κ-Selenocarrageenase activity and response surface test results
Polysaccharides can only be stained with Lugol’s iodine solution (a mixture of potassium iodide and iodine components), while oligosaccharides degraded by carrageenase cannot be stained. As shown in Fig.1, the transparent zones around intracellular and extracellular enzymes appeared on the plate, thus proving that κ-selenocarrageenan was degraded by κ-selenocarrageenase. The intracellular and extracellular enzyme activities of Xi13 were 6.079 ± 0.5 U/mL and 3.254 ± 0.3 U/mL, respectively.
Design Expert 8.0.6 software was used to conduct quadratic multiple regression analysis of variance on the experimental results. The quadratic regression equation is:
Y=8.33+0.43A-0.39B-0.38C-0.46AB-0.46AC-0.18BC-1.95A2-1.25B2-0.44C2 (1)
The model’s R2 value was 0.9730 with a 95% confidence level, indicating that the quadratic polynomial model (Equations (1)) fits the experimental data well. Furthermore, the lack of fit (p=0.1552>0.05) was non-significant, indicating that the model was acceptable and suitable for the optimization of enzyme production in the fermentation of Xi13. The positive linear effects of A (κ-selenocarragenan concentration), B (Ca2+ concentration), and C (temperature) on Y (enzyme activity) were found to be significant (p<0.05). All of the quadratics were significant items (p<0.05). Yet those of BC was non-significant (p>0.05) (Table 3). The model fitted well with the actual results, and may be used to analyze and predict the enzyme activity of Xi13.
The Expert Design software was used for further analysis and prediction, and the response surface map and contour map were drawn as Fig.2. κ-Selenocarrageenase activity increased with the three factors, with its peak activity approaching the midpoint of the response plot. As shown in Fig.2, the optimum concentration of κ-selenocarragenan for the enzyme production was 1.59%. The results were similar to marine strain on Pedobacter sp. NJ-02 (Zhu and Ning, 2016). When the concentration is lower than 1.59%, the enzyme activity increases with the increase of the concentration, because the effect of the increase of the concentration on microbial growth, metabolism and inductive effect of κ-selenocarrageenase is greater than the adverse effect due to the increase of the viscosity of the reaction medium. When the concentration is higher than this value, however, the enzyme activity decreases with the increase of the concentration, because the adverse effect due to the increase of the viscosity on microorganisms is greater than that of the increase of the concentration. Generally, when fermentation encounters high viscosity media containing macromolecules, the large mass transfer resistance will seriously affect the growth and metabolism of microorganisms (Salehi et al., 2021). A high concentration of κ-carrageenan will result in a high viscosity of the medium because the viscosity significantly increases with the increases of the concentration. A high viscosity will lead to a large mass transfer resistance of the substrate, such as oxygen and κ-carrageenan, thereby reducing the enzyme production rate of strain Xi13. Ca2+ concentration was found to have significant effects on κ-selenocarrageenase production and cell growth. The optimum concentration of Ca2+ is 3.69 mmol/L, which was consistent with a previous report by Guo et al.(J. Guo et al., 2016), showing the optimum concentration of Ca2+ for production of κ-carrageenase by Thalassospira sp. Fjfst-332 was 0.1g/L.
Three factors corresponding to the highest enzymatic activity of the response surface map were 1.6% κ-selenocarragenan (theoretical value 1.59%), 3.7 mmol/L Ca2+ (theoretical value 3.69 mmol/L), and the temperature 33℃ (theoretical value 32.52℃), respectively. Three parallel fermentation experiments were repeated, and the enzyme activities were 8.372, 8.407 and 8.468 U/ml, respectively, and the average value was 8.416 U/mL, which is in good agreement with the predicted value (8.495 U/ml), indicating that the model could predict the κ-selenocarrageenase production (Table 4) very well.
Enzymatic properties of κ-selenocarrageenase
As shown in Fig. 3(a), with the increase of temperature, the catalytic capacity of κ-selenocarrageenase increased first and then decreased, and the activity of κ-selenocarrageenase reached the highest value at 40℃. The relative enzyme activity at 70℃ was more than 70% lower than that at the optimum temperature (Fig.3(a)). Fig.3(b) shows that the relative enzyme activity completely lost at 45℃ for 6 h. While at 35℃ and 40℃ for 6 h, the relative enzyme activity still retained 40.78% and 18.56%, which are better than those at 45℃ and means the κ-selenocarrageenase has higher thermal stability. The optimum temperature for thermostable κ-carrageenase production of Pseudoalteromonas SP. QY203 was 45℃. (Li et al., 2013) Sun et al. (Sun et al., 2010) isolated a strain Tamlana sp. HC4 producing carrageenase whose optimum reaction temperature was measured to be 30℃. Compared with above researches, the thermal stability of κ-selenocarrageenase from Xi13 needs to be further improved.
The pH is an important factor that affects the enzyme activity. As shown in Fig.3(c), the relative activity of κ-selenocarrageenase was maximized at pH 8.0, which was ~1.7 fold that at pH 5.5, indicating that the enzyme had better tolerance to alkaline environment. This is probably because the spatial structure of the protein changed under acidic conditions, leading to the loss of its function. However, the relative enzyme activity value at pH 10 was only about 30% of that at the optimal pH 8.0. It is suggested that the strong alkali may cause the change of spatial conformation of the protein and thus leading to the inactivation of the protein, resulting in inhibited rate of enzymatic reaction. It also could be observed that the relative enzyme activity remained above 70% when kept at 4℃, pH 8.0-9.0 for 3.0 h, indicating the optimum pH of κ-selenocarrageenase from Xi13 was 8.0 (Fig.3(d)). The results were similar to previous findings on κ-carrageenases of Pseudoalteromonas sp. ZDY3 (Zhao et al., 2021) and Tamlana sp. HC4 (Sun et al., 2010).
As shown in Fig 3(e), the activity of κ-selenocarrageenase can be promoted by Na+, Ca2+, K+ and Mg2+, while Ca2+ has the best effect, which can increase the activity by 72.43%. On the contrary, Mn2+, Zn2+, Ba2+ inhibited the enzyme activity, and the relative enzyme activity of solution added Mn2+only retained 33.21%. Cu2+ had no obvious effect on enzyme activity. However, Mg2+ and Ba2+ significantly stimulated the κ-carrageenase activity of Pseudoalteromonas porphyrae (Guang-Lei Liu et al., 2011), which is inhibited by Ca2+, Zn2+ ,Cu2+ and Na+. The reason may be that ions have different effects on the functional domains of proteins due to different stereoscopic structures of kinds of carrageenases.
It can be seen from Fig. 3(f), the surfactant Tween-80 promotes the activity of κ-selenocarrageenase because it can increase the contact between enzyme and substrate and interact with enzyme molecules through hydrogen bonding and hydrophobic interaction (You-Yan Liu et al., 2000). EDTA inhibites the enzymeactivity, indicating that the enzyme was ion dependent. DTT and β-mercaptoethanol has different inhibitory effects on the enzyme, which may be due to the presence of thiols and the protective effect of catalytic amino acid (Cys). SDS is a denaturing agent, which makes enzyme conformation change greatly and thus inhibits its activity. However, PMSF shows no significant effect on enzyme activity, indicating that serine and histidine are not involved in the active center.
Optimization of vacuum freeze-drying process
The relative enzyme activity of κ-selenocarrageenan prefrozen solution at -20℃, -80℃ and liquid nitrogen is shown in Fig.4(a). It can be seen from Fig.4(a) that the optimum activity was reached at -80℃, while the enzyme activity decreased when the temperature deviated from -80℃. When the temperature reaches between the freezing point and the eutectic point of the product, the enzyme needs to cool quickly. When the temperature is too low, the pre-freezing rate will slow down, which will easily lead to protein denaturation (Pikal, 2004). As can be seen from Fig.4(b), the relative enzyme activity increased gradually when the pre-freezing time was from 6 h to 12 h, and reached the optimum activity at 12 h. Prolonging the pre-freezing time would reduce the enzyme activity, thus 12 h should be chosen as the optimal pre-freezing time. In the process of vacuum freeze-drying, the enzyme activity increased with the increase of the thickness of enzyme solution layer when it was less than 5mm (Table 5). However, the enzyme activity began to decrease when the thickness was more than 5mm. As the thickness increases, the mass transfer resistance of water vapor will increase, which will slow the leakage of water vapor, thus reducing the damage caused by rapid dehydration to proteins. Otherwise, when the feed liquid layer is too thick, the heat transfer resistance and the mass transfer resistance of water will be increased, resulting in the extension of the drying stage and loss of enzyme activity (Du et al., 2013). Trehalose as a lyophilized protective agent can stabilize the structure and function of proteins. With the increase of trehalose concentration, the enzyme activity reached the highest at the concentration of 2.0% (Fig. 4(c)). When the concentration exceeded 3%, the enzyme activity tended to be stable. Too much protective agent will increase the viscosity of the solution, prolong the freeze-drying time and waste energy, so the concentration of 2.0% trehalose was chosen. The changes of κ-selenocarrageenase activity and water loss rate with the increase of drying time are shown in Fig. 5. The enzyme activity at the starting point is the κ-selenocarrageenase activity that the enzyme solution is only frozen but not dried. It can be seen that there is no loss of the enzyme activity during the freezing process, and the main stage of loss is in the process of water sublimation. Because thermodynamic destabilization will occur due to the loss of hydrogen bonding between protein and water during drying stage, which will lead to the decrease of enzyme activity (Pikal, 2004).
Stability of lyophilized κ-selenocarrageenase
The optimum catalytic temperature of free enzyme is the same as that of lyophilized enzyme, and the change trend of their activities is similar (Fig.6(a)). However, the relative enzyme activities of free enzyme and lyophilized enzyme were 13.56% and 60.17%, respectively after holding at 40℃ for 6.0 h (Fig.6(b)). The thermal stability of lyophilized enzyme was better than that of free enzyme at 40℃ due to the protective effect of lyophilized protectant on protein. The optimum pH of lyophilized enzyme was pH 8.0, which was the same as that of free enzyme (Fig.6(c)). However, the free enzyme was more sensitive to the change of pH than the lyophilized enzyme. When the pH changes slightly, the free enzyme activity decreases or increases rapidly. The relative enzyme activity of lyophilized enzyme remained above 80% when kept at 4℃ and pH 7.0-9.0 for 3.0 h (Fig.6(d)). The results indicate that the pH stability of lyophilized enzyme is higher than that of free enzyme.
There are various factors such as temperature, pH, air and light that contribute to storage stability of enzyme. After 30 days of storage at -20℃ and 4℃, the residual enzyme activity was 34.22% and 15.02%, respectively. By contrast, the enzyme activity was completely lost when kept at 25℃ and 37℃ for 30 days (Fig. 7(a)). As the enzyme belongs to the protein, the spatial structure of the protein is likely to change and its activity will be lost due to the high temperature during storage. Therefore, -20℃ is chosen as the storage temperature of lyophilized enzyme. Not only temperature but also air significantly affects the storage stability of enzyme. The residual enzyme activities of lyophilized enzyme were 26.31% and 13.34% at -20℃ and 4℃ when exposed to air and protected from light for 30 days (Fig.7(b)), respectively, which were about 10% lower than those under vacuum conditions. Therefore, vacuum storage should be selected because air has an adverse impact on the storage of enzyme preparations. Fig. 7(c) shows that the residual enzyme activity of lyophilized enzyme stored at -20℃ in light for 30 days is 16.56%, which was lower than that under dark condition. Since light provides energy to accelerate enzyme inactivation, enzyme preparations should be stored away from light. In order to control the microbial content, sterilization is necessary, such as UV irradiation, which inevitably affects enzyme activity. When the UV irradiation time was within 0-40 min, the activity of lyophilized enzyme remained in a stable range, and the residual enzyme activity was above 40%. The activity began to decrease at 40min of UV irradiation, and decreased to 13.03% of that at 90min (Fig. 7(d)), indicating that the activity could be maintained if the irradiation time was kept within 40 min.
Optimization of enzyme immobilization
The morphology of sodium alginate beads with different concentrations of sodium alginate was shown in Fig.8. It was observed that the particle size of 3% sodium alginate beads with low hardness is more uniform, and the beads is easy to drop and their roundness is appropriate. There is no obvious tailing on the surface of the 3% sodium alginate beads, which has good viscoelasticity, good transparency and excellent mechanical strength. However, the particle size of other immobilized enzymes prepared by using different concentrations of sodium alginate is not uniform with low roundness.
Too high concentration of sodium alginate would limit the diffusion of both substrate and product. Instead, too low concentration of sodium alginate would lead to the loss of enzyme molecules from the gel space. As shown in Fig.9(a) the relative enzyme activity increased with the increase of the concentration of sodium alginate up to 3.0%. When the concentration exceeds 3.0%, however, the enzyme activity decreased. Because the gel formed between sodium alginate and free calcium ions, and then the enzyme was limited to react with κ-selenocarrageenan. The crosslinking process can restore damaged reticular structure, avoid enzyme shedding and improve its reusability (Seenuvasan et al., 2013). Glutaraldehyde is a protein denaturing agent and also a crosslinking agent. In the range of 0.1%-0.5% glutaraldehyde, the activity of immobilized enzyme increased continuously (Fig.9(b)). Excessive glutaraldehyde concentration can lead to excessive crosslinking and inactivation of the enzyme. Therefore, the enzyme activity decreases when glutaraldehyde concentration exceeds 0.5%. Alginate can combine with calcium ions to form a special "EGG-box" structure to immobilize the enzyme (Braccini and Pérez, 2001). When the concentration of calcium ions is too low, the embedding effect is poor. On the contrary, excessive calcium ions covering the gel surface will hinder the interaction between enzyme and substrate. Therefore, the enzyme activity reached the highest when the concentration of CaCl2 reached 3% (Figure 9(c)). As can be seen from Fig.9(d), the enzyme activity reached the maximum at the fixed time of 2 h and the cross-linking time of 1.5 h. Too long fixed time may aggravate the action of calcium ions on the enzyme, hinder the reaction of substrate and lead to the decrease of enzyme activity. If the fixation time is too short, the gap of the gel is enlarged, and the enzyme is not firmly bound to the substrate.
The enzyme activity recovery rate, i.e. the residual enzyme activity, was 62.83%. Fig.10 shows the morphology of alginate sodium-immobilized enzyme. Milky white immobilized enzyme has uniform particle size and smooth surface. The freeze-dried alginate sodium-immobilized enzyme had loose network structure (Fig.10(c)).
Stability of immobilized κ-selenocarrageenase
As shown in Fig.11(a), the immobilized κ-selenocarrageenase exhibited higher thermal stability with optimum temperature of 50℃ which is 10℃ higher than that of free enzyme. At the same temperature, the thermal stability of the immobilized enzyme was better than that of the free enzyme. When κ-selenocarrageenase molecules were immobilized by sodium alginate, the carrier would produce steric and shielding effects. The movement of the enzyme molecules is blocked by the alginate gel network, which greatly increases the ability of the enzyme to resist the external environment. The immobilized κ- selenocarrageenase has higher stability at elevated temperatures than the free enzyme as reported (Li et al., 2013; Shen et al., 2018). The optimum pH of both free and immobilized enzymes was 8.0 (Fig.11(c)). When the free enzyme and immobilized enzyme were placed in the buffer solution of pH 2.0-9.0 for 3.0 h, the free enzyme was more sensitive. The activity of immobilized enzyme preparation remained above 80% after being placed at 4℃, pH 7.5-9.0 for 3.0 h, showing good tolerance to alkaline environment (Fig.11(d)).
As shown in Fig.11(e), the loss rate of immobilized and free enzymes increased with the extension of storage time at -20℃. After 20 days of storage, the residual activity of the free enzyme was 20.31%, however, the activity of the immobilized enzyme was almost completely lost. The reason may be that the water in the immobilized enzyme is frozen at -20℃, forming many ice grains. On the one hand, the ice grains can reduce the stability of enzymes and thus affect the enzyme activity. On the other hand, ice particles change the internal structure of sodium alginate particles, affecting the internal diffusion and thus the overall speed of the catalytic reaction. Instead, at 4℃, the activity loss rate of immobilized enzyme was significantly lower than that of free enzyme. This may be because the spatial configuration of the enzymes embedded in sodium alginate is more stable and the mechanical strength is enhanced. After 30 days, the residual enzyme activity of the immobilized enzyme remained above 40%, while the free enzyme was inactivated, indicating that the storage stability of the immobilized enzyme was better than free enzyme at 4℃. The immobilized enzymes benefit the repeated, continuous, or batch uses of enzymes, and as a result lessen production cost. The reusability of the immobilized enzymes is an indispensable parameter for industrial applications. Fig.11(f) shows the activity of immobilized enzyme after repeated use. After recycling used for four times, the immobilized enzyme activity decreased to 58.28%. The experimental results showed that the alginate immobilized enzyme was feasible and could be used for many times.
Selenium content of the enzymatic degradation product by κ-selenocarrageenase preparations
Selenium content in κ-selenocarrageenan oligosaccharide which is a degradation product of κ-selenocarrageenan catalyzed by lyophilized κ-selenocarrageenase preparation and immobilized κ-selenocarrageenase, was determined respectively. As shown in Table 6, the average selenium content of κ-selenocarrageenan was 55.73 µg/g. The organic selenium content of the degradation products by lyophilized κ-selenocarrageenase and immobilized κ-selenocarrageenase were 43.45 µg/g and 47.06 µg/g, respectively.