Study of target spacing of thermo-sensitive carrier on the activity recovery of immobilized penicillin G acylase

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Study of target spacing of thermo-sensitive carrier on the activity recovery of immobilized penicillin G acylase

T

⁎⁎

Ke Lia,b, Guolei Shana,b, Xiaobing Maa,b, Xinyu Zhanga,b, Zhenbin Chena,b, , Zhenghua Tangc,d, ⁎ Zhen Liue, a

State Key Laboratory of Gansu Advanced Non-ferrous Metal Materials, Lanzhou University of Technology, Lanzhou, Gansu, 730050, China School of Material Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, 730050, China c Guangzhou Key Laboratory for Surface Chemistry of Energy Materials, New Energy Research Institute, School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou, 5100067, China d Guangdong Provincial Key Laboratory of Atmospheric Environment and Pollution Control, Guangdong Provincial Engineering and Technology Research Center for Environmental Risk Prevention and Emergency Disposal, School of Environment and Energy, South China University of Technology, Guangzhou Higher Education Mega Centre, Guangzhou, 510006, China e Department of Physics and Engineering, Frostburg State University, Maryland, 21532, United States b

A R T I C LE I N FO

A B S T R A C T

Keywords: Reactivity ratio Penicillin G acylase Carrier Activity recovery rate Target spacing

The immobilized penicillin G acylase (PGA) is an important industrial catalyst, the activity recovery rate of it directly affects enterprise efficiency. How to improve the enzyme activity recovery rate has been a research focus in this field. Based on the above problems, this work further improved the activity recovery rate by adjusting the target spacing for the first time. Glycidyl methacrylate (GMA) was used as the immobilized target and methyl methacrylate (MMA) as the copolymer monomer. According to the copolymer composition equation of P(MMAco-GMA), the thermo-sensitive copolymers, PDEA-b-PHEMA-b-P(MMA-co-GMA) with different target spacings, were synthesized rapidly and efficiently via reversible addition-fragmentation chain transfer (RAFT) polymerization method. The error range between the theoretical and actual values of MMA and GMA in the copolymers carrier was (0–4)%, which demonstrated that the reliability of using composition equation to accurately and quickly synthesize copolymers with specific spacing. Studies on the thermo-sensitive showed that the low critical solution temperature (LCST) of the copolymer carrier decreased with the increase of hydrophobic monomer. Most importantly, the activity recovery rate increased with the increase of target spacing, and when the molar ratio of MMA to GMA in the copolymer was 8.75:1, the recovery of activity of immobilized PGA could be up to 63.50%, which was 21.70% higher than that of pure GMA. This work provided an important idea for improving the activity of immobilized PGA.

1. Introduction Immobilized penicillin G acylase (PGA) has become an important catalyst in industrial applications because it could catalyze penicillin G potassium (PG) to form 6-aminopenicillanic acid (6-APA) [1–4], which is an important intermediate for semi-synthetic penicillins and has been widely used in pharmaceutical field. Because the reusability and the activity recovery rate of the immobilized PGA directly affects the economic benefits of the enterprise. The reusability and the activity recovery rate of the immobilized PGA are both closely related to the properties of the solvent [5–8], immobilized method [9–13] and the

properties of carrier [14,15] etc. Therefore, how to design a suitable carrier type needs to consider the above factors. For solvent, the effect of different types of solvents on the activity of free PGA has been reported [5,15,16] and concluded that free PGA activity and stability were better maintained in pH = 7.8 phosphatebuffer solution [17–19]. For immobilized method, it is mainly related to carrier type and can be divided into two kinds of physical and chemical method, although the activity of immobilized PGA by chemical method can loss more than that of physical adsorption method, it can greatly improve the reusability of immobilized PGA. Because the reusability plays an important role in economic cost and environmental pollution.

⁎

Corresponding author. Corresponding author at: State Key Laboratory of Gansu Advanced Non-ferrous Metal Materials, Lanzhou University of Technology, Lanzhou, Gansu, 730050, China. E-mail addresses: [email protected] (Z. Chen), [email protected] (Z. Liu). ⁎⁎

https://doi.org/10.1016/j.colsurfb.2019.03.064 Received 25 January 2019; Received in revised form 11 March 2019; Accepted 27 March 2019 Available online 01 April 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

the best binary block ratio, the copolymer carriers, PDEA-b-PHEMA-b-P (MMA-co-GMA) with different target spacings, were synthesized rapidly and efficiently by referring to the copolymer composition of P (MMA-co-GMA). Their structures were characterized by 1H NMR and the composition ratios of MMA to GMA in copolymer were also calculated by the area of the characteristic functional group in the hydrogen spectrum. The molecular weight distribution of the copolymer was determined by gel permeation chromatograph (GPC). Then, the relationship between the transmittance of copolymer solution and temperature was determined by UV-VIS spectrophotometer. Finally, the relationship between the activity recovery rate of immobilized PGA and the target spacing was explored.

Therefore, chemical method is favored by many researchers and widely used. It’s reported that epoxy group can be combined with amino group on PGA through covalent bond under the mild reaction conditions, which can reduce the deformation degree of immobilized PGA active center conformation. At present, glycidyl methacrylate (GMA) containing epoxy group is widely used of the immobilized PGA targets. For carrier, because PGA is a hydrolytic enzyme, in order to facilitate the catalytic transformation of immobilized PGA, hydrophilic carriers are often used to construct the carrier microenvironment. At present, the research on immobilized PGA carrier mainly focuses on the type of carrier, which could be divided into inorganic carriers, organic carriers and composite carriers. Because organic synthetic copolymer carriers [11,20–23], which have the advantages of simple synthesis, variety of functional groups and controllable molecular weight, etc. Magnetic nano-composite [12,24–27] carriers are a kind of composite carriers, it combines the advantages of magnetic materials and organic materials, especially in the performance of carrier recovery. Based on the advantages, the above two carrier types have received extensive attention from researchers in recent years. However, there's a strange phenomena about that the activity of immobilized PGA decreased [21,28] when the immobilized targets were linearly arranged and dense, either for organic or composite carriers. Besides, it was found that researchers only stayed in the analysis stage, and did not adopt corresponding methods to further improve the activity recovery rate of immobilized PGA. In order to further improve the enzyme activity recovery rate of immobilized PGA, the relationship between enzyme molecules and the target should be first explored. Therefore, it is crucial to select the appropriate carrier type. Although magnetic nano-composites [28,29] as an important carrier type of immobilized PGA have excellent recycling performance, which is the molecular weight of the complex is limited due to the easy aggregation of magnetic nanoparticles. In addition, due to the limitations of characterization methods, it is difficult to characterize the structure and composition of copolymer in composites, which will hinder the exploration of the relationship between the enzyme molecule and the target. Therefore, it is necessary to design appropriate carrier structure, which not only has good reusability, but also can explore the relationship between enzyme molecules and the target through characterization methods. According to the reports [30,31], thermo-sensitive copolymer used as the carrier of PGA has some advantages, such as the immobilized PGA could achieve the catalytic conversion in the sol state; the carrier could be recovered in the gel state. In addition, the synthesis process is simple, and the structure and molecular weight can be characterized by nuclear magnetic resonance spectroscopy (1HNMR). Based on the above finding, in this work, pH = 7.8 phosphate buffer solution was as solvent, N,N-diethylacrylamide (DEA) was used as thermo-sensitive monomer, hydrophilic monomer β-hydroxyethyl methacrylate (HEMA) was used to construct microenvironments, glycidyl methacrylate (GMA) was used to immobilized PGA via covalent bonding, methyl methacrylate (MMA) as a copolymer was introduced to adjust the target spacing. And most of all the experiment could utilize the monomer reactivity ratios and composition ratio to achieve precise and controllable copolymer structure, and its structure is characterized by 1HNMR, which is beneficial to explore the influence of target spacing on the activity of immobilized PGA. The research content of target spacing on immobilized PGA was as follows: First, the reactivity ratios of MMA and GMA at 80 °C with solvent N,N-dimethylformamide (DMF) was investigated. The monomer composition ratio in P(MMA-co-GMA) was calculated by the area of the characteristic functional group in the hydrogen spectrum. Then, copolymerization curve of P(MMA-co-GMA) was simulated according to the monomer feeding ratios and reactivity ratios, and the accuracy of the theoretical value was verified by actual values. Besides, PDEA-bPHEMA with different HEMA contents was synthesized via RAFT polymerization method, the influence of HEMA contents on the immobilized penicillin G acylase (PGA) activity was explored, based on

2. Experimental 2.1. Materials Acenaphthylene (AR) was collected from Shanghai Su Ren Responsible Co., Ltd. methyl methacrylate (AR), Glycidyl methacrylate (AR), β-hydroxyethyl methacrylate (AR), ethyl acetate (AR), n-hexane (AR), absolute ether (AR), petroleum ether was all obtained from Lionon Bohua Pharmaceutical Chemical Company. N,N-dimethylformamide (AR), methanol (AR) were collected from the Beijing chemical plant. All of them were used as received. 2-Cyano-2-propyl dodecyl trithiocarbonate (CPDTC, AR) was provided by Shanghai McLean biochemical technology co. LTD., Penicillin G acylase (PGA, the original free enzyme activity was 33470U/g), 6-aminopenicillanic acid (98%, HPLC), penicillin G (98%) was provided by Shanghai Maclin Reagent Co. LTD. Distilled water was prepared in our lab. Azobisisobutyronitrile (AR) was provided by Yantai Shuangli Chemical Co. Ltd., and was recrystallized with 95% ethanol before usage. 2.2. Determination of reactivity ratios A series of MMA and GMA were respectively added to the corresponding devices in a ratio of 8:2−2:8, and the total molar weight of MMA and GMA were 0.01 mol. Then, the molar ratio of azodiisobutyronitrile (AIBN) to total monomer, was that n(AIBN)/n (MMA + GMA) = 0.03%, and N,N-dimethylformamide (DMF) as a solvent was added with n(MMA + GMA)/n(DMF) = 50.00%. After nitrogen purge for 15 min, the devices were sealed and placed in a 80 °C oil bath with magnetic stirring. After a certain reaction time (the reaction time should ensure that the conversion rate was less than 10%), the samples were precipitated by methanol. Finally, the samples were separated and put into a 40 °C vacuum drying under the vacuum degree of 0.08 to constant, P(MMA-co-GMA) was obtained. The content of MMA and GMA in the copolymer were characterized by 1HNMR, then, the reactivity ratios of MMA and GMA was obtained by Fineman-Ross method. Conversion rate [32,33] could be obtained by Eq. (1):

Conversion rate =

ms × 100% m1 + m2

(1)

Where, ms represented the mass of P(MMA-co-GMA), m1, m2 represented the mass of MMA and GMA, respectively. 2.3. Preparation of PDEA-b-PHEMA-b-P(MMA-co-GMA) AIBN was used as initiator, CPDTC as RAFT reagent, acenaphthylene as fluorescent material and ethyl acetate as solvent, the mass of monomers were added in a test tube in turn according to the molar ratios of AIBN: CPDTC: DEA = 1:20:500, the concentration of solution was 50.00% (mDEA/methyl acetate). After nitrogen purge for 30 min (air was expelled from the test tube), the test tube was sealed and put into an oil bath of 80 °C under magnetic stirring. After the reaction was over, n-hexane was used as precipitating agent and ethyl acetate as solvent, PDEA was obtained and dried for use [31]. 154

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

the residual liquid was withdrawn, and immobilized PGA was rinsed with phosphate buffer until the wash solution plus PG solution after reaction with PDAB colorimetric measured absorbance of less than 0.003. Then, the final obtained precipitate was dried in a 35 °C vacuum oven under the vacuum degree of 0.08 to a constant, and the immobilized PGA was obtained and the mass of immobilized PGA was weighed.

Then, PDEA was used as RAFT reagent to synthesize PDEA-bPHEMA, and the molar ratio was that AIBN:PDEA:HEMA = 1:4:100, DMF was used as solvent and the concentration of solution was 10.00%. After nitrogen purge for 15 min, it was sealed and placed in a 80 °C oil bath with magnetic stirring for 36 h, then the product was precipitated by anhydrous ether. Finally, it was separated and put into a 40 °C vacuum drying under the vacuum degree of 0.08 to constant, and PDEA-bPHEMA was obtained. Thereafter, PDEA-b-PHEMA was used as RAFT reagent to synthesize PDEA-b-PHEMA-b-PGMA and PDEA-b-PHEMA-b-P(MMA-co-GMA). Meanwhile, the molar ratio of AIBN, PDEA-b-PHEMA and GMA could be expressed as n(AIBN): n(PDEA-b-PHEMA): n(GMA) = 1:4:56. For PDEA-b-PHEMA-b-P(MMA-co-GMA), different molar ratios of MMA were added separately to the reactors according to the reactivity ratios of MMA and GMA obtained in section 2.2, DMF was used as solvent and the concentration of solution was 10.00%. After nitrogen purge for 15 min, it was sealed and placed in a 80 °C oil bath with magnetic stirring for 48 h, then the product was precipitated by the mixture of anhydrous ether and DMF (Vanhydrous ether:VDMF = 5:1). Finally, the product was separated and put into a 40 °C vacuum drying under the vacuum degree of 0.08 to constant, and PDEA-b-PHEMA-b-PGMA [30] and PDEA-b-PHEMA-b-P(MMA-co-GMA) were obtained.

2.8. Determination of activity recovery rate and loading capacity The immobilized PGA obtained in section 2.7 was added in a litter bottle which had loaded with 5.00 mL, 5.00% PG, then, the bottle was set in the 37 °C constant temperature bath oscillator for 5 min. Thereafter, 0.10 mL of the solution was diluted with a certain amount of phosphate buffer. Thereafter, pipetting 0.50 mL of the diluted solution and injecting it in a cuvette which had loaded 3.50 mL of 4.00% PDAB solution, the reaction was kept for 3 min, then the absorbance was measured at 420 nm, and immobilized enzyme activity and activity recovery rate [28] was calculated using Eqs. (2), (3) and (4):

Immobilized enzyme activity =

C×V (m1 − m 0) × t

(2)

2.4. Structure characterization of copolymer

Activity recovery rate =

0.50 mL, 20 mg/mL deuterated chloroform (CDCl3) solution of P (MMA-co-GMA), and 0.50 mL, 20 mg/mL PDEA, PDEA-b-PHEMA, PDEA-b-PHEMA-b-PGMA and PDEA-b-PHEMA-b-P(MMA-co-GMA) were prepared, respectively. And then, 1HNMR for each compound was determined by a nuclear magnetic resonance spectroscopy (AV-400, Bruker, USA), the molecular structure was characterized according to chemical shift of hydrogen in each compound.

Enzyme loading (mg / g ) =

Immobilized enzyme activity × 100% Free enzyme activity (m1 − m 0) × 103 m0

(3)

(4)

Where, C represented the concentration of 6-APA (mmol/L), V represented the volume of the reaction system (mL), t represented the reaction time (min), m1 represented the mass of immobilized PGA carrier dried in a 35 °C vacuum oven under the vacuum degree of 0.08, m0 represented the mass of copolymer carrier dried in a 35 °C vacuum oven under the vacuum degree of 0.08. (The above equations for calculating the activity recovery rate of immobilized PGA was different from the equations in our previous work [30,31,34], and the activity recovery rate calculated by above equations was lower than the previous ones. The reasons for the above phenomenon have been explained in detail in Attachment 1)

2.5. Characterization of molecular weight distribution DMF (5 mg/mL) solution of PDEA-b-PHEMA, PDEA-b-PHEMA-b-P (MMA-co-GMA) were prepared, respectively, and the molecular weight distribution for each compound was detected by a gel permeation chromatograph (GPC, Waters 1525/2414/2487, Fairburn industrial development co. LTD, Shanghai, China) which was precalibrated with narrowly distributed polymethyl methacrylate. During this process, DMF in which 0.05 mmol/L lithium bromide was contained was adopted as the mobile phase; the injection volume was 10 μL at 1 mL/ min flowrate and 25 °C.

3. Results and discussion 2.6. Characterization of thermo-sensitive performance of copolymer 3.1. Structures of copolymers 10.00 mg samples were weighed and dissolved with trace DMF, and then diluted it with distilled water to 10.00 mL and obtained the solution with a concentration of 0.01 mg/mL. Then, the transmittance of the solution was determined (uv-240 UV–vis spectrophotometer, Japan, shimatsu) with a heating rate of 0.5 °C and a detection wavelength of 500 nm. The LCST of the copolymer was determined by the temperature corresponding to the initial change point of the slope of the transmittance curve.

The 1HNMR spectra of P(MMA-co-GMA) and PDEA-b-PHEMA-b-P (MMA-co-GMA) were shown in Fig. 1(a), (b), respectively. The 1HNMR spectra of CPDTC, PDEA, PDEA-b-PHEMA, PDEA-b-PHEMA-b-PGMA were shown in Fig.A1, Fig.A2, Fig.A3 and Fig.A4, respectively (which were shown in Attachment 2). The composition of copolymer could be judged according to the corresponding chemical displacement, the characteristic peak had been marked by the corresponding letter in the hydrogen spectrum according to the position in the chemical formula. Besides, the characteristic peaks and peak areas of PDEA, PGMA, PHEMA and PMMA in the PDEA-b-PHEMA-b-P(MMA-co-GMA) have been shown in Table A1(which was shown in Attachment 2), and x, k, h, c represented the characteristic peaks of PDEA, PGMA, PHEMA and PMMA, respectively, the ratio of the number of hydrogens represented by x, k, h and c was 4:1:2:3. For PDEA-b-PHEMA-b-P(MMA-co-GMA) series, which has different spacing of GMA, but the molecular weight of PDEA-b-PHEMA in PDEA-b-PHEMA-b-P(MMA-co-GMA) was a constant, therefore, the molar ratio of PMMA to PGMA was expressed as: n (PMMA):n(PGMA)= C/3: K.

2.7. Preparation of immobilized PGA 0.0300 g copolymer carrier was weighted (FA1004 N, Shanghai precision instruments co. LTD) and put in a 35 °C vacuum oven under the vacuum degree of 0.08 (DZF-6090-L, Shanghai binglin instrument co. LTD) for 48 h to a constant weight, and then, the mass of the dried carrier was weighed as the mass of immobilized carrier. Thereafter, it was added in bottles with 5.00 mL, 5.00% free PGA, and placed in a constant temperature water bath oscillator (HH-W600 Digital Electric Constant Temperature Water, Changzhou) at 37 °C for 16 h. After that, 155

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

Fig. 1. 1HNMR spectra of copolymer. a) 1HNMR spectra of P(MMA-co-GMA); b) 1HNMR spectra of PDEA-b-PHEMA-b-P(MMA-co-GMA).

was linearly increased with the polymerization time at the study range and the variation threshold of PDI was (1.10, 1.24), which illustrated that it successfully achieved the activity control of molecular weight and molecular weight distribution through RAFT polymerization.

3.2. Characterization of molecular weight and polydispersity index of copolymers Because GPC is based on the size exclusion effect using the size exclusion method to determine the molecular weight of a copolymer sample, a standard sample of known molecular weight must be used as a calibration curve. To dissolve this problem, 1HNMR was conscript to characterize the molecular weight, while GPC was used to analyze the relative molecular weight distribution. Because the molar ratio PDEA, PHEMA, PMMA and PGMA in the copolymer could be expressed as X/ 4:H/2:C/3:K. According to the hydrogen spectrum of PDEA (Attachment 2), the relative molecular weight of PDEA could be calculated, which was 1794 g/mol. Therefore, according to the relative molecular weight of PDEA and the molar ratio of each monomer in copolymer, the relative molecular weight of copolymer could be obtained. The relationships of the relative molecular weight, polydispersity index of copolymer and polymerization time were shown in Fig. 2. It was shown that the relative molecular weight of copolymer

3.3. Reactivity ratios of monomers The 1HNMR spectra of P(MMA-co-GMA) was shown in Fig. 1(a), Where c and k represented the characteristic peaks of MMA and GMA in the copolymer, respectively, and the corresponding peak areas were represented by C and M. Therefore, the molar ratio of MMA to GMA in P (MMA-co-GMA) was C/3 M. According to this method, the molar ratio of MMA and GMA in P(MMA-co-GMA) could be obtained. The reactivity ratios of MMA and GMA were obtained by using the differential equation of Fineman-Ross binary copolymer [35–37], which was Eq. (5):

156

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

Fig. 3. Regression equation of reactivity ratio of P(MMA-co-MMA).

meant the rate of polymerization growth of GMA radicals with their own monomers is faster than that of with MMA. Besides, the copolymerization curve [38] could be obtained according to Eq. (8):

F1 =

F1 = (5)

2

If Y =

R (ρ − 1) , ρ

2

Y = r1X − r2

X=

(6) R2 , ρ

(8)

r1 f1 r1 f1 + f2

(9)

Because r1(MMA)×r2(GMA)=0.9918≈1, which meant that the polymerization reaction of P(MMA-co-GMA) was close to the general ideal copolymerization. According to Equation (10), the relationships between the feeding ratio of MMA, GMA and the composition ratio in P (MMA-co-GMA) were simulated, respectively. In addition, the actual value was calculated and compared with the theoretical value, which were shown in Fig. 4. Results showed that the error range was 00.013%, which revealed that it could accurately simulate the monomer constituent ratio in random copolymer via Eq. (9).

Where, the x1/x2 represented the feeding ratio of MMA to GMA, dx1/ dx2 represented the constituent ratio of MMA to GMA in the copolymer, r1 and r2 represented the reactivity ratios of MMA and GMA, respectively. dx x If ρ = dx 1 , R = x 1 , Eq. (5) could be simplified as Eq. (6):

R (ρ − 1) R2 = r1 − r2 ρ ρ

+ 2f1 f2 + r2 f22

Where, f1, f2 represented the feeding ratios of MMA, GMA to monomer mixture at a certain moment, respectively; F1 and F2 represented the constituent ratio of PMMA, PGMA to copolymer at the same certain moment, respectively. In addition, for random copolymer, if r1×r2 = 1 or r2 = 1/r1, it means that the polymerization type of the random copolymer is general rational copolymerization. And copolymerization curve [38] could be obtained according to Eq. (9):

Fig. 2. The relationship of the relative molecular weight, polydispersity index of copolymer and polymerization time. a) PDEA-b-PHEMA; b) PDEA-b-PHEMAb-P(MMA-co-GMA).

d x1 x r x + x2 = 1 × 1 1 dx2 x2 r2 x2 + x1

r1 f12 + f1 f2 r1 f12

Eq. (6) could be simplified as Eq. (7): (7)

The parameters of P(MMA-co-GMA) were shown in Table A2(which was shown in Attachment 2). According to X and Y in Table A2, the regression equation of reactivity ratio of P(MMA-co-GMA) was performed, which were shown in Fig. 3. For P(MMA-co-GMA), r1(MMA) = 0.6891, r2(GMA) = 1.4393. The monomer reactivity ratios in P(MMA-co-GMA) was analyzed as follows: the slope represented the reactivity ratio of MMA, r1(MMA) = 0.6891, intercept represented the reactivity ratio of GMA, r2(GMA) = 1.4393. Because r1=K11/K12, r2=K22/K21, K11 and K22 were the chain propagation rate constant of MMA and GMA free radicals with their own monomers, respectively. K12 was the chain propagation rate constant of MMA free radical with GMA and K21 was the chain propagation rate constant of GMA free radical with MMA. For r1(MMA) = 0.6891 < 1, which meant the rate of polymerization growth of MMA radicals with their own monomers was slower than that of with GMA. For r2(GMA) = 1.4393 > 1, which

Fig. 4. The composition curve of P(MMA-co-GMA). 157

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

Fig. 5. The influence of HEMA content on the activity of immobilized PGA.

Fig. 6. The influence of MMA content on the LCST of copolymer.

3.4. The influence of HEMA content on the activity of immobilized PGA

that phase separation at low temperatures and the transmittance of copolymer would decrease.

As could be seen from Fig. 5, with the increase of HEMA content in PDEA-b-PHEMA, the activity of immobilized PGA increased first and then decreased; and it reached the maximum when HEMA content was 10%. The phenomenon could be attributed to the effect of molecular polarity on the conformation of the active center of immobilized PGA. Because the conformation of PGA active center was composed of hydrogen bond, hydrophobic interaction and other weak interactions, which was easy to be destroyed under the influence of polar molecules. Although DEA and HEMA were both hydrogen bond receptors and hydrogen bond donors, which all could compete for water molecules with amino acid residues in the active center of PGA. However, because the polarity of DEA was greater than that of HEMA, it could destroy the conformation of the active center of PGA more seriously. Therefore, when PDEA was used as the carrier, the activity of immobilized PGA was lower than that of the carrier adding appropriate HEMA. However, with the increase of HEMA, the molecular chain increased. Because the copolymer was in a random group structure, a PGA molecule could be surrounded by multiple HEMAs, the probability of hydrogen bond formation between PGA and HEMA was increased [39], which also led to the gradual decrease of activity of immobilized PGA.

3.6. The influence of target spacing on activity recovery rate of immobilized PGA In section 3.3, the molar ratio of monomers in P(MMA-co-GMA) has been simulated according to the copolymerization equation, and the error range between the theoretical value and the actual value was (00.013)%. In the same way, the molar ratio of MMA to GMA in the copolymers was also simulated, and the error range between the theoretical value and the actual value was (1–4)%, which was shown in Table 1. The error range was slightly larger than that of the random copolymer, it could be attributable to the different polymerization methods and types of copolymer. Fortunately, it proved that the monomer composition ratio in the block copolymer obtained by RAFT polymerization method based on the copolymerization equation of the random copolymer was feasible and accurate. It could be found that the activity recovery rate of immobilized PGA gradually increased with the increase of GMA target spacing (Fig. 7). The above phenomena could be attributed to the effect of immobilized target spacing [28]. Because the active center of PGA was constructed with hydrogen bond, van der Waals force and other weak interaction, which was easily destroyed [7]. Besides, the volume of PGA was large (7 nm × 5 nm × 5.5 nm) [43]. When the immobilized targets density was high, on the one hand, it could lead to the conformation of the

3.5. Thermo-sensitive performance of copolymer It could be found that with the increase of GMA and MMA in the copolymer, the temperature at the initial change in the slope of a transmissivity curve of the copolymer gradually decreased, which meant that the low critical solution temperature (LCST) of the copolymer decreased (Fig. 6). Besides, it could also found that with the increase of MMA, the phase transition interval became smaller, and the value of the transmittance reaching the equilibrium value increased. The above phenomenon could be attributed to the following reasons: For the thermo-sensitive copolymer PDEA-b-PHEMA-b-PGMA and PDEA-b-PHEMA-b-P(MMA-co-GMA), which contained a certain proportion of hydrophilic groups, when the temperature was low, the hydrophilic groups in the copolymer and the water molecules in the solvent formed hydrogen bonds, at this time, the copolymer was dissolved in the solvent and the solution was transparent. However, because the GMA and MMA was hydrophobic monomer, the increase of hydrophobic monomer proportion in copolymer leaded to the increase of the proportion of hydrophobic groups, which would result in a lower solubility of the copolymer. With the increase of temperature, the hydrophobic interaction between molecules and within molecules would be enhanced [40–42]. As the solubility decreased, so did the temperature at which the phase separation occurred. therefore, with the increase of hydrophobic monomer GMA and MMA, copolymer occurred

Table 1 The relationship between parameters of immobilized PGA and target distance. Composition ratio of compound

Error/%

a

Enzyme loading(mg/ g)

b

Immobilization yield (%)

c Enzyme activity(U/ g)

0 0.8627 2.0628 3.7228 8.7505

/ 1.32 2.21 3.71 2.65

201 156 77 54 33

46.7 36.2 17.9 12.5 7.6

14017 15620 17916 19211 21256

The carriers were the series of PDEA-b-PHEMA-b-P(MMA-co-GMA); The composition ratio of compound of meant that the molar ratio of MMA to GMA in copolymer; The activity of free PGA was 33,470 U/g; The initial amount of enzyme was 12.90 mg in each preparation. Immobilization yield represented the mass ratio of the PGA immobilized on the carrier to the amount of the initial PGA. a the error was ( ± (5–14)%). b the error was ( ± (1–2)%). c the error was ( ± (5–10)%). 158

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

References [1] A. Illanes, C. Altamirano, M.E. Zuñiga, Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products, Biotechnol. Bioeng. 50 (2015) 609–616. [2] S.M. Atyabi, S.H. Gigloo, M. Salimi, S.B. Momen, A. Akbarzadeh, D. Nrouzian, Enzymatic synthesis of ampicillin by immobilized penicillin g acylase of E.cOli, Biosci. Biotechnol. Res. Asia 9 (2016). [3] S. Deng, X. Ma, E. Su, D. Wei, Efficient cascade synthesis of ampicillin from penicillin g potassium salt using wild and mutant penicillin g acylase from Alcaligenes faecalis, J. Biotechnol. 219 (2016) 142–148. [4] Z.D. Kneževićjugović, Ž. MG, E.S. Džunuzović, S.M. Jakovetić, A.B. Stefanović, K.B. Jeremić, S.M. Jovanović, An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidylmethacrylate-co-ethyleneglycoldimethacrylate) as a potential industrial biocatalyst, Biotechnol. Prog. 32 (2016) 43–53. [5] G. Massolini, E. Calleri, A. Lavecchia, F. Loiodice, D. Lubda, C. Temporini, G. Fracchiolla, P. Tortorella, E. Novellino, G. Caccialanza, Enantioselective hydrolysis of some 2-aryloxyalkanoic acid methyl esters and isosteric analogues using a penicillin G acylase-based HPLC monolithic silica column, Anal. Chem. 75 (2003) 535. [6] F.N. Novikov, O.V. Stroganov, I.G. Khaliullin, N.V. Panin, I.V. Shapovalova, G.G. Chilov, V.K. #x, vedas, Molecular modeling of different substrate‐binding modes and their role in penicillin acylase catalysis, FEBS J. 280 (2013) 115–126. [7] C.E. Mcvey, M.A. Walsh, G.G. Dodson, K.S. Wilson, J.A. Brannigan, Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism, J. Mol. Biol. 313 (2001) 139–150. [8] A. Slade, A.J. Horrocks, C.D. Lindsay, B. Dunbar, R. Virden, Site-directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105. Effect on conformation and catalytic activity, EJBio 197 (2010) 75–80. [9] Serra, Immacolata, Albertini, M. Alessandra, Assessment of immobilized PGA orientation via the LC-MS analysis of;tryptic digests of the wild type and its 3K-PGA mutant assists in the;rational design of a high-performance biocatalyst, Anal. Bioanal. Chem. 405 (2013) 745–753. [10] F.B. Anspach, G. Altmann-Haase, Immobilized‐metal‐chelate regenerable carriers: (I). Adsorption and stability of penicillin G amidohydrolase from Escherichia coli, Biotechnol. Appl. Biochem. 20 (2011) 313–322. [11] Z.D. Knežević-Jugović, Ž. MG, S.M. Jakovetić, A.B. Stefanović, E.S. Džunuzović, K.B. Jeremić, S.M. Jovanović, An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) as a potential industrial biocatalyst, Biotechnol. Prog. 32 (2016) 43–53. [12] R. Liu, J. Fan, Y. Zhang, P. Wang, X. Shen, Immobilization and characterization of penicillin G acylase (PGA) immobilized on magnetic Ni₀.₅Zn₀.₅Fe₂O₄ nanoparticles, J. Nanosci. Nanotechnol. 16 (2016) 182–188. [13] R. Liu, D. Chen, H. Fu, P. Lv, D. Zhang, Y. He, A facile preparation process of magnetic aldehyde-functionalized Ni0.5Zn0.5Fe2O4@SiO2 nanocomposites for immobilization of penicillin g acylase (PGA), J. Nanosci. Nanotechnol. 17 (2017) 893–899. [14] A. Illanes, A. Fajardo, Kinetically controlled synthesis of ampicillin with immobilized penicillin acylase in the presence of organic cosolvents, J. Mol. Catal., B Enzym. 11 (2001) 587–595. [15] M. Arroyo, R. Torresguzmán, M.I. De, M.P. Castillón, C. Acebal, Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition, Enzyme Microb. Technol. 27 (2000) 122–126. [16] A. Banerjee, K. Chatterjee, G. Madras, Effect of solvents on the enzyme mediated degradation of copolymers, Mater. Res. Express 2 (2015). [17] M. Sorri, K. Jounio-Ervasti, Purification and properties of penicillin acylase from Kluyvera citrophila, J. Agric. Chem. Soc. Jpn. 39 (2008) 1655–1661. [18] K. Langowska, C.G. Palivan, W. Meier, Polymer nanoreactors shown to produce and release antibiotics locally, Chem. Commun. 49 (2012) 128–130. [19] R. Gotti, J. Fiori, E. Calleri, C. Temporini, D. Lubda, G. Massolini, Chiral capillary liquid chromatography based on penicillin G acylase immobilized on monolithic epoxy silica column, J. Chromatogr. 1234 (2012) 45–49. [20] V. Pashova, V. Dakov, G. Georgiev, Immobilization of penicillin-amidase on poly (Glycidylmethacrylate) or on a copolymer of Glycidylmethacrylate and 2Hydroxyethylmethacrylate both grafted onto poly(Acrylonitrile) fibres, Biotechnol. Biotechnol. Equip. 6 (2014) 21–24. [21] L. Ke, B.C. Zhen, L.L. Dong, Z. Lin, Z. Tang, W. Zhe, Z. Yu, L. Zhen, Design and synthesis study of the thermo-sensitive copolymer carrier of penicillin G acylase, Polym. Adv. Technol. (2018). [22] D. Liu, Z. Chen, J. Long, Y. Zhao, X. Du, Immobilization of penicillin acylase on macroporous adsorption resin CLX1180 carrier, Adv. Polym. Tech. (2016). [23] Q.L. Ye, Y.X. Li, Studies on a phenolic resin as a carrier for the immobilization of penicillin acylase, Polym. Adv. Technol. 8 (2015) 727–730. [24] X. Li, L. Tian, Z. Ali, W. Wang, Q. Zhang, Design of flexible dendrimer-grafted flower-like magnetic microcarriers for penicillin G acylase immobilization, J. Mater. Sci. 53 (2018) 937–947. [25] X.M. Ling, X.Y. Wang, P. Ma, Y. Yang, J.M. Qin, X.J. Zhang, Y.W. Zhang, Covalent immobilization of penicillin G acylase onto Fe3O4@Chitosan magnetic nanoparticles, J. Microbiol. Biotechnol. 26 (2016) 829–836. [26] R.J. Liu, Covalent immobilization and characterization of penicillin G acylase on magnetic NiFe2O4 nanorods prepared via a novel rapid combustion process, Chem. Biochem. Eng. Q. 30 (2017) 511–517. [27] X. Jin, Q. Wu, Q. Chen, C.X. Chen, X.F. Lin, Immobilization of penicillin G acylase

Fig. 7. The influence of target spacing on activity recovery rate of immobilized PGA. n(MMA)/n(GMA) represented the constituent ratio of MMA to GMA in PDEA-b-PHEMA-b-P(MMA-co-GMA).

active center of immobilized PGA change due to mutual extrusion [28]. On the other hand, the higher target density increased the probability of immobilized PGA multipoint fixation, as a result, the methylene space structure of the active center of immobilized PGA was destroyed due to multi-point [44,45]. Therefore, the activity recovery rate was lower. With the increase of target spacing, the deformation degree of the immobilized PGA activity center conformation caused by mutual extrusion and multi-point fixation was gradually reduced, the recovery rate of enzyme activity was gradually increased. 4. Conclusion The reactivity ratios of MMA and GMA were obtained via FinemanRoss binary copolymer composition differential equation under 80 °C, solvent of DMF. Besides, the copolymerization curve of the random copolymer was simulated according to the monomer reactivity ratios. Then, based on the optimal binary block proportion, the copolymer carrier, PDEA-b-PHEMA-b-P(MMA-co-GMA) that adjacent GMA have different spacing, for PGA was synthesized accurately and quickly according to the copolymerization equation of P(MMA-co-GMA), and the structure and molecular weight of copolymer were characterized by 1 HNMR and GPC, respectively. Results illustrated that the activity control of molecular weight and molecular weight distribution through RAFT polymerization was successfully achieved. The relationship between the activity recovery rate of immobilized PGA and the n(MMA)/n (GMA) in copolymer was explored, results showed that the activity recovery of immobilized PGA increased with the increase of targets spacing, and when the molar ratio of MMA to GMA in the copolymer was 8.75:1, the recovery of activity could be up to 63.50%, which was 21.70% higher than that of pure GMA. It proved that the activity recovery of immobilized PGA could be effectively improved by adjusting the target spacing. This work was the first to break through the low recovery rate of immobilized PGA by precisely regulating the spacing between immobilized targets, which provided a new idea for the development of industrial immobilized PGA carrier. Acknowledgment This work was supported by National Natural Science Foundation of China (Grant No. 51563015). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.03.064. 159

Colloids and Surfaces B: Biointerfaces 179 (2019) 153–160

K. Li, et al.

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

on a composite carrier with a biocompatible microenvironment of chitosan, J. Chem. Technol. Biotechnol. 83 (2010) 1710–1716. P. Xue, W. Su, Y. Gu, H. Liu, J. Wang, Hydrophilic porous magnetic poly(GMAMBAA-NVP) composite microspheres containing oxirane groups: an efficient carrier for immobilizing penicillin G acylase, J. Magn. Magn. Mater. 378 (2015) 306–312. L. Xue, T. Lei, Z. Ali, W. Wang, Q. Zhang, Design of flexible dendrimer-grafted flower-like magnetic microcarriers for penicillin G acylase immobilization, J. Mater. Sci. 53 (2018) 937–947. K. Li, Z.B. Chen, D.L. Liu, L. Zhang, Z. Tang, Z. Wang, Y. Zhao, Z. Liu, Design and synthesis study of the thermo-sensitive copolymer carrier of penicillin G acylase, Polym. Adv. Technol. 29 (2018) 1902–1912, https://doi.org/10.1002/pat.4299. K. Li, X.T. Liu, Y.F. Zhang, D. Liu, X.Y. Zhang, S.M. Ma, J.M. Ruso, Z. Tang, Z.B. Chen, Z. Liu, The engineering and immobilization of penicillin G acylase onto thermo-sensitive tri-block copolymer system, Polym. Adv. Technol. 30 (2019) 86–93, https://doi.org/10.1002/pat.4446. T. Qian, J. Wang, T. Cheng, X. Zhan, Q. Zhang, F. Chen, A novel block copolymer with excellent amphiphobicity synthesized via ARGET ATRP, J. Polym. Sci. Part A: Polym. Chem. 54 (2016) 2040–2049. T. Terashima, T. Sugita, M. Sawamoto, Single-chain crosslinked star polymers via intramolecular crosslinking of self-folding amphiphilic copolymers in water, Polym. J. (2015) 47. X. Wang, Z. Chen, K. Li, X. Wei, Z. Chen, J.M. Ruso, Z. Tang, Z. Liu, The study of titanium dioxide modification by glutaraldehyde and its application of immobilized penicillin acylase, Colloids Surf. Physicochem. Eng. Asp. 560 (2019) 298–305, https://doi.org/10.1016/j.colsurfa.2018.10.001. N. Roka, O. Kokkorogianni, M. Pitsikalis, Statistical copolymers of N‐vinylpyrrolidone and 2‐(dimethylamino)ethyl methacrylate via RAFT: monomer

[36]

[37] [38] [39]

[40] [41]

[42]

[43] [44]

[45]

160

reactivity ratios, thermal properties, and kinetics of thermal decomposition, J. Polym. Sci. Part A: Polym. Chem. (2017) 55. S. Sundararajan, A.B. Samui, P.S. Kulkarni, Synthesis and characterization of poly (ethylene glycol) acrylate (PEGA) copolymers for application as polymeric phase change materials (PCMs), React. Funct. Polym. 130 (2018) 43–50. N.S. Pujari, M. Wang, K.E. Gonsalves, Co and terpolymer reactivity ratios of chemically amplified resists, Polymer (2017) 118. 嵌段共聚物P(AM-DMBAC)的制备及其污泥脱水研究, in, 重庆大学, 2017. M. Kozanecki, J. Ulański, P. Wojciechowski, M. Kryszewski, M. Boudeulle, E. Duval, Analysis of the hydrogen bonding in (2-hydroxypropyl)cellulose-poly(acrylic acid) composites by Raman spectroscopy, Macromol. Symp. 141 (1999) 185–195. M. Kasimova, A. Yazici, Y. Yudin, T. Rohacs, V. Carnevale, Role of hydrophobic solvation in TRPV1 temperature sensitivity, Biophys. J. 114 (2018) 481a. A. Licea‐Claveríe, E. Rogel‐Hernández, R. Salgado‐Rodríguez, L.A. Castillo, J.M. Cornejo‐Bravo, F. Arndt, The use of hydrophobic spacers in the development of new temperature‐ and pH‐sensitive polymers, Macromol. Symp. 207 (2004) 193–216. P. Bharmoria, K.S. Rao, T.J. Trivedi, A. Kumar, Biamphiphilic ionic liquid induced folding alterations in the structure of bovine serum albumin in aqueous medium, J. Phys. Chem. B 118 (2014) 115. 刘冬蕾, 青霉素酰化酶固定化载体的设计合成, in, 兰州理工大学, 2017. O. Romero, J.M. Guisán, A. Illanes, L. Wilson, Reactivation of penicillin acylase biocatalysts: effect of the intensity of enzyme-support attachment and enzyme load, J. Mol. Catal., B Enzym. 74 (2014) 224–229. V. Stepankova, S. Bidmanova, T. Koudelakova, Z. Prokop, R. Chaloupkova, J. Damborsky, Strategies for stabilization of enzymes in organic solvents, ACS Catal. 3 (2013) 2823–2836.