Recyclable Î²-Glucosidase by One-Pot Encapsulation with Cu-MOFs

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Recyclable #-glucosidase by one-pot encapsulation with Cu-MOFs for enhanced hydrolysis of cellulose to glucose Lei Wang, Wenjing Zhi, Jing Wan, Juan Han, Chunmei Li, and Yun Wang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05489 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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ACS Sustainable Chemistry & Engineering

Recyclable β-glucosidase by one-pot encapsulation with Cu-MOFs for enhanced hydrolysis of cellulose to glucose Lei Wanga, Wenjing Zhia, Jing Wana, Juan Hanb, Chunmei Lia, Yun Wanga,* a

School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu

Road, Zhenjiang 212013, Jiangsu Province, China b

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,

Zhenjiang 212013, Jiangsu Province, China *

Corresponding author: Yun Wang. E-mail: [email protected]

Abstract Enhancing the stability and reusability of enzymes by encapsulating them within a powerful class of porous materials termed metal-organic frameworks (MOFs) has been demonstrated by recent studies. However, in all of these reports, the stability of MOFs under acidic conditions was a major issue to enzymes with catalytic activity at low pH. The objective of this study is to encapsulate the β-glucosidase (β-G) into the Cu-MOF due to its acidic stability, using a co-precipitation approach. Indeed, the as-prepared β-G@Cu(PABA) biocomposite with high encapsulation efficiency exhibited enhanced resistance to acidic and thermal condition, as well as organic solvents. Importantly, the β-G@Cu(PABA) biocomposite showed superior reusability, retaining 90% of activity even after ten cycles. Using the hydrolysis of carboxymethylcellulose (CMC) as a mode, the mixture of β-G@Cu(PABA) biocomposite and co-immobilized cellulase afforded 98% glucose yield, which was 2-fold enhancement when compared with co-immobilized cellulose alone. The β-G@Cu(PABA) biocomposite and co-immobilized cellulase could been easily recovered and recycled to still retain 70% productivity in the eight cycles for hydrolyzing CMC. This discovery for the first time highlights the potential of Cu-MOF in preserving the enzymes under acidic conditions. The developed

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β-G@Cu(PABA) biocomposite by co-precipitation approach affords a valuable platform for enzyme-based industrial applications. Keywords:

Metal-organic

framework,

cellulose,

immobilized enzymes


biocatalysis,

β-glucosidase,

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Introduction Biofuel ethanol, the ethanol produced from renewable cellulose (an almost inexhaustible polymeric raw material), has attracted wide attention as an alternative and green liquid energy in recent years.1,2 However, conversion of cellulose into glucose is an important step of biofuel ethanol production,3 which mainly depends on the degradation capacity of a complex cellulase system including endo-β-1,4glucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (β-G). The EG and CBH act synergistically to degrade cellulose to cellobiose, which is further hydrolyzed into glucose by β-G.4 During enzymatic degradation of cellulose, the β-G is considered as a key enzyme because it can reduce the cellobiose inhibition for EG and CBH.5 Thus, the additional supplementation of sufficient β-G into the cellulase system can enhance overall cellulose hydrolysis rate and improve glucose yield.6,7 Despite this encouraging result, the role of native β-G on industrial application has so far been mainly limited to their high cost, poor stability, difficult separation and recycling.8,9 Therefore, it is imperative to search a new approach to make the industrial application feasible via improving β-G stability and reducing β-G cost by recycling. One approach that aims to address this challenge is to encapsulate the β-G within a protective shell that shields the β-G from the external environment. Recently, metal-organic frameworks (MOFs) have been effectively used as protective shells for enzymes.10-12 As an emerging class of excellent porous nanomaterials, MOFs are comprised of metal ions and organic building blocks linked together by strong coordination bonds. More importantly, MOFs possess high surface areas and large pore volumes that facilitate the transport of enzyme substrates through porous network.13 And it also can offer exceptional thermal and chemical protection for enzymes, compared to other traditional porous materials such as, mesoporous metal oxides and silica.14,15 This superior properties point toward MOFs as potential candidate materials for the protective coatings of enzymes. Indeed, this concept is supported by recent investigations that illustrate the stability of encapsulated enzymes has been improved with the protection of the MOF layer.16



Most recently, zeolitic imidazolate framework-8 (ZIF-8) have been primarily reported as ‘containers’ for the biomolecules encapsulation because of their excellent thermal and chemical stability, as well as negligible cytotoxicity.17 However, ZIF-8 is not appropriate for the β-G encapsulation. Since the optimum pH value for hydrolysis reaction by β-G is around 5, this acidic environment can dissolve the framework of ZIF-8.18 Here, we reported for the first time that the encapsulation of β-G in the Cu-MOF could be a valuable strategy to enhance the β-G stability and reusability. Because it could survive at pH of 5 that almost matched with the optimum pH value for hydrolysis reaction.19,20 This alternative will highlight the potential of Cu-MOF in preserving the enzymes under acidic conditions. So far, a breakthrough in the one-pot synthesis of protein-embedded MOFs has been reported since the pioneering work by Liang group.21 This discovery has not been applied to directly embed the β-G in Cu-MOF by a co-precipitation approach. In this study, we demonstrated that co-precipitation of Cu-MOF formed a protective shell, and allowed for more expeditious diffusion of the substrates and products through the Cu-MOF due to their smaller crystal size.22 In addition, the successful encapsulation of β-G by Cu-MOF highlighted the versatility of this co-precipitation method. Here, the β-G@Cu(PABA) biocomposite maintained a relatively high activity against high temperature and organic solvent, which was superior to the system of β-G immobilized on polymer and inorganic material. During a continuous operation, the β-G@Cu(PABA) biocomposite offered significant advantages over the free β-G such as high activity and recyclability. In order to afford excellent performance for the hydrolysis of cellulose, the additional β-G@Cu(PABA) biocomposite was added to co-immobilized cellulase system to increase glucose yield.

Materials and methods Materials β-glucosidase (β-G, from almonds, powder, a specific activity of ≥ 6 U/mg) was purchased from Sigma-Aldrich. Cellulase, a mixture of EG, CBH and β-G (from T. viride, CMCase activity of 15 U/mg), was obtained from Sinopharm Chemical


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Reagent

Co.

Ltd.

(Shanghai,

China).

Cupric

acetate

monohydrate

，

para-aminobenzoic acid and polyvinyl pyrrolidone (PVP, average mol wt 30 k) were purchased from Sinopharm (Shanghai, China) Chemical Reagent Co. Ltd. and was of analytical grade. P-nitrophenol (p-NP), p-nitrophenyl-β-D-glucoside (p-PNG) were obtained from Aladdin (Shanghai, China). Fluorescein5 (6)-isothiocyanate (FITC), N-Hydroxy-

succinimide

(NHS)

and

N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) were purchased from Macklin Biochemical Co, Ltd. (Shanghai, China). All other chemicals (analytical grade) were purchased from Sinopharm (Shanghai, China) Chemical Reagent Co. Ltd. and used without modification. The water used throughout the experiments was deionized. Pierce BCA protein assay kit was purchased from Thermo Scientific (Shanghai, China). Synthesis of Cu(PABA) Metal-organic frameworks (Cu(PABA)) were constructed from metal ions and organic components by co-precipitation method. Typically, copper acetate (50 mM) and para-aminobenzoic acid (PABA) (12.5 mM) were dissolved in acetate buffer solution (50 mM, pH 7.0), respectively. And then the above solutions were mixed and incubated for 8 h at room temperature in an incubator. Subsequently, the resulting powder was obtained by centrifugation, washed three times with buffer solution of acetic acid-sodium acetate (pH 5.0) and saved at 4 °C. Preparation of β-G@Cu(PABA) β-G@Cu(PABA) biocomposite was constructed by using the co-precipitation method as follows. A mixture was prepared by adding copper acetate solution (50 mM, 500 μL) into the solution of β-G containing PVP (2 mg/mL, 50 μL). A separate solution of PABA (12.5 mM, 500 μL) dissolved in the acetate buffer saline solution (pH 7.0, 50 mM) was also prepared. These two solutions were combined and incubated for 8h at room temperature in an incubator for the formation of PABA coatings. Then the biocomposite was collected by centrifugation-wash cycles for three times and saved at 4 °C. The synthesis of PMAAc-cellulose was based on previously reported methods.23



Labeled β-G with FITC β-G (20 mg) was dispersed in 5 mL of NaAc-HAc buffer solution (50 mM, pH 5.0) under 4 ºC. And then EDC (10mg) and NHS (5 mg) were mixed with solution containing β-G for 1 h. FITC solution (20 μg/mL, FITC in NaAc-HAc buffer solution) was added into the above solution and stirred for 4 h at 4 °C in the dark. This containing FITC-β-G solution was dialyzed (MWCO: 10k) for 24 h at 4 ºC in the dark to remove the unreacted EDC, NHS and FITC. Characterization methods The morphologies of the Cu(PABA) and β-G@Cu(PABA) biocomposite were taken on JSM-6010 PLUS/LA scanning electron microscope (SEM). For scanning electron microscope (SEM), the samples was prepared by a suitable amount of ethanol suspension of the Cu(PABA) and β-G@Cu(PABA) were dropped onto a silicon wafer and dried at room temperature, respectively. Powder X-ray diffraction (XRD) patterns were recorded by an X-ray diffractometer (XRD-6100Lab, Japan) with scattering angles (2θ) of 10-80 °C at a scan rate of 7 °/min. The fluorescence images of Cu(PABA) and FITC-β-G@Cu(PABA) biocomposite under bright field and fluorescence fields were obtained with a TCS SP5 II Laser Confocal Microscope. UV-Vis absorption spectra were acquired on a UV-Vis spectrometer (Shimadzu, Japan). Fourier transform infrared (FTIR) measurements were carried out on a Nicolet Nexus 470 Spectrometer in the range of 400- 4000 cm-1 by KBr pellets. Thermal gravimetric analysis (TGA) was performed in the STA449C Thermal Analyzer. The dried sample was filled into a crucible, and collected from 20-600 °C under a continuous stream of nitrogen gas (heating rate of 10 °C /min). Activity Assay The activity of β-G@Cu(PABA) was assessed by using p-PNG as a substrate in a colorimetric method. p-PNG (40 μL) was mixed well with 440 μL of NaAc-HAc buffer solution (50 mM, pH 5.0) in a 1.5 mL EP tube, and preheated 10 min in a water bath at 50 °C. β-G solution (2 mg/mL, 20 μL) was then quickly added to above described solution with gentle shaking, and reacted accurately at 50 °C for 10 min. Then, the reaction was immediately stopped by the addition Na2CO3 solution (1 M,


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500 μL) under vigorous shaking, and allowed to stand for half an hour. The absorbance of the supernatant was monitored at 410 nm under a UV-Vis spectrophotometer, and p-NP was used as a standard. The enzymatic assays were performed for three times, respectively. In the enzyme activity assay using free β-G, the amount of free enzyme introduced into solution was adjusted to be equal to the amount of enzyme loaded into β-G@Cu(PABA), as determined according to the encapsulation capacity. The weight percentage of β-G in the Cu(PABA) was measured by Pierce BCA protein assay kit. The encapsulation efficiency was defined as the ratio of β-G amount embedded in the β-G@Cu(PABA) composites to the initial amount of β-G. The encapsulation capacity was defined as the ratio of β-G amount embedded in the β-G@Cu(PABA) composites to the weight of the β-G@Cu(PABA) composites. One enzyme activity unit (U) was defined by the amount of enzyme that is required to decompose the substrate of p-PNG to produce 1 μmol of p-NP per minute by β-G at 50 C and pH 5.0. The relative enzyme activity was defined as the percentage of the measured enzyme activity value and the highest enzyme activity value in the same group of experiments.

Encapsulation efficiency (%) 

m - C1V1  100% m

Encapsulation capacity (mg/g) 

Enzyme activity ( U) 

m  C1V1 W

CV t  1000

(1)

(2)

(3)

Here, m (mg) represents the mass of β-G initially added to the solution; C1 (mg/mL) and V1 (mL) are the β-G concentration and the volume of supernatant, respectively. W (g) is the weight of the β-G@Cu(PABA) composites. C (μM) represents the concentration of p-NP; V (mL) is the volume of the reaction solution; t (min) is the time of reaction. Catalytic Activity of the β-G@Cu(PABA) To obtain the optimal pH and temperature of free and immobilized β-G, These β-G enzymes were incubated in different ranges of pH (3.0-7.0) and temperature



(30-80 °C) for 10 min. The activities were measured by the above mentioned method. Their activities were determined to draw on relative activity by taking the optimum activity of free enzyme as 100%. Stability and reusability of β-G@Cu(PABA) The thermostability of β-G and β-G@Cu(PABA) were assessed in NaAc-HAc buffer solution (50 mM, pH 5.0) in a rocking incubator over the temperature range of 30-80 °C for 2 h with a one-hour interval. And their pH stabilities were analyzed in different NaAc-HAc buffer solution (50 mM, pH 3.0-8.0) in a rocking incubator at 25 °C for 2 h. To evaluate the tolerance of polar solvents, free β-G and β-G@Cu(PABA) biocomposite were incubated in a certain proportion of organic solvent-water systems such as acetonitrile (AN), methanol (MeOH), 1,4-dioxane (1,4-Diox), ethyl acetate (EAC), and ethanol (EtOH) for 1h. The storage stabilities of free and immobilized β-G were examined in NaAc-HAc buffer solution (50mM, pH 5.0) at 4 °C for 40 days with a five-day interval. In order to measure the reusability of the β-G@Cu(PABA), the β-G@Cu(PABA) was obtained through centrifugation after each reaction, rinsed with NaAc-HAc buffer solution (50 mM, pH 5.0), and utilized for the next round. The reaction repeated 10 times and the initial activity of enzyme was defined as 100 %. Michaelis−Menten kinetics The kinetic parameters (Km and Vmax) of free β-G and immobilized β-G were obtained by monitoring the rate of p-PNG decomposition spectrophotometrically at 410 nm. V0 

V max[S] Km  [S]

(4)

Here, V0 is the initial catalytic rate. Vmax is the maximum rate conversion. [S] is the initial substrate concentration, and Km is the Michealis-Menten constant. Catalytic Hydrolysis Application and Recycling of β-G@Cu(PABA) The experiments were taken in a 1.5 mL EP tube with different ratios of β-G@Cu(PABA) biocomposite and immobilized PMAAc-cellulase by adding the carboxymethylcellulose sodium solution (CMC,10 mg). The mixture was shaken at


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50 °C in a temperature-controlled shaking incubator, the amount of glucose was measured continuously at two hours interval by the dinitrosali cylic acid (DNS) method.23 Similar

procedure

was

employed

to

continuous

hydrolysis

of

carboxymethylcellulose to glucose. After the above reaction was completed, β-G@Cu(PABA) biocomposite was obtained after centrifuging at room temperature. Subsequently, the supernatant containing PMAAc-cellulase and glucose was collected, and then centrifuged at 5 °C, followed by the collection of PMAAc-cellulase. The supernatant containing soluble glucose was also separated by centrifugation, and measured by DNS method to calculate the yield. The resulting β-G@Cu(PABA) and PMAAc-cellulase were resuspended. The catalytic hydrolysis reaction was performed again after the addition of CMC. The cycle step is repeated to obtain the saccharification rate corresponding to the number of cycles.

Results and discussion Acid stability of Cu(PABA) material To choose properly acid-resistant MOFs for β-G encapsulation, the effect of pH on stability of Cu(PABA) and ZIF-8 was evaluated. As displayed in Figure S1, these two materials were incubated under acetate buffers of pH 7.0 and 5.0 for 20 h to investigate the residual weight. Both Cu(PABA) and ZIF-8 material retained their original weight after incubating under pH of 7.0. After 20 h incubation at pH 5.0, 98% of its initial weight was preserved for Cu(PABA) material. While ZIF-8 was completely disassembled after 20 h incubation at pH 5.0. The same phenomenon was reported by the previous publication, in which a pH change from 7.4 to 6.0 was sufficient to dissolve the protective ZIF-8 layer.20 This investigation proved that the framework of Cu(PABA) was not significantly affected by acidic conditions (pH 5.0). Since the optimum pH value for hydrolysis reaction by β-G was around 5.0, Cu(PABA) material was selected as protective layer to properly encapsulate the β-G. Furthermore, we performed energy dispersive X-ray spectroscopy (EDS) analysis on the Cu(PABA) treated after acetate buffer of pH 5.0 (Figure S2a) and pH 7.0 (Figure



S2b). The presence of C, O, N and Cu elements provided a direct proof for the preservation of the Cu(PABA) framework. This conclusion was also verified via XRD analysis (Figure S3). Preparation of β-G@Cu(PABA) Biocomposite A typical one-pot synthesis of β-G@Cu(PABA) biocomposite was presented in Scheme 1.

Scheme 1. Synthesis strategy of the β-G@Cu(PABA) biocomposite. The formation of Cu(PABA) shell to provide protection of β-G was mainly influenced by concentration of metal cations, organic ligands and β-G, incubation pH and time. Therefore, the activity and encapsulation capacity of β-G during growth process of biocomposite were investigated in detail to gain the optimum experimental condition. Both activity and encapsulation capacity of β-G were systematically enhanced to 81.89% and 162.95 mg/g, respectively, with the increase in the concentration of Cu2+ ions up to 50 mM and organic PABA ligands up to 12.5 mM (see Table S1 and S2). However, after the addition of 62.5 mM of Cu2+ ions resulted in the emergence of a decreased activity and encapsulation capacity of β-G, because Cu2+ ions had negative effects on the activity of β-G. Such idea was also observed when incubating free β-G with the solution of Cu2+ ion (see Table S3). Similar phenomenon could be found upon addition of 18.75 mM of PABA ligands. In order to obtain high activity and encapsulation capacity of β-G, 50 mM of Cu2+ ions and 12.5 mM of PABA ligands were selected as the optimum concentration for further evaluation. Nevertheless, as presented in Figure S4, the enzymatic activity gradually enhanced to 82% when the concentration of β-G increased to 2 mg/mL, but dropped down with continuous increase of β-G. The result of encapsulation capacity of β-G


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was consistent with the activity of β-G, which gave 85% encapsulation efficiency at β-G concentration of 2 mg/mL. Taking the above results into account, the following study of the activity of β-G was evaluated by using 2 mg/mL of β-G. It was well known that pH value was another crucial factor in activity and encapsulation capacity of β-G. And therefore, the incubation pH in the range of 5.5 to 8.0 was performed. The maximum activity and encapsulation capacity of β-G were obtained when the pH value of acetate buffer solution was 7 (the mixture solution of Cu2+ ion, PABA ligand and buffer solution at pH 5.3) (Figure 1a). We speculated that the acetate buffer solution at pH 7.0 was favorable for the formation of the Cu(PABA) protective layer, and did not cause denaturation of the β-G during the co-precipitation process. The effect of incubation time for the activity and encapsulation capacity of β-G was also examined with the range of time from 4 to 14 h (Figure 1b). The results revealed that the activity and encapsulation capacity increased gradually, and then reached a stable constant of 82% and 163 mg/g at the incubation time of 8 h, respectively. Such results indicated that the complete encapsulation in this reaction system needed 8 h.

Figure 1. (a) Effect of immobilization pH and (b) immobilization time on relative activity and encapsulation capacity of β-G@Cu(PABA) biocomposite. The error bars represent the standard deviations from triplicate experiments. Characterization of β-G@Cu(PABA) Biocomposite The self-assembly process of β-G@Cu(PABA) biocomposite was manifested by means of SEM images. As depicted in Figure 2a, the pure Cu(PABA) had a regular smooth ellipsoid structure, and the length was about 10-20 μm. The SEM image of β-G@Cu(PABA) biocomposite (Figure 2b) displayed the same morphology and



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length as pure Cu(PABA), demonstrating that the embedded β-G had not influence on the morphology of Cu(PABA). From the XRD patterns (Figure 2c) could be clearly seen that there was no significant difference with regard to the crystalline structure between the pure Cu(PABA) and β-G@Cu(PABA) biocomposite because the positions of all diffraction peaks matched well. The results indicated that the structural integrity of Cu(PABA) could be retained in the presence of β-G. However, the corresponding intensities of the diffraction peaks were lower than that of Cu(PABA). This further demonstrated that the degree of crystallinity of Cu(PABA) was decreased and tuned by β-G.24

Figure 2. (a) SEM images of the pure Cu(PABA) and (b) β-G@Cu(PABA) biocomposite. (c) XRD patterns of pure Cu(PABA) and β-G@Cu(PABA) biocomposite. To further ascertain that β-G was indeed encapsulated by Cu(PABA) crystalline shell instead of absorbed on the external surface, β-G tagged with fluorescein isothiocyanate (FITC) was used to induce the formation of FITC-β-G@Cu(PABA) biocomposite. The resultant biocomposite was washed with water to remove any surface adsorbed β-G, and detected under confocal laser scanning microscopy. Subsequently,

homogenously

green

fluorescence

of

FITC-β-G@Cu(PABA)

biocomposite was observed, as shown in Figure 3d, suggesting that FITC-β-G was encapsulated during the growth process of biocomposite. The same result was also


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confirmed by TEM images of β-G@Cu(PABA) biocomposite (Figure S5), whereas the β-G aggregates were not emerged on surface of the encapsulated β-G@Cu(PABA) biocomposite. Additional evidence of β-G encapsulation in Cu(PABA) was garnered by FT-IR spectra (Figure 4a). The spectrum of β-G@Cu(PABA) biocomposite was quite similar to that of Cu(PABA), with the exception of the peak at 1655 cm-1 that attributed to the stretching vibration of C=O (-CONH) bond due to the presence of β-G.24 Another characteristic of β-G was also presented at 1430 cm-1, corresponding to a combination between of NH bending and CN stretching vibrations. The peaks at 1606 cm-1 and 1550-1390 cm-1 were ascribed to the stretching vibration of carboxyl and entire aromatic groups, respectively. The intensity of the characteristic peaks of the Cu(PABA) at 1606 cm-1 and 1550-1390 cm-1 was clearly decreased. This was mainly because Cu(PABA) formed on β-G surfaces was in small quantity.25,26 Furthermore, no new peak and a significant peak shift appeared in the spectrum of biocomposite, which demonstrated that the embedment of β-G in Cu(PABA) was via coprecipitation process with no formation of covalent bonding. The TGA was also performed to confirm the presence of β-G in the biocomposite. As revealed in Figure 4b, the Cu(PABA) displayed a slight weight loss step of 5% from 100 to 200 °C, which was ascribed to the removal of water molecules. However, a gradual weight-loss profile occurred from 250 °C to 350 °C in the β-G@Cu(PABA) biocomposite, which was mainly attributed to the decomposition of β-G.24 The loss amount of β-G was calculated to be approximate 13%. The ratio was consistent with the β-G encapsulation efficiency determined through the absorbance at 562 nm using Pierce BCA protein measurement (16%), as mentioned above. The thermal decomposition of Cu(PABA) crystals was present over 350 °C, implying the good thermal stability of Cu(PABA). Three intense peaks in the XRD pattern of residual component were consistent with the data published by the Joint Committee on Powder Diffraction Standards (JCPDS no. 71-4610) for pure crystalline copper (Figure S6). Hence, crystalline copper was obtained as a residual component. Nitrogen adsorption-desorption isotherms and Barrett-Joyner-Halenda (BJH) pore diameter distribution were then employed to assess the porosity of Cu(PABA)



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(Figure S7) and β-G@Cu(PABA) biocomposite (Figure 5). The pore diameter distribution analysis of Cu(PABA) gained by isotherms revealed mesopores of Cu(PABA) (around 10-60 nm).27 The mesoporous structure of Cu(PABA) was ideally sized, facilitating β-G (molecular dimensions: ∼6.2 nm × 11.8 nm × 7.7 nm) encapsulation. As illustrated in Figure 5, there was a significant hysteresis loop in a relative pressure range of 0.5 to 1.0, suggesting β-G@Cu(PABA) biocomposite inherited the mesoporous structure of Cu(PABA). Indeed, the conclusion was supported by the corresponding pore diameter distribution (inset in Figure 5), in which the pore size of the biocomposite was mostly 40 nm. These interconnected channels within the biocomposite preserved the accessibility of the encapsulated β-G, thus allowing easier diffusion of substrates to the active sites of β-G.

Figure 3. (a) Confocal microscope images of bright field of Cu(PABA) and (c) FITC-β-G@Cu(PABA),

and

(b)

fluorescent

field

of

Cu(PABA)

and

(d)

FITC-β-G@Cu(PABA).

Figure 4. (a) FT-IR spectra of β-G, β-G@Cu(PABA) biocomposite and Cu(PABA). (b) TGA curves of β-G, Cu(PABA) and β-G@Cu(PABA).


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Figure 5. N2 adsorption–desorption curves and pore diameter distributions (insert) of β-G@Cu(PABA). Catalytic Activity of β-G@Cu(PABA) Biocomposite To obtain highest catalytic activity of β-G@Cu(PABA) biocomposite, The effects of reaction pH value and temperature on catalytic activity of β-G@Cu(PABA) were evaluated. No catalytic activity was obtained in the presence of Cu(PABA) framework alone as catalyst at different pH values and temperatures. As displayed in Figure 6a, the highest catalytic activity of the pristine β-G and β-G@Cu(PABA) biocomposite was exhibited at pH 5.0. When the pH of the solution was adjusted to 4.5 or 5.5, the catalytic activity of pristine β-G drastically decreased to 50% and 80% of its highest activity, respectively. Whereas, the β-G@Cu(PABA) biocomposite had a relative higher activity than that of pristine β-G at pH 4.5 or 5.5, which was attributed to confinement effect provided by the Cu(PABA) shell that restricted the β-G from aggregation. Previous reports showed that the MOFs shell can provide confinement effect for encapsulation enzymes under acidic condition.26 The temperature-dependent

activity

of

the

pristine

β-G

and

β-G@Cu(PABA)

biocomposite was also recorded in the temperature range of 30 to 80 °C (Figure 6b). The pristine β-G exhibited the maximum activity at the temperature of 50 °C, while above 50 °C a rapid decrease in activity was seen for the pristine β-G; In contrast, the β-G@Cu(PABA) biocomposite essentially remained the maximum activity at 60 °C before gradually decreasing. A significant improvement in optimum temperature of β-G@Cu(PABA) over that of pristine β-G might be explained due to the rigid



structure of Cu(PABA) that prevented enzyme conformational transition at elevated temperature that leaded to denaturation.28

Figure 6. (a) Effects of reaction pH and (b) temperature on catalytic activity of free β-G and β-G@Cu(PABA). The error bars represent the standard deviations from triplicate experiments. The kinetic parameters of the β-G@Cu(PABA) biocomposite were further investigated to assess whether mass transport was hindered by the Cu(PABA) shell. Based on the catalytic rates as the function of substrate concentrations, the Lineweaver-Burk plot was accomplished with a good linear relationship, as shown in Figure S8. The Km and Vmax value of free β-G ascertained from Lineweaver-Burk plot was found to 3.86 mM and 1.48 mM·min-1, respectively. Compared to free β-G, β-G@Cu(PABA) biocomposite showed a decrease in the Km value (2.46 mM) and Vmax value (0.64 mM·min-1) (see Table S4). These results suggested that β-G@Cu(PABA) biocomposite had a higher affinity toward the substrate, which was possibly due to the microenvironment created by the Cu(PABA) shell.28 However, the decline in Vmax value may be caused by increased mass transport limitations. Stability and Reusability of β-G@Cu(PABA) Biocomposite Exposing enzymes to harsh environments (for example, acidic conditions, high temperatures and organic solvents) normally result in the loss of activity. Thus, the β-G@Cu(PABA) biocomposites under harsh conditions were also conducted to obtain an insight into the efficiency of Cu(PABA) shell to enhance the stability of β-G. As demonstrated in Figure 7a, an increase in the pH value from 3.0 to 8.0 firstly led to a


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significantly increase of the activity of free β-G and then decreased after incubation at different pH for 2 h. The β-G@Cu(PABA) biocomposite demonstrated a similar behaviour in the pH range of 3-8. Notably, free β-G exhibited 30% and 60% of its initial activity after incubating under pH of 4.0 and 6.0 for 2 h, respectively, which was less than 80% and 90% of activity for β-G@Cu(PABA) biocomposite under the same conditions. These data showed that the β-G@Cu(PABA) biocomposite extended the bioactive pH range of β-G compared to the free β-G. Further, the β-G@Cu(PABA) biocomposite against high temperature for 2 h was also investigated to assess its thermal stability (Figure 7b). Even at higher temperatures of 60 °C, the β-G@Cu(PABA) biocomposite could maintain more than 80% of activity. In contrast, the free β-G lost over 60% of activity at 60 °C. The enhanced thermal stability of β-G@Cu(PABA) biocomposite indicated that β-G@Cu(PABA) biocomposite could be applied over a wider temperature range amongst enzymatic reactions. The XRD pattern of β-G@Cu(PABA) after the 60 °C reaction, in Figure 8, showed the preservation of the Cu(PABA) framework. To examine the tolerance of polar solvents including acetonitrile(AN), methanol(MeOH), 1,4-dioxane(1,4-Diox), ethyl acetate(EAC), and ethanol(EtOH), the β-G@Cu(PABA) biocomposite were incubated in these polar solvents for 2 h (see Figure S9). The β-G@Cu(PABA) biocomposite almost did not deactivate under all above polar solvents, while a approximately 40% and 70% drop in activity of native β-G was observed on different solvents, respectively. These results clearly indicated that the Cu(PABA) coating was able to protect β-G from organic solvents attack. Long-term storage stability of β-G@Cu(PABA) biocomposite was also performed after storage at 4 °C, and then examined its residual enzyme activity (Figure 7c). As expected, the β-G@Cu(PABA) biocomposite retained nearly 90% of its initial activity for 30 days, while the native β-G quickly lost the activity and nearly completely lost its activity for 30 days. The excellent durability enabled β-G@Cu(PABA) biocomposite with great potential for industrial applications. The XRD pattern of β-G@Cu(PABA) after storage at 4 °C for 40 days, in Figure 8, showed the preservation of the Cu(PABA) framework.



The recyclability of the β-G@Cu(PABA) biocomposite was evaluated as displayed in Figure 7d. The activity of β-G@Cu(PABA) biocomposite was decreased only 10% after recycling 10 times. The loss of activity was mainly caused by long-time use of the biocomposite and active site clog of β-G. In addition, Cu(PABA) shell made the biocomposite easier to reuse by centrifugation after each cycle. All the above-mentioned results demonstrated the feasibility of extending application range of β-G, and dramatically enhancing the stability of β-G by using the Cu(PABA) shell as the protective layer.

Figure 7. (a) Incubation pH stability of β-G and β-G@Cu(PABA). (b) Incubation temperature stability of β-G and β-G@Cu(PABA). (c) Storage stability of β-G and β-G@Cu(PABA). (d) Cycle stability of β-G and β-G@Cu(PABA). The error bars represent the standard deviations from triplicate experiments.


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Figure 8. XRD patterns of β-G@Cu(PABA) biocomposite after the 60 °C reaction (biue) and after storage at 4 °C for 40 days (red). Hydrolysis

of

Cellulose

by

Co-immobilized

Cellulase

and

Additional

β-G@Cu(PABA) Biocomposite In

order

to

enhance

the

glucose

yield

for

the

hydrolysis

of

carboxymethylcellulose (CMC), β-G@Cu(PABA) biocomposite was supplemented into co-immobilized cellulase system and the mixture was used as a catalyst due to a relatively low amount of β-G secreted by Trichoderma viride. As shown in Figure 9a, hydrolysis for 8 h by using co-immobilized cellulose alone gave glucose in 48% yield, and by the mixture of β-G@Cu(PABA) biocomposite and co-immobilized cellulase achieved 68% glucose yield at the same time. The addition of β-G@Cu(PABA) biocomposite to the catalytic system can improve the glucose yield. Thus, different quantity of β-G@Cu(PABA) biocomposite was then added to co-immobilized cellulase system to investigate the glucose yield. The mixture afforded 68% glucose yield in a ratio of 1:4, as well as improved glucose yield to 98% in a ratio of 1:1. The significantly increased yields of glucose by co-immobilized cellulase and β-G@Cu(PABA)

biocomposite

was

benefitted

from

the

β-G@Cu(PABA)

biocomposite effectively reduced the inhibition of cellobiose on catalytic activity of EG and CBH. Thus, addition of β-G@Cu(PABA) biocomposite to the co-immobilized cellulase system could favour to enhance productivity for industrial applications. The reaction rate for the mixture of co-immobilized cellulase and β-G@Cu(PABA) biocomposite was slightly higher than for free cellulose and β-G,



which was mainly caused by a higher substrate affinity of β-G@Cu(PABA) biocomposite (Figure 9b). The result was in good agreement with the data of Km obtained from Michaelis-Menten kinetics analysis which had been discussed previously. The mixture of co-immobilized cellulase and β-G@Cu(PABA) biocomposite also displayed an increase in the productivity which could be explained due to the enhanced stability of β-G@Cu(PABA) biocomposite during the hydrolysis process of CMC to glucose.

Figure 9. (a) Time courses for the hydrolysis of CMC with different ratios of β-G@Cu(PABA) and PMAAc-cellulase. (b) Time courses for the hydrolysis of CMC with free and immobilized enzymes. The error bars represent the standard deviations from triplicate experiments. The excellent storage stability of co-immobilized cellulase and β-G@Cu(PABA) mixture system was confirmed by the hydrolysis of CMC. Expectedly, 83 % glucose yield was achieved after storage of co-immobilized cellulase and β-G@Cu(PABA) mixture for 25 days (Figure 11a). Free cellulose and β-G produced glucose in a 22% yield after 25 days of storage. The continuous hydrolysis of CMC with the mixture of co-immobilized cellulase and β-G@Cu(PABA) biocomposite was carried out. This simulation of cyclic regeneration process was demonstrated in Figure 10. The productivity of co-immobilized cellulase and β-G@Cu(PABA) biocomposite began to decrease after five cycles due to the loss of activity and sample (Figure 11b). However, this biocatalyst mixture still maintained over 70% productivity in the eight cycles, demonstrating that the biocomposite could be efficiently recovered and reused. Thus,


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the recyclability of the biocomposite was potentially beneficial for improving the efficiency of the enzymatic preparation of glucose and reducing the production cost.

Figure 10. (a) The hydrolysis cycle of CMC with co-immobilized cellulase and β-G@Cu(PABA) biocomposite. (b) DNS determination of glucose yields.

Figure 11. (a) Storage stability and (b) Reusability of β-G@Cu(PABA) and PMAAc-cellulase for the hydrolysis of CMC. The error bars represent the standard deviations from triplicate experiments.

Conclusions In conclusion, we have reported for the first time the development of Cu-MOF protective coatings for β-G encapsulation using a co-precipitation approach in aqueous medium, which exhibited relatively high encapsulation capacity of 163 mg/g.



The Cu-MOF was stable under acidic condition, which was essential for the stabilization and activity performance of β-G@Cu(PABA) biocomposite. Our results showed that the β-G@Cu(PABA) biocomposite extended the bioactive pH and temperature range of β-G, and exhibited better pH, thermal and storage stability compared to the free β-G. This good performance of the β-G@Cu(PABA) biocomposite made it possible to serve as a external supplementation to improve the yield of glucose by the hydrolysis of cellulose. The synthesized β-G@Cu(PABA) biocomposite illustrated a slightly higher rate, as well as excellent reusability (73% of its initial activity) after eight cycles of hydrolysis of cellulose. Accordingly, employing Cu-MOF as a coating to protect the enzymes under acidic conditions provides a new promising tool for the further exploitation of biocomposites for industrial applications. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxxx/acsami.xxxxxxx. Photograph and weight of Cu(PABA) and ZIF-8 dissolved in the solutions of pH 5.0 and pH 7.0, EDS, XRD patterns, TEM, N2 adsorption–desorption curves and pore diameter distributions of Cu(PABA), and Lineweaver-Burk plots; effect of protein concentration on relative activity and encapsulation capacity of β-G@Cu(PABA) biocomposite, effects of organic solvent on the activity of free β-G and β-G@Cu(PABA), effects of different concentrations of metal ions and organic acids on the retaining activity and encapsulation efficiency of immobilized enzyme, effects of metal ions on the activity of free β-G, and Kinetic parameters of free β-G and β-G@Cu(PABA).


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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Present Addresses School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu Province, China Notes The authors declare no competing financial interest.

Acknowledgements This work is supported from the National Natural Science Foundation of China (NO. 21507047 and 21676124) and the Programs of Senior Talent Foundation of Jiangsu University (NO.14JDG053).

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For Table of Contents Use Only

Synopsis: β-G was encapsulated by acid-resistant Cu(PABA), which showed superior stability and reusability for the hydrolysis of CMC to glucose.


Page 28 of 28

Recyclable Î²-Glucosidase by One-Pot Encapsulation with Cu-MOFs

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