The Microwave-assisted Synthesis of Polyethersulfone ( PES ) as A Matrix in Immobilization of Candida antarctica Lipase B ( Cal-B )

Candida antarctica lipase B (Cal-B) has been widely used in the hydrolysis reaction. However, it has some weaknesses, such as: forming of the heavy emulsion during the process, which is difficult to resolve and has no reusability. Therefore, it needs to be immobilized into a suitable matrix. One of the suitable supporting materials is polyethersulfone (PES) and its synthesis becames the objective of this paper. The PES was synthesized via a polycondensation reaction between hydroquinone and 4,4'dichlorodiphenylsulfonein N-methylpyrrolidone (NMP) as solvent using Microwave Assisted Organic Synthesis (MAOS) method at170 °C for 66 minutes using an irradiation power of 300 watt. The synthesized PES was characterized by FTIR and 1H-NMR (500 MHz, DMSO-d6). Then the PES membrane was prepared from 20% of the optimized mixtures of PES, PSf (polysulfone), and PEG (polyethylene glycol) dissolved in 80% NMP. The Cal-B was immobilized on the PES membrane by mixing it in a shaker at 30 °C and 100 rpm for 24 h using phosphate buffered saline (PBS). The identification of the immobilized Cal-B was done by using FTIR-ATR spectroscopy and SEM micrographs. The results of Lowry assay showed that the ‘Cal-B immobilized’ blended-membrane has a loading capacity of 91 mg/cm2 in a membrane surface area of 17.34 cm2. In this work, the activity of immobilized Cal-B was twice higher than the native enzyme in p-NP (p-Nitrophenolpalmitate) hydrolyzing. The results indicated that the synthesized PES showed a good performance when used as a matrix in the immobilization of Cal-B. Copyright © 2017 BCREC Group. All rights reserved


Introduction
Candida antarctica Lipase B (Cal-B) has a superiority in industrial scale.It was used as a biocatalyst and shows a high degree of selectivity during the esterification and transesterification reactions as in the hydrolysis of triglyceride to form fatty acids and glycerol [1][2].However, the weakness of lipase catalysis is the forming of heavy emulsion during the process which is difficult to resolve and has no reusability [3].Therefore, the immobilization technique can be used to improve lipase utilization and reusability by maintaining its catalytic effectiveness.In addition, the materials to be used as matrix for the lipase immobilization should have good mechanical characteristics.Polymeric membrane is one of the materials used as matrix for immobilized system [4].Polyethersulfone (PES) is one of the synthetic polymers which can be used as ultrafiltration membrane and other functional membranes [5].PES have several good characteristics such as high resistance to heat and chemicals, and have good mechanical strength [6].So, the PES has been used as a matrix in many immobilization systems for Candida rugosa, Comamonas testostroni, Bovine Serum Albumin (BSA), heparin, and TiO2 [11][12][13][14][15]. Furthermore, the synthesized PES derivative was used as one of the components in the matrix used for the Cal-B immobilization and showed good results in Cal-B catalysis activity and has better performance compared to previous studies [21][22][23][24][25].
Many methods have been developed to synthesize PES.Some studies reported that the PES was synthesized by using conventional method, i.e. refluxing the reaction mixtures for three hours in various conditions [7,11,16].Since the optimum condition in the synthesis of PES is crucial to obtain better yields of product in shorter reaction time, the modification of experimental condition for synthesizing PES became the main objective of this research.The synthesized PES was then used as a matrix for Cal-B immobilization.

Synthesis of PES
In this procedure, 2.78 g of DCDPS and 4.14 g of K2CO3 were placed in a three-necked flask and mixed with 30 mL of toluene and 20 mL of NMP.The flask equipped with a condenser was placed in a MAS-II Sineo® microwave oven reactor, and then subsequently a Dean-Stark trap apparatus was attached to the condenser.Afterwards, the hydroquinone was added gradually into the reaction mixtures and the reaction was carried out in the microwave cavity using an irradiation power of 300 Watt at 170 o C for 66 minutes with continuous nitrogen gas purging.After the completion of reaction, the hot green thick solution was directly precipitated in a mixture of water and methanol (1:1 in volume).The precipitated product was washed in hot water, then in methanol and ethanol.Finally, it was dried at 70 °C under vacuum condition.

Preparation of blended-PES membrane
A mixture of PES and polysulfone with a total polymer concentration of 18% was blend with a 2% of PEG and dissolved in 80% of NMP.The blending process was carried out in a glass vial by stirring at room temperature for 4 days.The casting solution was poured onto a 10×20 cm glass plate and casted to form a thin layer approximately 2 mm in thickness.After that, the glass plate was immersed into a demineralized water bath at room temperature to form a thin layer membrane.The membrane was further dried at room temperature for 24 hours.

Candida antarctica Lipase B immobilization
The Cal-B solution was prepared by dissolving 50 mg Cal-B in a 1 mg/mL phosphate buffer solution (PBS) at pH 7.0.The membrane with a surface area of 4.7 cm 2 was placed in a 50 mL beaker glass, and the 5 mL Cal-B solu-tion was added at 30 o C and agitated at 100 rpm for 24 h.The membrane was removed from the solution, subsequently washed with demineralized water.The solution and washing solution was kept in the beaker glass for enzyme loading determination.The membrane was dried at room temperature for approximately 3 hours to be further analyzed (FTIR-ATR and SEM).

Enzyme loading determination
Determination of enzyme loading was done by Lowry assay protocol [17].A complex reagent consisted of 3 components was prepared prior to analysis.The A Lowry component was a solution with a concentration of 2% (w/v) Na2CO3 and 0.6% (w/v) NaOH, while the B solution was 2% (w/v) CuSO4.2H2O in demineralized water and the C solution was 2% (w/v) sodium-potassium tartrate in demineralized water.Then the three components were mixed in a volume ratio of 100:1:1.The BSA standard solutions were prepared with a variation of 0, 2, 4, 6, 8, and 10 mg/L.One mL of standard, or sample solution or washing solution was added to 1 mL of 2 N NaOH solution, and subsequently hydrolyzed at 100 °C.The hydrolyzed samples or standard solutions were allowed to cool to room temperature, and then added by 5 mL of complexing reagent.Folin ciocalteu reagent was added to each solution after settling for 10 minutes, and then subsequently placed in the dark room for 60 minutes.Afterwards, the absorbance of each solution was measured at the lmax (660 nm) using UV-vis spectrophotometer.
The amount of enzyme loading was calculated as: (1) where, Mil is the amount of immobilized lipase on PES membrane surface (mg), Mls is the lipase stock mass (mg), Mul is the lipase mass (mg), and Lm is the membrane surface area (cm 2 ).The mass of lipase stock and lipase were calculated from the absorbance of protein by Lambert-Beer equation.

Lipase activity determination
Immobilized lipase activity was determined based on Cal-B and immobilized Cal-B ability to catalyze the hydrolysis reaction of p-NPP as the substrate.The stock of substrate solutions were prepared by mixing 47.5 mL PBS (1 M, pH 7), 2.0 mL ethanol (absolute grade), and 0.5 mL acetonitrile containing 2.0 mg p-NPP.The Cal-B solution was prepared by dissolving 50 mg Cal-B in 1 mg/mL buffer phosphate solution (PBS) at pH 7.0.An amount of 900 mL substrate solution was added into each of 300 mL Cal-B solution and immobilized Cal-B membranes.They were gently stirred and then incubated for 15 minutes in a shaker at 30 °C and 100 rpm.The immobilized Cal-B membrane and its solution were separated by decantation technique and Cal-B solution was used as a control.
The activities of Cal-B immobilized were investigated by determining the absorbance of pnitrophenol (p-NP) as the hydrolyzed product of p-NPP.The concentration of p-NP as the product was determined by UV/vis spectrophotometry analysis using lmax of 420 nm.In this study, Cal-B was immobilized onto PES membrane, therefore the unit activity of the immobilized Cal-B was defined as mmol p-NP/cm 2 /min.The activity of Cal-B and immobilized Cal-B membrane were calculated by a formula: (2) where, CpNP is the concentration of released p-NP, which is taken from the hydrolysis product's absorbance and 15 is the reaction time which had been used.

FTIR-ATR measurement
Attenuated Total Reflectance FTIR-ATR method was used to investigate the alteration of membrane structure caused by the immobilization process.The FTIR-ATR measurements of 'Cal-B immobilized' membrane and PES blended membrane were performed at room temperature.The membrane samples were puton Zinc Selenide (ZnSe) glass in close contact.The ATR-FTIR spectrum was taken from 400 to 4000 cm -1 .

SEM measurement
The surfaces of Cal-B immobilized in PESblended membranes were observed by SEM using an accelerating voltage of 10 kV and 1000x magnification.

Synthesis of PES
The PES was synthesized according to polycondensation reaction.Polycondensation reaction was carried out through the reaction be- and hydroquinone that having two active groups, which are -Cl and -OH groups, respectively [19].Water molecules as side product of this reaction was eliminated by adding toluene in azeotrope condition and the reaction was performed using reactor equipped with Dean-Stark trap apparatus to remove water from the system.Keitoku et al. [18] and Handayani et al. [17] have synthesized PES by conventional methods, which needed three hours reflux due to slow heat transfer process.MAOS methods using oven microwave oven reactor and a Dean-Stark trap apparatus attachment is the renewable of this research.It has an efficient direct thermal heating which needed only 66 minutes using a power of 300 W. This method gave the advantages from the point of view of using quite low frequency to generate dipole movement of reaction mixtures directly [17][18]20].Dielectric heating process of microwaveassisted reaction was derived from NMP as solvent used in this reaction which has a dielectric constant (e) of 34.22 [20].Dielectric heating process was also supported by the polarizability structure of hydroquinone and DCDPS having a total dielectric constant (e) of 27.72, which promoted the activation of depolarization process of molecules rotation directly and increased the kinetic energy to create molecular collision of the reactants [17][18][19][20].Hence, MAOS method is faster than conventional method in performing reactions including the polymerization reaction [20].

Characterization of PES polymer by FTIR and 1 H-NMR
The FTIR analysis of the synthesized product (PES) was performed to identify the new functional group peaks indicating the formation of new bonds in the polymer.The FTIR spectrum of the synthesized PES was presented in Figure 1.The FTIR spectrum of the synthesized PES showed the characteristic peaks of PES at 1234.44 cm -1 and 1103.28 cm -1 corresponded to the C-O-C bonds; while peaks at 1184.29 cm -1 and 1149.57cm -1 represented the symmetric bond vibration of sulfonate (-SO2-) groups and peaks at 1010.70 cm -1 showed the aromatic vibrations [23][24].The elucidation structure of the synthesized PES was performed using 1 H-NMR measurement and its spectrum was presented in Figure 2. The 1 H-NMR spectrum of the synthesized PES (500 MHz, DMSO-d6) showed aromatic proton signals at chemical shift of 7.98 ppm (ortho towards -SO2-groups, meta towards -Obonds); 7.99 ppm (ortho towards -SO2-); 7.25 ppm (proton of aryl ether); 7.18 ppm (meta tow a r d s e n d g r o u p s o f 4 , 4 'dichlorodiphenylsulfone residue); and 7.16 ppm (ortho towards -O-bonds).Thus, based on the analyzed 1 H-spectrum, it was confirmed that PES have been successfully synthesized using MAOS method [23,25].

Immobilized lipase
The characterization of 'Cal-B immobilized' PES-blended membrane was carried out by ATR-FTIR method to investigate the interaction between PES membranes and Cal-B.The ATR-FTIR spectrum of 'Cal-B immobilized' PES blended membrane and PES membrane were shown in Figure 3.After immobilization, a new peak associated to the stretching vibrations of -N-H and -OH groups appeared at 3196.5 cm -1 .The spectrum of the 'Cal-B immobilized' membrane also indicated the increase of peak's transmittance referred to the -NH deformation at 1654.31 cm -1 , while the increase of transmittance at 1482.64 cm -1 corresponded to -CH2 deformation [23].So, the presence of aforementioned characteristic peaks showed intermolecular interactions occurred between the Cal-B and the PES/polysulfone-blended membrane.

SEM imaging
Figure 4 showed the morphology of the original PES/polysulfone-blended membrane and the 'Cal-immobilized' one.The PES mem-brane showed some particulates with a diameter range of 0.425-0.876mm.
Figure 4(b) showed that white agglomerated granules of Cal-B were adsorbed on the membrane surface.The gravity force allowed the enzyme to fill the vacancies of membrane pores [27,28].Chauhan et al., Gupta et al., and Wang et al. have done lipase immobilized onto several kinds of membranes, and they found that the membrane surfaces had some beads in clusters [24][25][26].Regarding the differences of SEM images shown in Figure 4, it can be concluded that lipase was successfully immobilized into the membrane [30].

Enzyme loading
The determination of enzyme loading has been carried out by Lowry assay with BSA standard.The results of Lowry assay showed  that the 'Cal-B immobilized' blendedmembrane has the loading capacity of 91 mg/cm 2 in 17.34 cm 2 of membrane surface area.The enzyme loading represented the quantity of Cal-B attached on the matrix (PES / polysulfone-blended membrane) by physisorption or covalent interaction [31].The negatively charged groups of PES might interact with electropositive groups of amino acid residues from the active binding sites of Cal-B.The possible intermolecular interactions between the Cal-B and PES/polysulfone-blended membrane are considered weak to moderate interactions, such as hydrogen bonding, van der Waals, London dispersion, and electrostatic interactions.Jesionowski et al. [32] and Feng et al. [33] explained that the moderate intermolecular interaction between enzyme and matrix is the most important factor of the immobilization system because it would minimize the disturbance of the active site of enzyme.Therefore, the enzyme would still have the catalytic capability [29,30].In this study, the physical intermolecular interactions between Cal-B lipase and the blended-membrane was proved to have a minimum disturbance on the activity of enzyme.

Lipase activity
The enzyme activities of 'Cal-B immobilized' blended-membrane were investigated by determining the absorbance of p-Nitrophenol (p-NP) based on the the release of p-NP hydrolyzed from p-NPP with the free enzyme as a control.The specific enzyme activity of the immobilized lipase membrane was found to be 80.43 mmol p-NP min -1 cm 2 , which was up to 2.5 times higher than the native enzyme (33.79 mmol p-NP min -1 cm 2 ).This result indicated that the synthesized PES showed good performance when used as matrix (blendedmembrane) in the immobilization of Cal-B, because the immobilized lipase was found to have a greater enzyme activity compared to its free form.

Conclusions
PES was synthesized by MAOS method and confirmed by the results of FTIR and 1H-NMR spectra analysis.The blended PES-polysulfone membrane was also prepared and confirmed structurally by ATR-FTIR spectra and its SEM image.The 'Cal-B immobilized' blendedmembrane was successfully prepared according to the analysis of FTIR-ATR spectra and its SEM image.The results of Lowry assay showed that the 'Cal-B immobilized' blendedmembrane had a loading capacity of 91 mg/cm 2 in 17.34 cm 2 of membrane surface area.The specific enzyme activity of the immobilized lipase membrane was found to be 80.43 mmol p-NP min -1 cm 2 , which was more than twice higher than the native enzyme (33.79 mmol p-NP min -1 cm 2 ).The results indicated that the synthesized PES showed good performance when used as matrix in the immobilization of Cal-B.

Figure 1 .Figure 2 .
Figure 1.The FTIR spectrum of PES synthesized by MAOS method