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Enhancing 6-APA Productivity and Operational Stability of Penicillin G Acylase via Rapid Surface Capping on Commercial Resins Dong Yang, Hua Liu, Jiafu Shi, Xueyan Wang, Shaohua Zhang, Hongjian Zou, and Zhongyi Jiang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02866 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Industrial & Engineering Chemistry Research

Enhancing 6-APA Productivity and Operational Stability of Penicillin G Acylase via Rapid Surface Capping on Commercial Resins Dong Yanga,b, Hua Liua,c, Jiafu Shib,c,d,*, Xueyan Wanga,c, Shaohua Zhanga,c, Hongjian Zoua,c, Zhongyi Jianga,c,* a

Key Laboratory for Green Chemical Technology of Ministry of Education, Key

Laboratory of Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China b

Tianjin Engineering Center of Biomass-derived Gas and Oil, School of Environmental

Science and Engineering, Tianjin University, Tianjin 300072, China c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin,

300072, China d

Key Laboratory of Biomass-based Oil and Gas (Tianjin University), China Petroleum and

Chemical Industry Federation, Tianjin 300072, China * Corresponding author: Zhongyi Jiang, [email protected]; Jiafu Shi, [email protected] KEYWORDS: Enzyme immobilization; Surface capping; Penicillin G acylase; Productivity; Stability 1

ACS Paragon Plus Environment


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ABSTRACT

In this study, immobilized penicillin G acylase (PGA) was prepared via a facile and rapid approach of generating TA-TiIV layer on PGA-adsorbed commercial resins (PGA@Resins). In brief, the TA-TiIV layer was constructed through coordination-enabled self-assembly of tannic acid (TA) and titanium (IV) bis (ammonium lactate) dihydroxide (Ti-BALDH). In comparison

to

PGA@Resins,

TA-TiIV-capped

PGA@Resins

exhibited

higher

6-aminopenicillanic acid (6-APA) productivity and enhanced operational stability along with comparable activity recovery during the catalytic hydrolysis of penicillin G potassium (PGK). Particularly, TA-TiIV-capped PGA@Resins exhibited relative activities of 103.7% and 81.51%, respectively, after 68-day storage and 20 cycles, indicating significantly enhanced storage and recycling stabilities compared to PGA@Resins (68.98% and 62.88%). Both immobilized PGA were further packed into glass column for hydrolyzing PGK in continuous flow reactor, where TA-TiIV-capped PGA@Resins displayed much higher 6-APA yield (initial yield: 49.22% vs. 28.99%; yield after 10 days: 17.39% vs. 6.11%) than PGA@Resins.

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1. INTRODUCTION

The family of β-lactam antibiotics is the most important class of clinically used antibiotics with higher than 50% of the global antibiotic market share.1,2 Penicillin, as a typical β-lactam antibiotic, occupies ~19% of the estimated worldwide antibiotic market share.3 6-aminopenicillanic acid (6-APA), an intermediate for production of semisynthetic penicillins and cephalosporins, are well-recognized as the worldwide largest selling β-lactam bulk intermediate. Primarily, 6-APA is synthesized through enzymatic4 or chemical deacylation5 of natural benzyl penicillin.6 For chemical methods, hazardous additives, such as phosphorous pentachloride, pyridine and nitrosylcholoride are often involved.7 On the contrary, enzymatic method is often implemented under mild conditions (aqueous phase, neutral pH, room temperature, no hazardous additives) along with high stereo-, regio-, and chemo-selectivity. Due to the merits of environmental friendliness, sustainability and high catalytic efficiency, enzymatic route for 6-APA production gradually becomes the preferred choice. To our knowledge, penicillin G acylase (PGA, E.C. 3.5.1.11) that shows high reactivity toward penicillin G potassium (PGK, precursor of 6-APA) is becoming a broadly used enzyme for 6-APA production.8 Nonetheless, the drawbacks of sensitivity to external stimuli and difficulty in recycling seriously restrict PGA for large-scale applications.

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To gain robust and recyclable PGA, a variety of scaffolds, e.g., magnetic silica nanoparticles, macrocellular silica monoliths, stimuli-response polymers, etc.,9-12 were fabricated for PGA immobilization in the past decades.13-16 In comparison, commercial resins were most competitive candidates in many application situations, primarily owing to their low cost, easy availability, high mechanical/chemical stability, ease of scaling up and recycling. As summarized in recent literatures, physical adsorption17 and covalent grafting18 were two dominant methods for enzyme immobilization on resins. Physical adsorption usually suffered from high enzyme leaching and low operational stability arisen from the weak hydrogen bonding and hydrophobic-hydrophobic interaction between enzyme and resin surface.19 For example, Jin et al.20 prepared immobilized PGA through directly adsorbing PGA on D301 resins. The immobilized PGA held a protein loading of only 27 mg (g dry carrier)

−1

and retained only 50% activity after 10 cycles. The treatment of

adsorbed enzyme on support through inter-enzyme (or enzyme-support) crosslinking21 or surface capping22 could, to some extent, address the above problems. However, commonly used cross-linkers (such as glutaraldehyde, genipin, etc.) often showed strong reactivity toward amine and hydroxyl groups of enzymes, which often caused the undesirable structural changes and inactivation of enzymes.20,23 By contrast, surface capping may be a better choice since enzyme can be physically retained by a thin film barrier. Several methods, including layer-by-layer assembly, biomimetic mineralization, bioadhesion, etc. have been utilized for surface capping, which all demonstrated the validity of this method.24 4


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In order to acquire a stable capping layer, multiple cycled assembling or long-time polymerization (>2 h) were often involved.25-28 Therefore, developing a facile, mild and efficient approach to engineering robust capping layer on commercial resins is particularly pursued albeit severe challenges.

Inspired by polyphenol-inspired chemistry, our group recently reported a new and relatively stable thin film (tannic acid-titanium (IV) thin film, denoted as TA-TiIV thin film) that could be capped on several kinds of substrates (CaCO3, chitosan, etc.).29,30 The thin film was acquired through a very simple and rapid process (~2 min) and maintained structure integrity against a broad range of pH values (4-10). Multi-point hydrogen bonds between TA and a material surface (adhesion) and stable coordination bonds between TA and TiIV ions (cohesion) both contributed the successful formation of the TA-TiIV thin film. The appropriate pore size/structure of TA-TiIV network could effectively inhibit the passing through of macromolecules, while, combined with the ultrathin property, small molecular substrates/products could freely diffuse across the film. Capping this thin film on the resins is expected to realize the enhancement of operational stability of immobilized enzyme without significantly altering enzyme activity.

Herein, we reported a facile and rapid approach to enhance 6-APA productivity and operational stability of immobilized PGA through forming TA-TiIV layer on the enzyme-adsorbed resins. Briefly, PGA was immobilized onto AB-8 macroporous resins (a

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kind of resins that composed of styrene-divinylbenzene copolymers) via physical adsorption (denoted as PGA@Resins), and then TA-TiIV coordination complex was formed on the resin surface. The catalytic activity, 6-APA productivity and operational stability (thermal, pH, storage and recycling stability) of TA-TiIV-capped PGA@Resins and PGA@Resins were systematically investigated, compared and discussed. The superiority of TA-TiIV layer in inhibiting PGA leaching and maintaining stable micro-environment for PGA was verified. When packed into glass column for hydrolyzing PGK in continuous flow reactor, TA-TiIV-capped PGA@Resins showed great potential for practical applications of such kind of immobilized PGA.

2. MATERIALS AND METHODS

2.1. Reagents and materials Penicillin G acylase (PGA, EC 3.5.1.11 from Bacillus megatherium, 1000 U mL-1) was purchased from Junfeng Bioengineering Co. Ltd. (Hangzhou, China). Tannic acid (TA, ACS reagent), titanium (IV) bis (ammonium lactate) dihydroxide (Ti-BALDH, 50 wt% aqueous solution) and tris (hydroxymethyl) aminomethane (Tris) were obtained from Sigma-Aldrich Chemical Co. Ltd. (USA). Hydrochloric acid (HCl) and AB-8 macroporous resins were obtained from Guangfu Co. Ltd. (Tianjin, China). Penicillin G potassium (PGK) was purchased from TCI Co. Ltd. (Shanghai, China).

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Other reagents were analytical grade and used without further purification. The water used in the experiments was purified by a three-stage Millipore Milli-Q purification system with a resistivity higher than 15.0 MΩcm.

2.2. Pretreatment of AB-8 macroporous resins

The physical properties of AB-8 macroporous resins were summarized in Table S1. The resins were pretreated to remove monomer and porogenic agents trapped inside the pores during the synthesis process. In brief, the resins were firstly soaked with 95 v/v% ethanol for 24 h, then washed thoroughly with deionized water for several times. Afterwards, the resins were immersed in 5 v/v% HCl for 3 h, and then rinsed by deionized water until neutral. Finally, the resins were soaked with 2 v/v% HCl for 3 h, washed thoroughly with distilled water until neutral.

2.3. PGA immobilization and activity assay

PGA was firstly immobilized onto AB-8 macroporous resins though physical adsorption. Briefly, pretreated resins (5.00 g, dry weight 1.67 g) were added to 15 mL Tris-HCl buffer solution (50 mmol L-1, pH 7.8) containing 5 mL PGA (25 mg mL-1), and gently stirred with mechanical mixing for 10 h at room temperature. The immobilized PGA was collected and washed three times with Tris-HCl buffer solution (50 mmol L-1, pH 7.8) to remove the free and loosely adsorbed PGA. Then, the as-prepared immobilized PGA was dispersed into a

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certain amount of deionized water with 1 min stirring to ensure complete dispersion. The mixture was stirred after the addition of TA and Ti-BALDH for 1 min for each addition. Afterwards, the immobilized PGA capped with TA-TiIV complex was washed with deionized water to remove excess TA and Ti-BALDH. The final concentration of TA was 0.06-0.24 mmol L-1 and the molar ratio of TA to Ti-BALDH was 1:10. The immobilized enzymes, denoted as PGA@Resins and TA-TiIV-capped PGA@Resins, respectively, were then stocked in at 4 oC for further use. The amount of PGA adsorbed on the resins was determined by the difference of protein concentrations in the solution before and after immobilization. Bradford method was applied for measuring the protein concentration with PGA in free form as the standard. Specifically, the absorbance at 595 nm was measured after mixing 1 mL enzyme solution or supernatant solution with 5 mL Coomassie Brilliant reagent for 3 min. The amount of enzyme immobilized onto the resins was calculated by mass balance with the following equation (Eq. (1)): Enzyme loading capacity (mg (g dry carrier)−1) = (m – CV)/Ws

(1)

where m is the initial amount of PGA introduced into the immobilization medium (mg); C (mg mL−1) and V (mL) are the enzyme concentration and volume of the supernatant solution, respectively; and Ws is mass of the initially added resins (g).

The catalytic activity of free and immobilized PGA was measured with a PGK (107.39 mmol L-1) Tris-HCl buffer solution (50 mmol L-1, pH 7.8). The hydrolysis of PGK by PGA

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yielded 6-APA. The produced 6-APA was determined by means of a spectrophotometric method with p-dimethylaminobenzaldehyde (PDAB) as a colored indicator. One unit of enzymatic activity was defined as the PGA amount required to produce 1 µmol of 6-APA per min. Activity recovery (%) was described as the activity of the immobilized enzymes compared to that of the free enzyme (Eq. (2)):

Activity recovery (%) = Aimmobilized/Afree × 100%

(2)

where Aimmobilized is the activity of immobilized PGA (U); Afree is the total starting activity of free PGA (U).

2.4. Operational stability evaluation

The thermal and pH stabilities were investigated by measuring the residual activity of immobilized PGA after incubation in Tris−HCl buffer (50 mmol L−1 pH 7.8) with different temperatures (30−70 oC) and pH values (4.0−10.0) for 2 h, respectively.

The recycling stability was investigated through measuring the relative activity of immobilized PGA during the process of circular reaction. The activity in the first cycle was taken to be 100%. Free and immobilized PGA was stored at 4 oC for a certain period of time. The storage stability was presented as the ratio of free or immobilized PGA activity after storage to the initial activity. The initial activity was taken to be 100%. 9



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The kinetic parameters were determined by measuring the initial reaction velocities of free and immobilized PGA (0.55 mg mL-1) with different substrate concentrations ranging from 1 to 40 mmol L-1.

A packed bed reactor was also constructed to perform the hydrolysis experiments in continuous mode. The immobilized PGA (1.8 g dry weight) was packed in a glass column (1 cm × 10 cm). The PGK solution (107.39 mmol L-1) was pumped through the column (flow rate: 1 mL min-1) in the top-down flow direction using a peristaltic pump (room temperature). The S0/Km ratios of PGA@Resins and TA-TiIV-capped PGA@Resins were 10.31 and 9.00, respectively. The 6-APA yield was determined every 12 hours.

2.5. Characterization The morphologies of AB-8 macroporous resins, PGA@Resins and TA-TiIV-capped PGA@Resins were observed using a field emission scanning electron microscope (SEM, Nanosem 430). FTIR spectra of AB-8 macroporous resins, PGA@Resins and TA-TiIV-capped PGA@Resins were obtained on a Nicolet-6700 spectrometer with a resolution of 4 cm-1 for each spectrum. The specific surface area and pore volume were recorded by nitrogen absorption-desorption isotherm measurements performed on a Micromeritics Tristar 3000 gas adsorption analyzer. The pore size distribution was determined by the nitrogen isotherms according to the Barret-Joyner-Halenda (BJH)

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method. Water contact angles were measured using contact angle measuring instrument (OCA20).

3. RESULTS AND DISCUSSION

3.1. Preparation and characterization of the immobilized PGA

Fig. 1. Optical images (a) and FTIR spectra (b) of AB-8 macroporous resins (A), PGA@Resins (B) and TA-TiIV-capped PGA@Resins (C).

Fig. 1a illustrates the color changes of AB-8 macroporous resins during enzyme immobilization. Pristine AB-8 macroporous resins showed a snow-white color. After PGA adsorption, the color of the resins changed from white to grey. The surface capping process

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further altered the color from grey to light yellow. Notably, this surface capping process was accomplished in a rather facile and rapid manner (just mixing of resins, TA and Ti-BALDH in aqueous solution for ~2 min). The color changes indicated the successful loading of PGA as well as the capping of TA-TiIV layer at macroscopic scale. It was well known that both adhesion and cohesion properties of an adhesive dominated the surface capping process. For the TA-TiIV complex, the adhesion capability was primarily originated from multiple hydrogen bonds between TA and resin, whereas the cohesion capability was resulted from the coordination bonds between TA and TiIV ions. Hence, the alternation of TA concentration may have some influence on the property of capping layer. As shown in Fig. S1, when TA concentration was increased from 0.06 to 0.48 mmol L-1, the color of the resins turned into deep yellow from pale yellow. This color changes meant, in this concentration range, higher amount of TA strengthened the cohesion capability of TA-TiIV complex through continuous formation of tris-coordination bonds between TA and TiIV ions, which finally promoted the growth of TA-TiIV layer on the resin surface.29 Then, to monitor the change of chemical compositions of the resins after PGA adsorption and surface capping, FTIR spectra of TA-TiIV-capped PGA@Resins and PGA@Resins were conducted. In Fig. 1b, the main characteristic bands at 3085, 3030, 2960 and 2930 cm−1 for all the resins were attributed to =CH2 group, C–H stretching of benzene ring, stretching of –CH3 and R–CH2–R group, respectively. The bands in the range of 1667-2000 cm−1 were assigned to the C–H stretch vibration of benzene ring, while the bands at 1602 and 1510 12


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cm−1 corresponded to C=C stretching vibrations of aromatic ring and carbon chain, respectively. The bands at or around 1490, 1440, 764 and 700 cm−1 were the typical indications of styrene.31 After PGA adsorption, the presnece of bands in the range of 3126-3600 cm−1 and at 1064 cm−1, which were origniated from the O–H stretching vibrations and C–N stretch vibration, indicated the successful immobilization of PGA on the resins. Clearly, the band located at 1657 cm−1 for TA-TiIV-capped PGA@Resins and PGA@Resins demonstrated a prominent C=O stretching, where the increased intensity suggested the successful capping of TA-TiIV complex on the resins.

Fig. 2. Nitrogen adsorption-desorption isotherm (a) and pore size distribution curve (b) of AB-8 macroporous resins, PGA@Resins and TA-TiIV-capped PGA@Resins.

The

cross-sectional

morphology

of

both

TA-TiIV-capped

PGA@Resins

and

PGA@Resins were almost unchanged in comparison to pristine resins (Fig. S2). This suggested both PGA adsorption and surface capping processes did not destroy the porous 13



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structure of the resin, maintaining numerous channels for substrate transfer. N2 adsorption/desorption isotherms for AB-8 macroporous resins, PGA@Resins and TA-TiIV-capped PGA@Resins were then conducted and shown in Fig. 2. All the isotherms were type II curves with H3 type hysteresis loops, indicating the resins also contained mesopores.32,33 The BET surface areas and pore volumes were summarized in Table 1. The specific surface area and pore volume of pristine resin were, respectively, 443.4 m2 g-1 and 1.31 cm3 g-1. After PGA adsorption, some mesopores in resins might be blocked, which caused a decreased specific surface area (342.8 m2 g-1) and pore volume (1.05 cm3 g-1). Interestingly, the specific surface area (314.3 m2 g-1) and pore volume (1.02 cm3) of TA-TiIV-capped PGA@Resins only decreased a little in comparison to PGA@Resins, indicating the TA-TiIV rarely entered the interior of PGA@resins.

Table 1. BET and BJH parameters of AB-8 macroporous resins, PGA@Resins and TA-TiIV-capped PGA@Resins. AB-8 macroporous resins

PGA@Resins

TA-TiIV-capped PGA@Resins

BET surface area (m2 g-1)

443.4

342.8

314.3

Pore volume (cm3 g-1)

1.31

1.05

1.02

3.2. Kinetics and activity assay of the immobilized PGA

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Several immobilization and kinetic parameters (Fig. S3) of the immobilized PGA were summarized and listed in Table 2. As illustration, the Vmax values of TA-TiIV-capped PGA@Resins and PGA@Resins were, respectively, 1.60 and 1.50 mmol (L min)−1, showing obvious decreased values compared to free PGA (35.33 mmol (L min)−1). Meanwhile, both TA-TiIV-capped PGA@Resins and PGA@Resins presented apparent higher Km values than free PGA, indicating lower affinity between enzyme and substrate after immobilization. The increase of Km for the immobilized PGA may be caused by diffusional restrictions. Vmax of immobilized PGA was much lower than that of free PGA, which could be due to the following aspects. The molecular size of PGA is ca. 7 nm that is only a bit smaller than the average pore diameter of the resins (13.5 nm). Hence, the active site of some immobilized PGA may not be accessible.34 Also, the reduced diffusion rate of substrate to the enzyme is another reason. This is expected based on the tortuous path during the diffusion of small molecules through porous network.35 In terms of two immobilized PGA, it was interestingly observed that TA-TiIV-capped PGA@Resins exhibited both higher Vmax and Km than PGA@Resins. Commonly, the TA-TiIV layer on TA-TiIV-capped PGA@Resins could cause the increase of mass transfer resistance, thus lowering the reaction velocity when compared to PGA@Resins. But, with the increase of the substrate concentration, gradually increased reaction velocities were obtained for both immobilized PGA. Accordingly, higher amount of phenylacetic acid was generated. Since both immobilized PGA were relatively hydrophobic (Fig. S4), partial phenylacetic acid 15



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without deprotonation may be adsorbed on the internal surface of resins after generated, which could suppressed the reaction in the forward direction. However, partially oxidized or deprotonated pyrogallol groups in TA may abstract the H+ from the as-generated phenylacetic acid, enhance its water solubility, and inhibit its adsorption onto resins, and finally ensure this by-product rapidly diffuse out of the resins. Notably, the abstraction of H+ could be evidenced by the slower decrease of pH value along with the reaction time for TA-TiIV-capped PGA@Resins (Fig. S5). Besides, we thought the inhibiting effect of phenylacetic acid was inconspicuous at lower substrate concentration but became more obvious at higher substrate concentration, which then promoted the reaction in the forward direction and resulted in higher Vmax and Km. Additionally, performance comparison of PGA immobilized on different spherical supports was shown in Table S2.

Table 2. Immobilization and kinetic parameters of free and immobilized PGA.

Name

Free PGA PGA@Resins

Loading capacity (mg (g dry carrier) −1) −

Kinetic parameters

Activity recovery (%)

Specific activity (U (mg enzyme) -1)

Km (mmol L−1)

Vmax (µmol (L min)−1)

−

62.84

3.35

35.33

72.60

4.42

2.59

10.41

1.50

68.50

3.91

2.53

11.93

1.60

IV

TA-Ti -capped PGA@Resins

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Fig. 3. 6-APA productivity as a function of the reaction time catalyzed by free and immobilized PGA (The amount of enzyme in both free and immobilized forms were 10 mg during the reaction.).

The 6-APA productivity as a function of the reaction time catalyzed by free and immobilized PGA was shown in Fig. 3. The hydrolysis reaction reached equilibrium in 20 min for free PGA, while 100 min were required for TA-TiIV-capped PGA@Resins and PGA@Resins. The equilibrium 6-APA productivity for TA-TiIV-capped PGA@Resins reached 0.90 mmol, which was higher than that of PGA@Resins (0.81 mmol). In accordance with the equilibrium 6-APA productivity, both immobilized PGA also exhibited much lower initial reaction velocity in comparison to free PGA. Specifically, the initial reaction velocity of free enzyme, calculated from the initial 5 min reaction, was 184.33 µmol min-1, whereas the velocities for TA-TiIV-capped PGA@Resins and PGA@Resins

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were 30.15 µmol min-1 and 34.85 µmol min-1, respectively. Compared to free PGA, the reduced 6-APA productivity and initial reaction velocities for both immobilized PGA could be as a result of the significantly increased mass transfer resistance of the resins. For both immobilized PGA, the higher 6-APA productivity and similar initial reaction velocity (or activity recovery) of TA-TiIV-capped PGA@Resins suggested that the capping layer neither destroyed the internal porous structure (as evidenced by SEM and BET results in Fig. 2 and S2) nor significantly increased the mass transfer resistance. On the contrary, this capping layer would improve the hydrophilicity of the resins (Fig. S4) and endow the resins with buffering effect (Fig. S5), where the by-product of phenylacetic acid could be deprotonated easily and diffuse rapidly out of the resins, thus promoting the reaction to the forward direction.36

3.3. Operational stability evaluation of the immobilized PGA

Operational stabilities, including thermal, pH, storage and recycling stabilities, are important criteria to evaluate a newly-developed biocatalyst. As shown in Fig. 4a, although similar decreased trends were observed with the increase of incubation temperature, both immobilized PGA exhibited higher thermal stability than free enzyme. Particularly, at 50 o

C, TA-TiIV-capped PGA@Resins and PGA@Resins could retain relative activities of

76.00% and 63.00%, respectively. By contrast, a relative activity of only 42.00% was left for the free enzyme. The improvement of thermal stability indicated the combination of

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surface capping and physical adsorption effectively inhibited the structure changes of PGA against the elevated temperature. After raising the incubation temperature to 70 oC, no activities were left for either free or immobilized PGA.

The activity retention after incubation of enzymes in different pH values was also investigated. In general, both TA-TiIV-capped PGA@Resins and PGA@Resins showed elevated pH stability in a broad range of pH values (Fig. 4b). Specifically, TA-TiIV-capped PGA@Resins maintained relative activities of 84.00% and 96.70%, respectively, under weak acidic conditions of pH 5.0 and 6.0. Under the same pH values, lower relative activities of 76.00% (pH 5.0) and 91.50% (pH 6.0) were observed for PGA@Resins. The enhancement of pH stability should be as a result of two aspects: 1) the pH-weak response property of TA-TiIV layer that inhibited the enzyme leaching, which was in accordance with our previous results, 29,30 and 2) the buffering effect of TA with –O-/OH pairs (Fig. S5). However, when pH value was decreased to 4.0, little activity was left for both free and immobilized PGA, which may be caused by the access of excessive H+ to enzyme active site that induced the structure changes of enzyme.

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Fig. 4. Thermal stability (a), pH stability (b), storage stability (c) and recycling stability (d) of free (a, b, d) and immobilized PGA (a, b, c, d).

The storage and recycling stabilities of the immobilized PGA were of particular importance for industrial application, which were also evaluated after incubating the reaction solution at 4 oC for a period of storage time (Fig. 4c). After storing for 68 days, TA-TiIV-capped PGA@Resins even retained 103.7% of its initial activity, while PGA@Resins and free PGA only retained 68.98% and 58.70% of their initial activities, respectively. For both immobilized PGA, the enzyme activities beyond 100% after several

20


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days' incubation might be owing to the migration of PGA from internal part to the surface of the resins along with the storing time. Since enzyme in both TA-TiIV-capped PGA@Resins and PGA@Resins were physically adsorbed, the detachment of PGA was inevitable during the long-term reaction. However, TA-TiIV layer could effectively inhibit the leaching of enzyme to the external reaction medium (Table S3), and then exhibit a much higher activity retention. Interestingly, the activity retention of surpassing 100% was observed for TA-TiIV-capped PGA@Resins, which might be ascribed to the migration of enzyme detached from internal part of the resins to the surface region, lowering the diffusion resistance during the reaction. For the recycling test (Fig. 4d), TA-TiIV-capped PGA@Resins still retained 81.51% of its initial activity after the 20th reaction cycle. In comparison, the PGA@Resins only retained 62.88% of its initial activity. The extraordinary storage and recycling stabilities of TA-TiIV-capped PGA@Resins were mainly resulted from favorable and stable micro-environment offered by the capping layer, which ensured this kind of immobilized enzyme great potential for industrial application.

3.4. Conversion of PGK enabled by immobilized PGA in continuous reactor

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Fig. 5. Schematic drawing of the continuous packed bed reactor enabled by the immobilized PGA (a); and catalytic performance for PGK hydrolysis using PGA@Resins and TA-TiIV-capped PGA@Resins in a packed bed reactor (b).

Continuous production of 6-APA enabled by immobilized PGA is highly desirable for industrial application. Therefore, we constructed a packed bed reactor to evaluate the catalytic performance of TA-TiIV-capped PGA@Resins and PGA@Resins in a continuous reaction mode. As shown in Fig.5a, the reaction enabled by TA-TiIV-capped PGA@Resins showed an initial 6-APA yield of 49.22%, which decreased slowly to 17.39% after 10 days of continuous conversion. In comparison, the initial 6-APA yield enabled by PGA@Resins was only 28.99%, and decreased to 6.11% after 10 days of continuous reaction. This result 22


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suggested that capping a layer on commercial resins through the present approach could offer a promising possibility to facilitate the use of PGA in continuous reactor. The decreased activity could be ascribed to the leaching of PGA from the resins during continuous reaction (Table S3). With respect to a biocatalyst, continuous reaction mode usually created a higher shearing force compared to bulk reaction, which may then led a washing out of TA-TiIV layer and PGA from the resins. Besides, during the hydrolysis of PGK, the produced phenylacetic acid could reduce the pH value of the reaction medium, which also contributed to the decrease of PGA activity.

4. CONCLUSIONS

In summary, a facile and rapid approach was developed to engineer the surface of commercial resins for simultaneously intensify the catalytic function and non-catalytic function of immobilized PGA. The capped TA-TiIV layer prevented the physically adsorbed PGA from leaching and improved the hydrophilicity and buffering function of the resins, acquiring

higher

6-APA

productivity.

The

immobilized

PGA,

TA-TiIV-capped

PGA@Resins, exhibited super-high storage and recycling stabilities, maintaining 103.7% and 81.51% of their initial activity after 68-day storage and 20 cycles. A packed bed reactor filled with TA-TiIV-capped PGA@Resins was also constructed, to well explore its potential application for massive production of 6-APA.

ASSOCIATED CONTENT 23



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Supporting information

The Supporting Information includes tables of physical properties of AB-8 macroporous resins, performance comparison of PGA immobilized on different spherical supports, enzyme leakage ratio and figures of optical images of TA-TiIV capped on AB-8 macroporous resins with different TA concentrations, Lineweaver-Burk plot for free and immobilized PGA, AB-8 macroporous resins SEM images, water contact angles and evolution of pH value in reaction medium as a function of reaction time. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding author

* E-mail address: [email protected] (Zhongyi Jiang), Tel.: +86-22-27406646, Fax: +86-22-27406646; E-mail address: [email protected] (Jiafu Shi)

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENT

The authors thank the financial support from National Natural Science Funds of China (21406163, 91534126), National Science Fund for Distinguished Young Scholars 24


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(21125627), Tianjin Research Program of Application Foundation and Advanced Technology (15JCQNJC10000), and the Program of Introducing Talents of Discipline to Universities (B06006).

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