Expression and purification of the K5-C and β-1, 3-xylanases
The diagrammatic sketch of the K5-C chimera was shown in Fig. 1a. The gene coding the SpyCatcher was fused to the K5V4F gene provided by the parent vector. After expressed in the host strain and purified, the purity and molecular weight of K5-C and β-1, 3-xylanases (Xyl3088) were characterized by SDS-PAGE. For the purity of K5-C and Xyl3088, SDS-PAGE yielded a clear thick band of 32 kDa and 49 kDa, respectively (Fig. 1b, c), which matched their theoretical molecular weight values of 32002 Da and 48853 Da calculated by ProtParam (http://web.expasy.org/protparam/). The final yield of K5-C and Xyl3088 measured by BCA protein assay was 273.05 ± 11.5 mg and 13.65 ± 1.4 mg from 1L culture, respectively. It means that the large-scale preparation of K5-C protein can be easily achieved by two rounds of ITC which requires only inexpensive reagents such as sodium chloride and simple centrifugation techniques[21, 27]. Thus, the expensive chromatographic purification processes were avoided. In addition, the K5-C has the ability of biomimetic silicification and can be used for low-cost mass production. It can be used in subsequent large-scale preparation of nanoparticles for immobilizing the targeted enzymes[22, 28]. Finally, the free β-1, 3-xylanases was purified by nickel column and the quantity-calculating results revealed the Xyl3088 comprised about 92% of the total soluble proteins after purification, which can be used for further investigation into the characteristics.
Synthesis of silica nanocomposites by K5C
When freshly prepared TMOS was loaded to K5-C solution under room temperature, white precipitation was mediated within seconds in PBS solution (Fig. 2a, right). While, no precipitation was observed in the negative control using the same reaction system without K5-C (Fig. 2a, left), indicating that K5-C is a vital template for silica synthesis. The scanning electron microscopy (SEM) images showed that the white precipitation mediated by K5-C were spherical with rough surfaces, and the diameters were from 200 to 600 nm (Fig. 2b). This suggested that high surface area for the anchoring of numerous SpyCatcher, which was beneficial to immobilizee enzymes by covalent bond. The transmission electron microscope (TEM) images further confirmed the morphology and size of the white precipitation (Fig. 2c). Besides, strong signals for C, O, N and Si in the white silica nanosphere were analyzed by energy dispersive spectrometer (EDS) experiment (Fig. 2d), indicating that K5-C fusion proteins were successfully encapsulated in the silica nanospheres (NPs). Hence, the silica NPs were organic-inorganic hybrid complexes, which can be applied for subsequent target enzyme immobilization.
Influences of K5-C concentration on the immobilization efficiency
To quantitatively analyze the mineralization activity of K5-C, the correlation between K5-C concentration and the yield of silica NPs was measured. As shown in Fig. 3a, the yield of silica was almost proportional to the amount of K5-C applied under premise controlling of the amount of silica precursor (TMOS). It was also consistent with some previous studies which silaffin, ELP(KV8F) and R5 were used to form silica nanospheres [28–30]. When the concentration of K5-C was 100 µ mol/L, 44.97% of TMOS was converted to silica NPs. Meanwhile, the specific activity of K5-C, R5 and ELP(KV8F) was listed in Table 1. Under the same reaction conditions, the specific activities of silaffin and R5 were only between 2 and 4, while the activities of K5-C and KV8F reached 99.93 and 97.18, respectively. These results proved that K5-C was a silica mineralized polypeptide with high catalytic activity and the introduction of lysine enhanced the activity of the polypeptides.
Table 1
Half-life of the free and immobilized β-1,3-xylanase at different temperature
Temperature | Free β-1,3-xylanase | Immobilized β-1,3-xylanase |
40 ℃ | 106.04 ± 10.75 | 113.23 ± 11.82*** |
45℃ 50℃ | 20.44 ± 1.98 17.28 ± 0.46 | 31.08 ± 2.06* 28.06 ± 1.25** |
t-test was performed on half-life of the free and immobilized β-1,3-xylanase at different temperatures. * indicate P < 0.05, **indicate P < 0.01 and ***indicate P < 0.001. |
To optimize K5-C immobilization efficiency, the concentration of K5-C varied from 100 to 500 µ mol/L. As can be seen in Fig. 3b, higher ratios of K5-C could self-immobilize in silica NPs with the increasing concentrations of K5-C. Approximately 99% of K5-C protein were immobilized when the concentration was 300 µ mol/L, and then the immobilization efficiency decreased with the increase of K5-C concentrations. This may be due to the exhaustion of TMOS, resulting in the excess of K5-C. It means that K5-C could almost self-immobilized in the silica NPs by completely consuming TMOS at 300 µ mol/L.
The capacity of immobilized K5-C for applications was further investigated by studying the extent of leaching[31]. As shown in Fig. 3c, the silica NPs could firmly entrapped the K5-C protein and negligible protein leakage was observed in the supernatant of silica NPs even after 56 h storage at 4℃, indicating that K5-C was strongly embedded inside the silica NPs, while the amine-based biosilica only retain 90% of the enzyme after 24 h of storage[31, 32]. Accordingly, K5-C@SiO2 NPs was a stable carrier displaying SpyCatcher on NPs surface, resulting in efficient assembly of the target enzymes in subsequent steps.
Purification and immobilization of β-1, 3-xylanases in one-step
To date, most of the enzyme immobilization techniques required prior purification of the target enzymes[
33–
35], which was a well-known process of costly and time-consuming[
36]. Hence, development of a simple and versatile strategy for simultaneous purification and immobilization of the target enzymes was highly desirable[
13]. Here, the silica NPs containing SpyCatcher on the surface as a carrier was loaded in the cell lysate of the chimeras of SpyTag fused β-1, 3-xylanases (X-T). The immobilization and purification results were shown in Fig.
4, the supernatant of X-T cell lysate after employing K5-C@SiO
2 revealed the loss of X-T band (51 kDa), while the amount and location of other impurities remained unchanged (Fig.
4, 2nd lane). Meanwhile, the loading of K5@silica NPs without SpyCatcher did not change the targeted X-T and other impurities (Fig.
4, 3rd lane), especially the target X-T. Accordingly, it clearly revealed that K5-C@SiO
2 can specifically and covalently react with the X-T(via SpyCatcher and SpyTag) from the cell lysate in one-step. The immobilization efficiency and activity recovery of the captured β-1, 3-xylanases (Xyl3088) reached 85.4% and 88.6%, respectively.
Self-immobilizing and purification systems possessing such tags offer advantages since they immobilize targeted enzymes under mild and non-toxic environment while retaining stereoselectivity and activity[37]. Besides, they would also save costs due to avoidance of crosslinkers and simplification of immobilization processes, such as purification and immobilization directly from crude cell lysate without any prior costly purification steps[38].
Effect of temperature and pH on the activity of the free and immobilized β-1, 3-xylanases
The ability of an enzyme may be modulated by its immediate microenvironment[39]. So, the activities of free and immobilized Xyl3088 were assayed at temperatures ranging from 25 to 75 °C (Fig. 5a). The relative activities of immobilized β-1, 3-xylanases (Xyl3088) were higher than those of the free ones over most of the temperature points, indicating that the immobilized Xyl3088 had more preferable temperature adaptability than the free one. The maximum activity of the free Xyl3088 was observed at 45 °C, while the optimum temperature of the immobilized Xyl3088shifted up to 50 °C. The rise in optimum temperature may be owe to the reducing conformational flexibility, which requires a higher activation energy for the molecule to reorganize its proper conformation to bind to the substrate [40], thus activity even at higher temperature is one of the main advantages of immobilized enzymes [41].
The optimum pH for the activity of an enzyme was mainly dependent upon the nature of its functional groups. Besides, binding enzyme on a solid matrix may increase the pH tolerance depending on the surface and residual charges of the solid matrix [42, 43].As shown in Fig. 5(b), the optimum pH of both the free and immobilized Xyl3088 were observed at 6.6. But the immobilized Xyl3088 was found to be more stable than the free Xyl3088 from pH 4.0 to 9.0, this may be due to an increase in net charge arising from the binding of the enzyme to the silica NPs[44].
The thermal stability, storage stability and reusability of the free and immobilized β-1, 3-xylanases
The activity of β-1, 3-xylanases (Xyl3088) was highly sensitive to temperature, therefore, improving the thermostability of them is very important for potential industrial application. The thermostability of the free and immobilized Xyl3088 were shown in Fig. 6(a), the immobilized Xyl3088 was more stable than the free one after incubation at 40 °C ,45 °C and 50 °C, respectively. This was further confirmed by the half-life test (Table 1). For example, the half-life of the free Xyl3088 reduced significantly in respect to the immobilized form (the half-life of the immobilized Xyl3088 in PBS buffer at 50 °C was about 28 min, which was 1.65-fold longer than the free one). The t-test result showed the p value was less than 0.01, revealing the differences between them were extremely significant. It also indicated that covalent immobilization might change the conformation of β-1, 3-xylanases, resulting in higher thermostability towards temperature compared with the free ones [45–47]. Meanwhile, the storage stability of Xyl3088 was evaluated by studying the residual activities after incubating at 30 °C (Fig. 6b). Compared with the free Xyl3088, the storage stability of the immobilized one was sharply increased. After three-day storage, the free Xyl3088 only remained 21% of its initial activity, while the immobilized Xyl3088 preserved 62% of its initial activity. This indicated that silica NPs could afford suitable microenvironment and impose the steric constraints to the β-1, 3-xylanase’s structure, preventing rapid denaturation. The improvement of thermostability was also suggestive of firm enzyme–support interactions, which perhaps arise from the enzyme entrapment that was possible owe to the one-step mild immobilization [31, 48].
The main advantage of immobilized enzyme was the reuse potential, which will save the cost of the enzyme. β-1, 3-xylanases immobilized on silica NPs retained 93.2% of its an initial enzymatic activity after five cycles of successive reusing. Even after twelve cycles, the enzyme maintained around 70.6% of its initial activity (Fig. 7). The slight, but gradual decrease of remanent activity after each cycle may be attributed to several mechanisms, including the incidental loss of silica NPs during centrifugation and transfer in each cycle, and enzyme denaturation or structural modification of β-1, 3-xylanases [32, 41, 49].
Kinetic parameters of the free and immobilized β-1,3-xylanase
The Michaelis-Menten parameters (Km) and the catalytic efficiency (Kcat/Km) of the free and immobilized β-1, 3-xylanases (Xyl3088) were calculated. A slight increase in the Km was seen from the immobilized β-1, 3-xylanases compared with the free ones, indicating that the decreased affinity between enzyme and substrate. This was due to the conformational changes of β-1, 3-xylanases by the immobilization carrier or a less accessibility of the active site of immobilized Xyl3088 to the substrate, especially the substrate was macromolecule β-1,3-xylan. Besides, the catalytic efficiency (Kcat/Km) decreased from 113.05 mg/ml/s for the free Xyl3088 to 73.77 mg/ml/s for the immobilized Xyl3088(Table 2). These results were consistent with most studies about enzyme immobilization. The decrease of active sites and the increase of mass transfer barriers may be responsible for the lower catalytic efficiency[50].
Table 2
The kinetic parameters of the free and immobilized β-1,3-xylanase
| Km(mg/mL) | Kcat(s− 1) | Kcat/Km(mg/ml/s) |
Free Xyl3088 | 5.23 ± 0.37 | 589.11 ± 4.39 | 113.05 ± 8.72 |
Immobilized Xyl3088 | 5.73 ± 0.45 | 513.15 ± 1.43 | 73.77 ± 5.53 |
Finally, the hydrolysates of the free and immobilized Xyl3088 were investigated by thin layer chromatography (TLC) to estimate their catalytic activities. As shown in Fig.S2, the amount and type of hydrolysates differ little, including xylose(X1), xylobiose(X2), xylotriose(X3), xylotetraose(X4), and other oligosaccharides[2, 7]. This revealed that immobilized carrier did not affect the catalytic process and hydrolysis products of β-1,3-xylanase. Wherein the immobilized Xyl3088 could be recycled to hydrolyze the β-1,3-xylan from the seaweed to produce xylose. It also helps develop cheaper and eco-friendly biorefinery processes that convert xylose into valuable chemicals such as 2,3-butanediol, furfural and xylitol.