Abstract
In order to improve the stability of oxalate decarboxylase (Oxdc), response surface methodology (RSM), based on a four-factor three-level Box-Behnken central composite design was used to optimize the reaction conditions of oxalate decarboxylase (Oxdc) modified with monomethoxy polyethyleneglycol (mPEG5000). Four independent variables such as the ratio of mPEG-aldehyde to Oxdc, reaction time, temperature, and reaction pH were investigated in this work. The structure of modified Oxdc was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy, the stability of the modified Oxdc was also investigated. The optimal conditions were as follows: the mole ratio of mPEG-aldehyde to Oxdc of 1:47.6, time of 13.1 h, temperature at 29.9 °C, and the reaction pH of 5.3. Under optimal conditions, experimental modified rate (MR = 73.69%) and recovery rate (RR = 67.58%) were matched well with the predicted value (MR = 75.11%) and (RR = 69.17%). SDS-PAGE and FTIR analysis showed that mPEG was covalently bound to the Oxdc. Compared with native Oxdc, the modified Oxdc (mPEG-Oxdc) showed higher thermal stability and better tolerance to trypsin or different pH treatment. This work will provide a further theoretical reference for enzyme modification and conditional optimization.
Keywords: Oxalate decarboxylase, Monomethoxy polyethyleneglycol, Response surface method (RSM)
Introduction
Oxalate decarboxylase (Oxdc, EC 4.1.1.2) was first found in wood-destroying fungi [1], which is one of the major enzymes that can catalyze the oxalate into formate and CO2 [2]. Since it was reported in 1955, Oxdc has been attracting a lot of interest, and has been widely applied in agriculture, food and industrial process, biological monitoring, human or animal health and other fields [3]. Research showed that Oxdc has been used to treat some oxalate diseases in vivo [4]. Studies of Jeong [5] showed that orally given recombinant Bacillus subtilis Oxdc expressed from E. coli has effectively reduced the urinary oxalate level in a hyperoxaluria rat model experiment, which demonstrated that Oxdc has potential application prospects in food and pharmaceutical industries to prevent and cure calcium oxalate stones.
A major problem with this kind of application is that Oxdc is unstable and is easily damaged by gastric acid or protease and this will limit its application in medical research. Therefore, it is necessary to modify Oxdc appropriately and optimize the reaction conditions, and finally achieve the purpose that the stability and performance of Oxdc can be improved in practical applications.
Chemical modification is often considered to be an effective way of obtaining multifunctional proteins with better stability and catalytic capacity. Research of Distel [6] showed that nonselective modification on the surface of organic-soluble enzyme by capric acid chloride proleather leads to its solubility up to 44 mg/ml in chloroform, and the activity of the modified enzyme has increased 4–22 multiples in different organic solvents. However, for clinical treatment of oxalate stones, the selected modifier that is used should be nontoxic.
Monomethoxy polyethyleneglycol (mPEG), which is a kind of linear polyether compound that is nontoxic, and has been widely used in the modification of protein drugs to reduce antigenicity and prolong the effective time in human body [7]. Research showed that mPEGy can be used to modify superoxide dismutase (SOD), asparaginase, and horseradish peroxidase to enhance its tolerance to acids, heat, and proteases [8, 9]. Therefore, mPEG was selected in this work and the modified conditions were optimized by response surface methodology (RSM). RSM is a collection of mathematical and statistical methods and is typically used to reduce the number of experimental trials, which, for evaluating the multiple parameters and their interactions, is helpful to determine the target value based on the investigation of the individual and interactive effects of the parameters [10]. It is faster and more accurate than the single-factor tests and was first put forward by Box [11].
In this work, the influence of various modifying conditions such as the molar ratio of Oxdc to mPEG, pH, reaction time, and temperature was optimized by using RSM based on the quadratic model Box–Behnken design (BBD), the stability of Oxdc and mPEG-Oxdc to heat, trypsin, and different pH were also investigated.
Experimental section
Materials
Recombinant E. coli (E.coli BL21(DE3)/pET32a/YvrK) was kept in our laboratory. mPEG5000, potassium oxalate, and hydrogenlyase were purchased from Sigma-Aldrich (USA). Ampicillin was obtained from AMRESCO (St. Louis, MO, USA); Isopropyl-β-d-thiogalactoside (IPTG) was purchased from Merck (Darmstadt, Germany) and the nicotinamide adenine dinucleotide phosphate (NADP) was purchased from Roche (Basel, Switzerland); formate dehydrogenase (FDH) was purchased from Sigma (USA); AKTA-FPLC was purchased from USA (GE Healthcare Amersham AB, Sweden). Other reagents with analytical grade were purchased from Beijing Solarbio Technology Co., Ltd (Beijing, China); all the reagents or chemicals were used without further purification.
Analytical methods
Expression and purification of recombinant Oxdc
The expression of Oxdc from recombinant E.coli BL21(DE3)/pET32a/YvrK was completed according to the methods of Lin [12]. Genetically engineered strain was fermented and incubated in LB medium, when the OD600 of the culture reached 0.6 (logarithmic growth period), the cells were induced with 0.4 mmol/l IPTG and 5 mmol/l MnCl2 for 4 h [12]. Then the E. coli was harvested by centrifugation (8000 r/min, 20 min, 4 °C), and finally resuspended in 50 mmol/l phosphate buffered saline (PBS, pH 8.0). The cells were disrupted with ultrasound and supernatant was harvested by centrifugation and filtered with a 0.22-μm micro-membrane filter. The crude enzyme extract was purified by AKTA-FPLC.
Determination of Oxdc
Enzyme activity of Oxdc and mPEG-Oxdc were determined according to the methods of Drazzenka S [13]; 0.08 mM potassium oxalate was mixed in 0.05 mM citric acid buffer (pH 4.0) at 37 °C (named solution A), Oxdc was added in above solution and the reaction lasted for 10 min. Then the reaction was terminated by adding isometric 0.20 mM dipotassium phosphate (named solution B). After this, formate dehydrogenase (FDH) with a concentration of 1 mg/ml and 60 mM nicotinamide adenine dinucleotide phosphate (NADP) were added in solution B, and finally scanning the time spectrum by TU − 1901 in absorbance at 340 nm.
Synthesis of mPEG-Aldehyde
The mPEG was dissolved in a DMSO/chloroform mixture according to literature methods [14, 15], followed by the addition of acetic anhydride and then stirred for 9 h at 20 °C. The above reaction solution was then precipitated in cold ether and filtered to give a white precipitate. The resulting product was dried under vacuum at 20 °C overnight to give a final white powder, which is a mixture of mPEG-aldehyde and unreacted mPEG [16–18].
Determination and characterization of mPEG-aldehyde
The products from Schiff reagent chromogenic reacted with the aldehyde has characteristic UV absorption at 560 nm, according to the relationship between absorbance and concentration, the standard curve was plotted, and then the hydroformylation rate of the product can be calculated as described previously [19].
Microscale of dry powder (mPEG or mPEG-aldehyde) was mixed with KBr press disks. Fourier transform infrared (FT-IR) spectra were obtained with a Bomem MB100 FTIR spectrometer equipped with a DTGS detector. The spectra were recorded between 400 and 4000 cm−1 for each sample, a total of 64 scans were collected at a resolution of 4 cm−1.
Modification of Oxdc with mPEG and determination of the modified rate
Oxdc and mPEG were added in 3 ml 50 mM phosphate buffer (pH 5.0) according to the molar ratio of mPEG-aldehyde/Oxdc = 50:1. The solution was shaken at 25 °C for 12 h and then the reaction was terminated by adding an equal volume of 10 mM glycine. The above solution was filtered by a dialysis bag (molecular intercept 8000–12,000) for 4 h at 4 °C. The determination of the modified rate of Oxdc was according to the TNBS methods as reported by A.F.S.A. HABEEB [20].
Characterization of Oxdc and PEGylated-Oxdc
The molecular weight of Oxdc and mPEG-Oxdc was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the methods of Laemmli [21] . Oxdc or mPEG-Oxdc samples were mixed with an equal volume of 0.10 mM Tris-HCl buffer (pH 6.8), which contained 2% SDS, 20% glycerol and 0.001% Bromophenol blue. In addition, Oxdc and mPEG-Oxdc were also characterized by FT-IR spectroscopy after freeze-drying.
Optimization of mPEG-modified Oxdc by response surface methodology
In order to obtain the optimal reaction conditions, RSM based on Box–Behnken design (BBD) was used in this study for maximizing the extent of modification and the recovery rate of enzyme activity. The factors of the main operating conditions including the molar ratio of mPEG-aldehyde/Oxdc, reaction time, pH value, and reaction temperature were studied by single-factor experiment (data were not shown here). A three-level-four-factor BBD in RSM was applied to optimize the operating parameters in the present study. Actual values of the parameters were selected at three levels, coded as −1, 0 and +1 for low, middle, and high values, respectively. All the reaction conditions are given in Table 1.
Table 1.
Coded and actual levels of factors in the experimental design
| Factor | Coded levels | ||
|---|---|---|---|
| −1 | 0 | +1 | |
| Mole ratio of mPEG-aldehyde/Oxdc | 40:1 | 50:1 | 60:1 |
| Reaction temperature (°C) | 24 | 32 | 40 |
| Reaction time (h) | 6 | 12 | 18 |
| Reaction pH | 3.0 | 5.0 | 7.0 |
Results and discussion
SDS-PAGE analysis results
As shown in Fig. 1, the molecular weight of Oxdc is about 42 kDa. The two or three bands that appeared on the profile of mPEG-Oxdc indicated that one or more mPEG molecules were only grafted onto one Oxdc molecule. Similar results were also put forward by Yang Qingyuan [22] according to the research with mPEG5000-modified superoxide dismutase (SOD).
Fig. 1.
SDS-PAGE analysis of the modified Oxdc. (M: Marker; 1: Oxdc; 2: modified for 6 h; 3: modified for 12 h; 4: modified for 24 h, 5: mPEG-aldehyde)
FTIR analysis of Oxdc and mPEG-Oxdc
The prepared samples were dried under vacuum and were mixed with KBr. Then the mixture was compressed into a disk. Spectral scanning was performed in the range of 4000 to 500 cm−1. As shown in Fig. 2, mPEG-Oxdc gave absorption bands at 1636.27 cm−1 according to the C = N stretching vibration [23], but no bands appeared for unmodified Oxdc. This demonstrated that Schiff base was performed between the aldehyde group of mPEG and a-NH2 group of Oxdc. In addition, mPEG and mPEG-Oxdc both showed a broad peak around 1100 cm−1, which was attributed to the characteristic stretching of C-O-C [24], but no peaks for Oxdc indicated that mPEG had grafted onto Oxdc successfully.
Fig. 2.
a Infrared spectrum of Oxdc, b mPEG-Oxdc, c mPEG
Mathematical model and optimization of reaction conditions
Mathematical model and analysis of ANOVA
The experimental MR and RR values of mPEG-Oxdc are shown in Table 2. Based on the Box–Behnken design (BBD) and results of the experiments, according to the multiple regression analysis, the coefficients of the regression equation were calculated using the software of design expert 8.0.6.1 (Stat-Ease, Minneapolis, Minnesota, United States (U.S.)). The following second-order polynomial equation was established to investigate the MR value of mPEG-Oxdc.
Table 2.
Experiment design and results of the Box–Behnken central composite design
| Run | mPEG-aldehyde/Oxdc A | Temperature B (°C) | Time C (h) | pH D |
Modified rate (%) | Recovery rate of enzyme activity (%) |
|---|---|---|---|---|---|---|
| 1 | 0 | −1 | 0 | −1 | 35.54 | 27.93 |
| 2 | 0 | 1 | 0 | −1 | 28.72 | 10.86 |
| 3 | 0 | 0 | 0 | 0 | 75.65 | 63.26 |
| 4 | 0 | 0 | 1 | −1 | 35.11 | 24.31 |
| 5 | −1 | −1 | 0 | 0 | 52.32 | 52.58 |
| 6 | 0 | 0 | 0 | 0 | 74.15 | 67.2 |
| 7 | 0 | −1 | 0 | 1 | 21.37 | 70.75 |
| 8 | −1 | 0 | 0 | −1 | 65.85 | 32.37 |
| 9 | 0 | 0 | −1 | 1 | 13.49 | 76.1 |
| 10 | −1 | 0 | −1 | 0 | 45.16 | 72.62 |
| 11 | 0 | 1 | 1 | 0 | 83.83 | 51.23 |
| 12 | 1 | 1 | 0 | 0 | 72.13 | 21.77 |
| 13 | 1 | 0 | 0 | −1 | 46.9 | 39.85 |
| 14 | 1 | 0 | 1 | 0 | 73.73 | 38.63 |
| 15 | −1 | 1 | 0 | 0 | 75.06 | 42.02 |
| 16 | −1 | 0 | 1 | 0 | 72.07 | 44.76 |
| 17 | 0 | 1 | 0 | 1 | 23.84 | 43 |
| 18 | 0 | 0 | 0 | 0 | 66.01 | 67.91 |
| 19 | 0 | −1 | 1 | 0 | 65.45 | 75.02 |
| 20 | 0 | 0 | 1 | 1 | 29.3 | 41.6 |
| 21 | 0 | 0 | −1 | −1 | 16.16 | 53.53 |
| 22 | 0 | 1 | −1 | 0 | 52.14 | 57.39 |
| 23 | −1 | 0 | 0 | 1 | 33.17 | 45.21 |
| 24 | 0 | −1 | −1 | 0 | 45.11 | 86.18 |
| 25 | 1 | −1 | 0 | 0 | 58.08 | 66.79 |
| 26 | 0 | 0 | 0 | 0 | 78.55 | 63.91 |
| 27 | 0 | 0 | 0 | 0 | 75.98 | 64.23 |
| 28 | 1 | 0 | 0 | 1 | 39.17 | 61.46 |
| 29 | 1 | 0 | −1 | 0 | 43.21 | 54.17 |
Modified rate (%) = 75.8-3.53×A+5.65×B+16.44×C-4.25×D+4.08×A×B+2.65×A×C-1.26×A×D+2.84×B×C+0.57×B×D-0.79×C×D-8.99×A2-8.90×B2-14.38×C2-39.14×D2 (R2=0.9521). Recovery rate(%)= 65.30-5.57×A-11.25×B-9.04×C+16.27×D-3.62×A×B+0.58×A×C-0.31×A×D-1.25×B×C-4.67×B×D-0.32×C×D-9.63×A2-5.72×B2+2.79×C2-16.04×D2 (R2=0.9028).
Analysis of variance (ANOVA) was performed to check the significance of the experimental data, and the results are shown in Tables 3 and 4.
Table 3.
Analysis of variance (ANOVA) for the modified rate of mPEG-Oxdc
| Source | Sum of square | Degree of freedom | Mean square | F value | Prob > F | Significance |
|---|---|---|---|---|---|---|
| Model | 14,396.43 | 14 | 1028.32 | 19.86 | < 0.0001 | ** |
| A | 149.88 | 1 | 149.88 | 2.89 | 0.1110 | |
| B | 383.64 | 1 | 383.64 | 7.41 | 0.0165 | * |
| C | 3241.31 | 1 | 3241.31 | 62.6 | < 0.0001 | ** |
| D | 216.24 | 1 | 216.24 | 4.18 | 0.0603 | |
| AB | 66.5 | 1 | 66.5 | 1.28 | 0.2761 | |
| AC | 28.14 | 1 | 28.14 | 0.54 | 0.4732 | |
| AD | 6.38 | 1 | 6.38 | 0.12 | 0.7309 | |
| BC | 32.21 | 1 | 32.21 | 0.62 | 0.4435 | |
| BD | 1.31 | 1 | 1.31 | 0.025 | 0.8758 | |
| CD | 2.46 | 1 | 2.46 | 0.048 | 0.8304 | |
| A2 | 524.32 | 1 | 524.32 | 10.13 | 0.0067 | ** |
| B2 | 513.58 | 1 | 513.58 | 9.92 | 0.0071 | ** |
| C2 | 1340.73 | 1 | 1340.73 | 25.89 | 0.0002 | ** |
| D2 | 9939.15 | 1 | 9939.15 | 191.94 | < 0.0001 | ** |
Table 4.
Analysis of variance (ANOVA) for the recovery rate of mPEG-Oxdc
| Source | Sum of square | Degree of freedom | Mean square | F Value | Prob > F | Significance |
|---|---|---|---|---|---|---|
| Model | 8529.2 | 14 | 609.23 | 9.29 | < 0.0001 | ** |
| A | 372.86 | 1 | 372.86 | 5.69 | 0.0318 | * |
| B | 1518.3 | 1 | 1518.3 | 23.16 | 0.0003 | ** |
| C | 979.94 | 1 | 979.94 | 14.95 | 0.0017 | ** |
| D | 3177.53 | 1 | 3177.53 | 48.47 | < 0.0001 | ** |
| AB | 52.27 | 1 | 52.27 | 0.80 | 0.3872 | |
| AC | 1.35 | 1 | 1.35 | 0.02 | 0.8881 | |
| AD | 0.38 | 1 | 0.38 | 5.77E-03 | 0.9405 | |
| BC | 6.25 | 1 | 6.25 | 0.095 | 0.7621 | |
| BD | 87.24 | 1 | 87.24 | 1.33 | 0.2681 | |
| CD | 0.41 | 1 | 0.41 | 6.25E-03 | 0.9381 | |
| A2 | 601.25 | 1 | 601.25 | 9.17 | 0.0092 | ** |
| B2 | 211.96 | 1 | 211.96 | 3.23 | 0.0938 | |
| C2 | 50.35 | 1 | 50.35 | 0.77 | 0.3956 | |
| D2 | 1667.85 | 1 | 1667.85 | 25.44 | 0.0002 | ** |
A value of Prob > F less than 0.05 indicates the model terms are statistically significant, since the statistical relation between the response and the selected variable would be at 95% confidence level when the P value for the model was lower than 0.05. The P value for the multiple regression was very small (Prob > F < 0.0001), which indicates that the established model could adequately represent the real relationship among the selected parameters. The model F-value given in Tables 3 and 4 implies the model was significant. In addition, the P value was also used to check the significance of each coefficient; the smaller P value, the more significant the corresponding coefficient.
It can be seen from the Table 4 that the coefficients of A, B, C, D, C2 and D2 were statistically significant to the RR of mPEG-Oxdc activity, and the coefficients of B, C, A2, B2, C2, and D2 were statistically significant to the MR due to the P values were very small (P < 0.05). Thus, the order of the significant for the enzymatic activity is pH (P value 0.0001 < <0.05) > reaction temperature > time > the mole ratio of mPEG-aldehyde/Oxdc. However, for the MR, the order of the significant should be reaction time > temperature > pH > the mole ratio of mPEG-aldehyde/Oxdc according to the analysis results. Data with a significance of p < 0.01 are marked with **, and data with p < 0.05 are marked with *.
The interaction of different factors
The response surfaces and the contour plots, which were based on the model equation, will provide a visualization of the reaction system for understanding the interactions between two variables and the effects on the responses [25]. As shown in Figs. 3 and 4, the nonlinear nature of all response surfaces and contour plots demonstrated that there were considerable interactions between independent variables and independent variables on the MR and RR value of mPEG. Moreover, the nonlinear nature of all contour plots for these values indicated that there was no direct linear relationship among the selected independent variables. Furthermore, the response surface is a convex line curve that opens downwardly, which indicated that the response values have the maximum value.
Fig. 3.
Response surface plots showing effects of any two variables on the modification rate at the center level of other variables. a Interaction of reaction temperature and molar ratio of mPEG-aldehyde/Oxdc. b Interaction of reaction temperature and reaction time. c Interaction of reaction time and molar ratio of mPEG-aldehyde/Oxdc. d Interaction of reaction pH and reaction time. e Interaction of reaction pH and molar ratio of mPEG-aldehyde/Oxdc. f Interaction of reaction pH and reaction temperature
Fig. 4.
Response surface plots showing effects of any two variables on the recovery rate of enzyme activity at the center level of other variables. a Interaction of reaction temperature and the molar ratio of mPEG-aldehyde/Oxdc. b Interaction of reaction temperature and reaction time. c Interaction of reaction time and molar ratio of mPEG-aldehyde/Oxdc. d Interaction of reaction pH and reaction time. e Interaction of reaction pH and molar ratio of mPEG-aldehyde/Oxdc. f Interaction of reaction pH and reaction temperature
As shown in Figs. 3a and 4a, it was obvious that both of the lower (<40:1) and higher (>60:1) mole ratios of mPEG-aldehyde/Oxdc have a negative effect on the MR value, but the RR value dropped with increasing mole ratio. This could be due to the high concentration of mPEG, as a certain steric hindrance occurred and influenced the enzyme activity [26]. Similar results were also reported from the study of Ikeda [27]. As the reaction temperature increased, the MR value rose, but the RR value decreased obviously; this result is similar to the study of Morpurgo [28]. It can be interpreted from Figs. 3a and 4a that the maximum and minimum MR values obtained from the mPEGlation experiments were 75.98% and 40.08%, respectively, under the conditions of mPEG-aldehyde/Oxdc = 50:1 at 32 °C and mPEG-aldehyde/Oxdc = 60:1 at 24 °C.
As shown in Fig. 3c and 4c. When the reaction time was prolonged, the MR value increased, but the RR value decreased apparently. This result is consistent with the research of Liu Enqi [29]. According to the response surface figures, the maximum and minimum MR value were 75.98% and 23.21%, respectively, under the condition of mPEG-aldehyde/Oxdc = 50:1, reaction for 12 h and mPEG-aldehyde/Oxdc = 60:1, reaction for 6 h.
The effect of reaction pH and the mole ratio of mPEG-aldehyde/Oxdc on the mPEGlation are shown in Fig. 3e and 4e, when the reaction pH was set to 5.0 as the center point. Both of the lower and higher values of mPEG-aldehyde/Oxdc and pH have a negative influence on the MR value, and the reaction pH showed the same influence on the RR value. This could be due to that the isoelectric point of Oxdc is around 5.0, where Oxdc was easy to gather and to be modified [30]. Based on the response surface figures, it can be calculated that the maximum and minimum MR values were 75.01% and 36.90% under the conditions of the mole ratio of mPEG-aldehyde/Oxdc = 50:1, pH = 5.0 and mPEG-aldehyde/Oxdc = 60:1, pH = 3.0.
Optimization of reaction condition and the model validation
Within the experimental conditions in this study, the optimal conditions for the mPEG modification of Oxdc were predicted using the optimization function of the design expert software. The optimal condition and the predictive value of MR and RR are shown in Table 5.
Table 5.
The results of experimental verification
| Term | mPEG-aldehyde/OxdcA | Temperature B (°C) | Time C(h) | pH D |
Modified rate (%) | Recovery rate of enzyme activity (%) |
|---|---|---|---|---|---|---|
| Predicted | 47.6 | 29.9 | 13.1 | 5.3 | 75.11 | 69.17 |
| Experimental | 47 | 30 | 13 | 5.3 | 73.69 | 67.59 |
aMean value of three experiments
Results (Fig. 5) showed that the modified rate (MR = 73.69%) and recovery rate of enzymatic activity (RR = 67.58%) were in good agreement with the predicted values (MR = 75.11%, RR = 69.17%) of the model within a 95% confidence interval. Moreover, the experimental value under the optimal conditions was also better than the results of single-factor experiments. This conclusion indicated that the model is correct and suitable to estimate the experimental value.
Fig. 5.
Predicted results of response surface model
Comparison of the stability of Oxdc and mPEG-Oxdc
Thermal stability
The same concentrations of Oxdc and mPEG-Oxdc were bathed in water at 65 °C to test their thermal stability. The highest enzyme activity in the same group was considered to be 100%, and the average was used to calculate the relative enzyme activity (REA) [31]. Results are shown in Fig. 6.
Fig. 6.
Comparison of the thermal stability of Oxdc and mPEG-Oxdc
As shown in Fig. 6, both the REA of Oxdc and mPEG-Oxdc is around 80% when the time reached 30 min, as the time ranged from 60 to 120 min. REA of Oxdc decreased sharply from 31.80% to 8.21%, but REA of mPEG-Oxdc still remained 50.09% when the time was prolonged to 120 min. This indicated that the thermal stability of mPEG-Oxdc was obviously improved. This could be due to that the mPEG is covalently combined with Oxdc to form the surface protective film, which can protect the internal structure of Oxdc.
Stability of pH treatment
The solution pH was adjusted to a range of 2.0 to 7.0, and the relative enzyme activity of Oxdc and mPEG-Oxdc at different pH treatments were measured. The highest activity of the enzyme in the same group was regarded as 100% [32]. Results are shown in Fig. 7.
Fig. 7.
Stability of Oxdc and mPEG-Oxdc to pH treatment
It can be seen from Fig. 7 that both of the optimum pH levels of Oxdc and mPEG-Oxdc are about 3.5; however, as the pH was ranged from 2.0 to 6.0, mPEG-Oxdc exhibits a higher relative enzyme activity (REA). This indicated that the enzyme activity of Oxdc was obviously improved after grafting with mPEG.
Comparison of the trypsin tolerance
In order to investigate the trypsin tolerance of Oxdc and mPEG-Oxdc, the enzymes were treated with trypsin for 0–24 h, and the highest relative enzyme activity (REA) in the same group was regarded as 100%. Results are shown in Fig. 8.
Fig. 8.
Comparison of the trypsin tolerance of Oxdc and mPEG-Oxdc
As shown in Fig. 8, after trypsin treating for 24 h, the REA of mPEG-Oxdc still retained 47.1%, while the REA of Oxdc only retained 20.6% under the same conditions. This suggested that the trypsin tolerance of mPEG-Oxdc is significantly higher than that of Oxdc. A similar result was also reported from the study of Mu [8].
Conclusions
Based on the experimental results in this study, the following specific conclusions were drawn:
SDS-PAGE and FTIR analysis of Oxdc and mPEG-Oxdc demonstrated that mPEG had grafted onto Oxdc successfully. The modified rate (73.69%) and recovery rate (67.58%) were matched with the predicted value (75.11% and 69.17%), which verified that the optimum strategy is feasible.
Optimization of reaction conditions for mPEG modified Oxdc was carried out by using Box–Behnken response surface methodology. The optimal conditions are: mole ratio of mPEG-aldehyde/Oxdc = 47.6:1, temperature of 29.9 °C, time of 13.1 h, and reaction pH of 5.3.
Comparison of the stability of Oxdc and mPEG-Oxdc indicated that the modified Oxdc (mPEG-Oxdc) showed the higher thermal stability and better tolerance to trypsin or different pH treatment. This work will provide the further theoretical reference for enzyme modification and conditional optimization.
Acknowledgements
This research was supported by Guangxi Higher Education Institutes Talent Highland Innovation Team Scheme (GJR201147-12), Guangxi Natural Science Foundation Project (2014GXNSFAA118045). The authors also would like to thank the teacher ChengYu Wei for the help of information collection and equipment provided.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
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