3.1. Expression and Purification of the Recombinant Fluorinase
A gene fragment of 909 bp encoding f1A was cloned and inserted into the vector pET28a, and
E. coli BL21 (DE3) with the recombinant plasmid pET28a-f1A was cultured in LB medium, after which the expression of fluorase was induced. The expression of recombinant f1A was analyzed by SDS-PAGE, and the results in
Figure 1A show that the molecular weight of the recombinant f1A protein is approximately 34.4 kDa, which is consistent with that calculated from the amino acid sequence [
38]. For the low levels of the
flA gene expression, it is necessary to optimize the expression conditions in
E. coli BL21. However, the concentration of IPTG has little effect on the soluble target protein expression, as the results showed (
Figure S1, ESI). With the progress of the IPTG concentration, the total protein expression for the whole cell increased, and the insoluble matter also increased. Additionally, when the temperature was 16 °C (
Figures S2 and S3, ESI), there was less induced insoluble matter than at 18 °C. Similarly, the induced soluble target protein decreased with declining temperature. The 12 h period was suitable for the induction (
Figure S4, ESI).
In cells, molecular chaperones are a class of proteins that assist intracellular molecular assembly and protein folding [
39]. One of their main functions is to recognize and stabilize the partially folded conformation of the polypeptide chain, thereby participating in the folding and assembly of the new peptide chain. In addition to the basic role of assisting protein folding, they can also participate in protein transport, assembly, aggregation, and degradation, which aids the nascent polypeptides to reach their final structure [
36,
40]. However, as shown in
Figure S5, molecular chaperones have not assisted and improved the correct expression of target protein when the commercial chaperone plasmid set pGro7 from Takara was used in
E. coli BL21 (DE3). Moreover, the pKJE7 chaperone decreased and inhibited the normal expression of fluorinase.
The reason for the above phenomenon may be that there are significant differences in the frequency of using different codons between different organisms, although the expression hosts and operons are the same [
41]. As shown in
Figure S6 (ESI) and
Figure 1B, when the expression host is Rosetta (DE3), the content of insoluble expressed fluorinase significantly decreases. Generally, the concentration of protein was 3 mg·mL
−1, which was higher than that in
S. cattleya NRRL 8057 [
37]. The reason may be that the recombinant plasmid pET28a-flA belongs to a heterologous expression, and the expressed host cell is completely different from the previous one. Recombinant proteins that are not expressed or truncated by the host due to codon preference force the host to express specific proteins, and it does not have a large number of tRNAs. As a result, the recombinant gene cannot be expressed, or the expression level is low, and the activity of the expressed enzyme is reduced. Recombinant plasmids with lower expression levels generally include rare codons. For the heterologous expression of our recombinant plasmid pET28a-flA to achieve overexpression, the biggest problem to be solved is the solution of rare codons. The flA gene is derived from fungi and contains rare codons. The flA gene is expressed using
E. coli as the host cell, and compared with typical
E. coli proteins, the amino acid composition of the corresponding protein is biased. Thus, it may face translation problems, including translation pauses, premature translation termination, and translation frameshifting to reduce expression of the protein’s quantity or quality. The tRNA genes of the engineering strain
E. coli Rosetta include rare codons AGG, AGA, AUA, CUA, CCC, and GGA. Therefore, codon usage optimization is critical in
E. coli [
42,
43].
3.2. Preparation and Characterization of [email protected]
Prior to preparing the
[email protected] hydroxyapatite nanoflowers (
[email protected]), we examined the effect of metal ions on fluorinase. Fluorinase was used without any metal ions and EDTA as the control group, and its catalytic synthetic activity was set as 100%. As shown in
Figure 2, 1.0 mM Ca
2+, Mn
2+, and Co
2+ exerts no influence on the activity of FDAS.
Hydroxyapatite (HAP),
[email protected] (
[email protected]), fluoride-substituted hydroxyapatite (FHAp), and
[email protected] hydroxyapatite nanoflowers (
[email protected]) were characterized by SEM, and the results are presented in
Figure 3. The formation of FDAS-hydroxyapatite is based on the mechanism of biomineralization. The specific process that occurs is the regulation and influence of the biological macromolecular fluorinase protein, calcium ions, phosphates, and amino and carboxyl groups on the fluorinase protein, which work together to trigger the formation of nanocrystalline nuclei. With the gradual increase of crystal nuclei, lots of protein molecules and primary crystals are agglomerated, and finally, a protein–hydroxyapatite complex is formed, which has a shape similar to a petal [
44]. For the formation of
[email protected] hydroxyapatite nanoflowers, the fluoride ions added to the solution can remineralize with apatite crystals and biomacromolecules in several different ways in the presence of their different concentrations and solution compositions. At low fluoride levels, F-OH exchange between apatite solution phases can easily occur. By comparing the structures of the fluorinated hydroxyapatite and the hydroxyapatite, it can be seen that the bond length of the fluorinated hydroxyapatite is shorter than that of the hydroxyapatite, which means that the FDAS-FHAp-NFs formed is more stable. As time passes, more consistent and regular FDAS-FHAp-NFs are produced, leading to the formation of a more complete structure, but with a shape different from the FDAS-HAp-NFs structure [
32].
In
Figure S7A–C (ESI), the effects of pH and crystallization time in the different buffers on the crystal sHApe are shown. It is apparent that pH has a great influence on the crystal form, and the crystal form is more regular at pH 7.5. Crystal growth is slow and may be affected by ambient temperature, since the enzyme activity of the fluorinase is also sensitive and greatly affected by the ambient temperature. To prevent the structure and activity of this enzyme from being damaged, the temperature of the long co-crystallization was selected to be 4 °C, and the time required for the entire bionic immobilization of enzyme protein is 24 h (
Figure S7D–E, ESI).
Figure S8 (ESI) presents the effect of fluorinase concentration on crystal growth. It can be seen from the SEM photograph that as the concentration of the enzyme gradually increases, the overall structure of the crystal gradually increases and becomes regular. When the enzyme concentration is not less than 70 μg·mL
−1,
[email protected] hydroxyapatite nanoflowers form is preferred more than at higher enzyme concentrations. When the concentration of enzyme is too high, the enzyme loading rate on the support will decrease.
Figure S9 (ESI) shows the effect of different F ion concentrations on the crystal formation. When the F ion concentration is not less than 20 ppm, the crystal form is relatively regular, but when the F ion concentration is as high as 3000 ppm, spherical calcium fluoride crystals are more easily formed. This may result from the biomineralization process that takes place under the control of organic matter, and the low concentration of organic matter would result in less regulation.
Figure 4 displays the XRD analysis of the
[email protected], F-HAP,
[email protected], and HAP. The XRD analysis of fluoridated hydroxyapatite nanoflowers confirmed the opinion, while the relative strength of each diffraction peak of hybrid nanocomposites matched the standard pattern of fluoride-substituted hydroxyapatite (Ca
5(PO
4)
3F, FHAP), octacalcium phosphate (Ca
8H
2(PO
4)
6•5H
2O, OCP), hydroxyapatite (Ca
10(OH)
2(PO
4)
6, HAp), CaHPO
4•2H
2O, and its other derivatives. The wide-angle XRD pattern of the enzyme, FHAp-hNFs, shows distinct diffraction peaks at 11.72°, 26.03°, 32.12°, 32.29°, 40.65°, 46.85°, 49.65°, 53.25°, which were the most prominent characteristic peaks of Ca
5(PO
4)
3F and Ca
10(OH)
2(PO
4)
6. However, the diffusion peak at 2θ = 31°–34° may demonstrate the presence of amorphous calcium phosphate [
31]. In addition, this broadening peak could also result from the appearance of smaller apatite crystals, which also include octacalcium phosphate (Ca
8H
2(PO
4)
6•5H
2O, OCP). Evidently, it can be seen from 2θ = 30°–34° that the crystal changes to Ca
5(PO
4)
3F after the addition of F ions when preparing the
[email protected] and
[email protected] The presence of Ca
5(PO
4)
3F in the pattern of enzyme FHAp-NFs can be easily verified by peaks at 31.87°, 32.2°, 39.15°, 39.98°, and 50.63° [
31,
44].
FT-IR was also used to characterize the obtained
[email protected] hydroxyapatite nanoflowers, and the result in the upper panel in
Figure 5 indicates that the remineralized complex, which was prepared using fluorinase, presents protein and phosphate minerals. The main absorption peak of the protein is the amide band, including band I, band II, and band III. Amide band I (1690–1630 cm
−1) is mainly due to the strong absorption of γ
C = O. Comparing this with that of the free enzyme, a red shift occurs to the amide of the immobilized enzyme. It is speculated that the enzyme may adhere to the inorganic support hNFs, which have some influence on the spatial structure of the enzyme. The amide band II (1420–1400 cm
−1) is the absorption band of the strong carbon–nitrogen bond stretching vibration, and the absorption band of the amide bond of the enzyme on the support shifted a little, which indicates that no change of the main structure occurs in the enzyme protein after the immobilization by biomineralization and the formation of h-NFs. In addition, the (PO
4)
3− belt and the γ
4 belt are absorption bands for the (PO
4)
3− stretching vibration. At the same time, the γ
4 band (600 cm
−1 and 558 cm
−1) demonstrates the interaction of calcium phosphate with the fluorinase protein after biomimetic mineralization [
31]. Comparisons of the FT-IR spectra of samples A, B, C, and D show that the shift of the bands will also be offset after the introduction of F. This shift may be the overlap of fluoridated hydroxyapatite and octacalcium phosphate (OCP), when the hydroxyapatite layer is grown on the OCP precursor [
45].
3.3. Optimum Temperature and pH of Free FDAS and [email protected] FHAp-NFs Activity
A total of 2 mL of reaction mixture containing 2 mg of FDAS, 2 mM SAM, 50 mM KF, and 20 mM potassium phosphate buffer (pH 7.5) was incubated at 37 °C for 0.5 h.
Figure 6A shows that the optimum temperature for enzyme preparations is 37 °C, which is similar to that of the free fluorinase from the marine-derived bacteria
Streptomyces xinghaiensis NRRL B24674 [
46]. Importantly,
[email protected] hydroxyapatite nanoflowers present a wider suitable temperature range for the catalytic reaction and approximately 4.8-fold higher catalytic activity than free fluorinase. The reason for this may be that F ions are added during the biomineralization process and F ions are also the raw materials required for the synthetic reaction. Moreover, the substrate, S-adenosyl-L-methionine (SAM), is a polyamino and polyhydroxy compound, and the support, apatite, is also full of hydroxy, which can form the hydrogen bond network by the F
− [
9,
33] (
Figure 6A). This network would enrich the substrate SAM around the support and enzyme, which further enhances the catalytic activity of fluorinase.
Figure 6B shows the optimum pH of the enzyme preparations. The optimal pH of both free FDAS and
[email protected] activity was approximately pH 7.5, and enzyme protein fractions (1 mg·mL
−1) were incubated at 37 °C with SAM (2 mM) and KF (50 mM) in different buffers and in a final volume of 2 mL for 12 h. When compared with a free enzyme over a broad range of pH (from pH 5.5 to 8.5),
[email protected] preparation demonstrated greater stability. Additionally, FHAp-NFs present more activity than free enzymes in most pH conditions. However, the free fluorinase from
Streptomyces xinghaiensis NRRL B24674 would undergo a remarkable decrease of activity in the catalytic synthesis under basic conditions (pH 8.0–9.5) [
46].
3.4. Thermal Stability of Free FDAS and [email protected]
To evaluate the effect of temperature on enzyme catalytic activity, the enzyme preparations were incubated at temperatures ranging from 30 to 50 °C for different time periods. As indicated in
Figure 7, the FDAS-FHAp-NFs proved to be more thermostable than the free enzyme, and approximately 80% of the initial activity remained after heating for 8 h at 30 °C. After heating at 50 °C for 8 h, more than 60% of the initial activity was retained. However, both the FDAS-HAp-NFs and free enzyme lost most of their initial activities. Obviously, an enzyme with support presents better stability than the free enzyme. When the enzyme was confined in the fluoridated hydroxyapatite, the thermal stability of fluorinase was further improved. That is to say, compared with hydroxyapatite (HAp), fluoride-substituted hydroxyapatite (FHAp) was more suitable for fluorinase to retain its catalytic activity.
The enhanced thermal stability of the FDAS-FHAp-NFs may be due to the substitution of F
− for OH
− on the support, which results in a reduction of the unit cell volume and a denser lattice. Subsequently, the electrostatic attraction between fluoride and adjacent ions would strengthen, and the thermal stability of the FDAS-FHAp-NFs would be greatly improved. Fluoride has been shown to be able to replace the columnar hydroxyl groups, which are distributed in the apatite structure [
32]. This reduces the volume of the formed crystals and, therefore, increases the rigidity of the confined enzyme and consequently, enhances the thermal stability of the enzyme preparation.
3.5. Synthesis of 5′-Fluorodeoxy Adenosine using FDAS Enzyme Preparations
Based on the above experimental research, FDAS preparations were used to catalyze the synthesis of 5′-fluorodeoxy adenosine with S-adenosyl-L-methionine as substrate at 37 °C (
Figure 8). It can be seen from
Figure 8 that the peak position of the substrate SAM is 2.3 min, and the peak position of the product is 3.2 min. Moreover, at the optimum pH of 7.5, the activity of FHAp-NFs was approximately 2-fold higher than that of the free enzyme.
To examine the effect of reaction time on the yield of target product 5′-fluorodeoxy adenosine (5′-FDA), at regular intervals, a 500 μL sample was taken from the incubation mixture and used for HPLC analysis. The reaction mixture was incubated at 37 °C with SAM (0.8 mM), KF (10 mM), and fluorinase (1 mg·mL
−1) in a total volume of 10 mL. The results derived from
Figure S10 show that the observed Km for F
− is 1.12 ± 0.21 mM and kcat/Km is 0.13 mM
−1·min
−1. However, the Km for F
− is 10 ± 2 mM and kcat/Km is only 0.059 mM
−1·min
−1 for the free enzyme [
47]. Therefore, the catalytic efficiency of the
[email protected] is two-fold higher than that of the free enzyme.
As shown in
Figure 9, more than 98% of S-adenosyl-L-methionine (SAM) substrate was transformed to product 5′-fluorodeoxy adenosine using
[email protected] after 25 h of reaction. In addition, the
[email protected] gave a better catalytic activity than that of the same amount of free FDAS in the synthesis of 5′-fluorodeoxy adenosine (5′-FDA) when S-adenosyl-L-methionine (SAM) and a fluoride ion were used as substrates. By comparison, it can be concluded that in the first two hours, the conversion rate of the immobilized enzyme was twice that of the free enzyme. With the extension of the reaction time, the product conversion rate gradually increases, but the free enzyme is still lower than the immobilized enzyme. This may result from the more remarkable loss in enzyme activity for the free enzyme than for the
[email protected] as the reaction time increases.