx-EstBASΔSP and free EstBASΔSP, and the influence of (c) temperature at 60 °C and (d) storage duration (at 4 °C) on the stability of Lx-EstBASΔSP and free EstBASΔSP.
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Figure 7. (a) Conversion of chloramphenicol and vinyl palmitate to chloramphenicol palmitate catalyzed by Lx-EstBASΔSP and free EstBASΔSP at 60 °C; (b) reusability of Lx-EstBASΔSP.
Figure 7. (a) Conversion of chloramphenicol and vinyl palmitate to chloramphenicol palmitate catalyzed by Lx-EstBASΔSP and free EstBASΔSP at 60 °C; (b) reusability of Lx-EstBASΔSP.
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Figure 8. FTIR spectra of chloramphenicol (black) and chloramphenicol palmitate (red).
Figure 8. FTIR spectra of chloramphenicol (black) and chloramphenicol palmitate (red).
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Table
Table 1. Effects of metal ions on free EstBASΔSP and Lx-EstBASΔSP activity.
Table 1. Effects of metal ions on free EstBASΔSP and Lx-EstBASΔSP activity.
Ion ResidualActivity (%) a
(1 mM)(5 mM)(10 mM)
F bL cF bL cF bL c
None100 ± 0.0100 ± 0.0100 ± 0.0100 ± 0.0100 ± 0.0100 ± 0.0
Mg2+105 ± 2.1108 ± 1.9103 ± 1.9104 ± 2.188.2 ± 1.999.8 ± 1.7
Zn2+96.1 ± 3.797 ± 2.085.1 ± 2.787 ± 2.068.5 ± 3.570 ± 1.2
Co2+92.7 ± 2.9100 ± 1.781.8 ± 3.596 ± 2.571.4 ± 2.474 ± 1.5
Ni2+91.5 ± 2.392 ± 2.590.6 ± 2.491 ± 2.383.2 ± 2.087 ± 2.2
Fe2+92.8 ± 3.493 ± 1.883.4 ± 2.684 ± 1.768.0 ± 1.776 ± 1.6
a Lx-EstBASΔSP and free EstBASΔSP were pre-incubated with the metal ions for 1 h at 4 °C. The presented results are the mean ± standard deviation. The activity toward pNPH without any metal ions was set to 100%. b F: Free EstBASΔSP, c L: Lx-EstBASΔSP.
Table
Table 2. The impact of detergents on the activity of Lx-EstBASΔSP and free EstBASΔSP.
Table 2. The impact of detergents on the activity of Lx-EstBASΔSP and free EstBASΔSP.
DetergentResidualActivity (%) a
1%10%
F bL cF bL c
None100 ± 0.0100 ± 0.0100 ± 0.0100 ± 0.0
Tween2094.0 ± 2.195 ± 1.940.5 ± 1.941 ± 1.1
Tween6094.5 ± 1.696 ± 2.145.6 ± 1.847 ± 1.9
Tween8094.8 ± 1.895 ± 2.353.3 ± 2.155 ± 2.1
SDS18.9 ± 1.220 ± 1.700
TritonX-10036.1 ± 3.738 ± 2.9ND d8 ± 0.9
a The assay was performed under optimum conditions. The residual activity was measured in Tris-HCl buffer (50 mM, pH 8.0) at 60 °C with pNPH as the substrate. Values represent the mean ± standard deviation (n = 3). The activity measured without additives was set to 100%. b F: Free EstBASΔSP. c L: Lx-EstBASΔSP. d ND: not detectable.
Table
Table 3. Effects of organic solvents on free EstBASΔSP and Lx-EstBASΔSP activity.
Table 3. Effects of organic solvents on free EstBASΔSP and Lx-EstBASΔSP activity.
Organic SolventsLogP aResidualActivity (%) b
F bL c
Control 100 ± 1.2100 ± 0.9
1, 4-dioxane−1.012.6 ± 0.780.2 ± 2.9
Ethanol−0.2452.5 ± 1.892.5 ± 1.6
Acetone−0.2325.0 ± 1.193.8 ± 2.6
Acetonitrile−0.15ND d92.0 ± 1.5
a The logP value is the partition coefficient of an organic solvent between water and n-octanol phases. After incubation of ESTBASΔSP for 70 h in one of four organic solvents, the residual activity was measured in Tris-HCl buffer (50 mM, pH 8.0) at 60 °C with pNPH as the test substrate. An enzyme sample incubated in the buffer alone served as a control (100% activity). b F: Free EstBASΔSP, c L: Lx-EstBASΔSP, d ND: not detectable.

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MDPI and ACS Style

Dong, F.; Tang, X.; Yang, X.; Lin, L.; He, D.; Wei, W.; Wei, D. Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate. Catalysts 2019, 9, 620. https://doi.org/10.3390/catal9070620

AMA Style

Dong F, Tang X, Yang X, Lin L, He D, Wei W, Wei D. Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate. Catalysts. 2019; 9(7):620. https://doi.org/10.3390/catal9070620

Chicago/Turabian Style

Dong, Fengying, Xudong Tang, Xiaohui Yang, Lin Lin, Dannong He, Wei Wei, and Dongzhi Wei. 2019. "Immobilization of a Novel ESTBAS Esterase from Bacillus altitudinis onto an Epoxy Resin: Characterization and Regioselective Synthesis of Chloramphenicol Palmitate" Catalysts 9, no. 7: 620. https://doi.org/10.3390/catal9070620

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