Surface functionalization of graphene oxide by amino acids for Thermomyces lanuginosus lipase adsorption

Journal of Colloid and Interface Science 546 (2019) 211–220

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science journal homepage: www.elsevier.com/locate/jcis

Regular Article

Surface functionalization of graphene oxide by amino acids for Thermomyces lanuginosus lipase adsorption Wenfeng Zhou a,b, Wei Zhuang a,b,⇑, Lei Ge c, Zhenfu Wang a,b, Jinglan Wu a,b, Huanqing Niu a,b, Dong Liu a,b, Chenjie Zhu a,b, Yong Chen a,b, Hanjie Ying a,b,⇑ a

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing 210009, China College of Biotechnology and Pharmaceutical Engineering, National Engineering Technique Research Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China c Centre for Future Materials, University of Southern Queensland, Springfield, Queensland 4300, Australia b

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 25 December 2018 Revised 18 March 2019 Accepted 20 March 2019 Available online 21 March 2019 Keywords: Lipase adsorption Graphene oxide Surface modification Freundlich isotherm model Amino acids

a b s t r a c t Graphene oxide (GO) with oxygen containing functional groups can be selectively modified by small biomolecules to achieve heterogeneous surface properties. To achieve a hyper-enzymatic activity, the surface functionality of GO should be tailored to the orientation adsorption of the Thermomyces lanuginosus (TL) lipase, and the active center can be covered by a relatively hydrophobic helical lid for protection. In this work, amino acids were used to interact with GO through reduction reaction, hydrophobic forces, electrostatic forces, or hydrogen bonding to alter the surface hydrophobicity and charge density. Characterization of the structure and surface properties confirmed that the GO samples decorated with phenylalanine (Phe) and glutamic acid (Glu) exhibited superior hydrophobicity than other modifications, whereas tryptophan (Trp) and cysteine (Cys) provided weaker reduction effects on GO. Moreover, the zeta potential of the samples modified by amino acids of lysine (Lys) and arginine (Arg) is higher than other modified samples. The adsorption amount of lipase on Glu-GO reached 172 mg/g and the relative enzymatic activity reached up to 200%. The thermodynamic data and the Freundlich isotherm model fitting showed that the lipase adsorption process on modified samples was spontaneous, endothermic and entropy increase. Ó 2019 Elsevier Inc. All rights reserved.

1. Introduction ⇑ Corresponding authors at: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 5, Xinmofan Road, Nanjing 210009, China. E-mail addresses: [email protected] (W. Zhuang), yinghanjie@njtech. edu.cn (H. Ying). https://doi.org/10.1016/j.jcis.2019.03.066 0021-9797/Ó 2019 Elsevier Inc. All rights reserved.

Graphene oxide (GO) is a derivative of graphene, which is prepared by multi-step oxidation, ultrasonic purification and the like methods from graphite [1]. With excellent large surface area,

212

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

stable nature, and tunable surface oxygen functionalities, GO has many applications in biocatalysis, biomedicine, catalysis, and composite materials [2]. In addition, in recent years, reduced GO has also been extensively studied in electrode materials. For example, Nath et al. [3] added carboxyl groups at the edge of GO, Parveen et al. [4] synthesized composites of GO and manganese dioxide, and Parveen et al. [5] anchored b-Ni(OH) onto GO. All these cases indicate that GO has the prospect of becoming a promising electrode material for energy storage applications. Studies on GO with planar structure and non-uniform distribution of functional groups as enzyme-immobilized carriers have been reported, displaying increased enzyme loading and reduced mass transfer resistance. Liu et al. [6] immobilized pectinase on GO, which effectively increased the enzyme loading rate and catalytic activity. Likewise, Gu et al. [7] immobilized papain on GO significantly improving the catalytic performance and thermal stability of the enzyme. Moreover, experiments by Hermanova et al. [8] have shown that the immobilization of lipase, from the rice root enzyme, on GO enhanced its organic solvent tolerance. However, the surface of GO contains different types of oxygen-containing functional groups (hydroxyl, epoxy, carbonyl, carboxyl, etc.)[2] and has strong hydrophilicity and a large amount of negative charge, repulsing the adsorption ofnegatively charged enzymes. Therefore, the regulation of GO surface functionality is necessary to improve the catalytic activity of the immobilized enzyme. Zhuang et al. [9] modified GO with PEGNH2 to make the catalytic activity of immobilized nuclease on functionalized GO higher than that of pristine GO. Vajihe Mehnati-Najafabadia et al. [10] used polyethylene glycol bisamine (PEGA) to modify GO in order to improve the catalytic performance of xylanase. Mathesh et al. [11] used ascorbic acid to reduce GO for enzyme immobilization, which showed enzymatic activity enhancement. The reducing agents commonly used in chemical reduction methods are hydrazine hydrate [12,13], pure hydrazine [14,15], benzenediol [16], sodium borohydride [17], metal hydrides [18,19] and similar ones. However, the above reducing agents are usually corrosive or toxic, and the reduction process is relatively intense and difficult to control. In contrast, amino acids can be used as a simple, green, controlled reducing agent to prepare RGO. There is related literature explaining that amino acids and GO are mainly controlled by electrostatic forces [20], hydrophobic forces [21], and hydrogen bonding [22]. Studies by Mallineni et al. [23] have either shown that amino acids are primarily adsorbed onto GO surface by electrostatic and p-p interactions or that amino acids cause a shift in the surface charge of GO. Furthermore, Tran et al. [24] demonstrated that non-aromatic and thiol-free amino acids are a green reducing agent used to prepare RGOs, and that amino acids mainly act on carboxyl groups, carbonyl groups, epoxy groups, etc. [24] on the surface of GO via a mild and controllable reduction process. In addition, Chen et al. [25] prepared RGO nanosheets by using L-Cys as a reducing agent, increasing the electrical conductivity of GO and introducing only non-toxic substances. Nonetheless, these works have not systematically studied the interaction between amino acids and GO and have not linked the heterogeneity of the carrier to the heterogeneity of lipase. Thus, controlling the surface heterogeneity of GO by means of amino acids reduction is very promising to prepare a carrier suitable for lipase immobilization. Amino acids can be classified into non-polar aliphatic amino acids, uncharged polar amino acids, aromatic amino acids, and charged polar amino acids. The uncharged polar amino acids (cysteine (Cys)), charged polar amino acids (glutamate (Glu), arginine (Arg), lysine (Lys)) and aromatic amino acids (tryptophan (Try), phenylalanine (Phe)) have been selected as GO reducing agents.

Different amino acids have different polar and charge properties, not only induce surface hydrophobicity to GO due to the carried hydrophobic tail groups, but also regulate the oxygen-containing functional groups on the surface of the GO by partial reduction [25] or adsorption [26]. Moreover, the essence of an amino acid molecule is its constituent unit of protein, which is also an essential substance for the living organisms, so it is safe, environmentally friendly, green and healthy. Thermomyces lanuginosus Lipase (TL lipase or LIP) is an alkaline thermostable lipase produced from Thermomyces lanuginosus. TL lipase exists in fat-containing tissues of animals, plants, and microorganisms (such as molds, bacteria, etc.). TL lipase can catalyze the hydrolysis of glyceride compounds, peptide synthesis, biosurfactant synthesis, and transesterification [27,28]. These characteristics make lipases widely used in food, cosmetics, agriculture, papermaking and other fields. TL lipases have the advantages of possessing high catalytic efficiency, high selectivity, and mild reactivity [29,30]. However, free lipase is not suitable for industrial use, but this can be achieved by means immobilization [31]. Immobilized lipase facilitates the separation of subsequent products and can enhance the catalytic activity and stability of lipases. The active center of TL lipase has a helical fragment (the lid) [11],which has suitable interfacial activation for immobilization. The lid is a triplet composed of three amino acids (serine-histidine-aspartate). The outer surface of the lid is relatively hydrophilic and the inner surface facing the catalytic site is relatively hydrophobic. Since the lipase has a nucleophilic inner core and a hydrophilic shell, it prohibits the substrate from accessing the basic active center in the closed configuration. When the lipase moves to the water-oil interface, the helical lid opens and the active center is exposed to the reactant for catalytic reactions. Based on the lipase structure, the combination of the lipase and the hydrophobic carrier is beneficial to enhance the half-life of enzyme, the catalytic activity and the stability of the enzyme, and the like. Various hydrophobic carriers, such as ionic liquids [32] and macroporous adsorptive resins [33], have been used to immobilize lipases and show better reusability compared to free enzymes. Therefore, the selection of a suitable carrier to achieve super-enzyme activity and recyclability is crucial to its practical catalytic application. Large surface area and a large number of functional groups are characteristics that make GO very suitable for the adsorption of lipase and the improvement of catalytic activity. The enzyme immobilization methods include [34] adsorption, covalent binding, entrapment, microencapsulation, and crosslinking. Among them, physical adsorption is more suitable for the immobilization of lipase on GO [8] due to the process simplicity and the excessive activation of enzymes. The main interaction force between lipase and GO is hydrophobic and electrostatic interactions. Therefore, the different charge properties and hydrophobicity of the carrier will affect the catalytic performance of lipase [35,36]. This study investigated the interaction of amino acids with GO and the effect of GO surface properties on lipase adsorption. We compared the reduction of GO by six different amino acids and evaluated the change of surface properties of GO by chemical reduction and electrostatic adsorption. RGO forms a certain degree of difference in charge properties and hydrophobic properties, mainly manifested by amino acid adsorption and difference in functional groups. We used different amino acid functionalized GOs for lipase adsorption, simulated the adsorption results and investigated the adsorption characteristics. The utilization of the subsequent GO to immobilize lipase may have a great influence on the synthesis process of 1,3-Dioleoyl-2-palmitoylglycerol (OPO).

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

2. Materials & methods

bilized enzyme(mg) on 1 g of support. The loading of the enzyme (AL (mg/g)) after washing was calculated based on Eq. (1):

2.1. Materials All chemicals were analytical grade. Thermomyces lanuginosus (TL lipase, =100,000 U/g) was purchased from Sigma-Aldrich (St. Louis, MO, USA). P-nitrophenyl palmitate(pNPP) and pnitrophenol(pNP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). GO powder (=99.95%) and amino acids(=98%) were purchased from Aladdin Industrial Corporation (USA). Sodium hydroxide (NaOH, AR), disodium hydrogen phosphate dodecahydrate (Na2HPO4
12H2O, AR) and sodium dihydrogen phosphate trihydrate (NaH2PO4
3H2O, AR) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd (Shanghai, China). Coomassie Brilliant Blue G250 (AR) was purchased from Macleans (Shanghai, China). Anhydrous ethanol was purchased from Yasheng Chemical Co., Ltd (Wuxi, China). Posphoric acid and isopropanol were supplied by the Sinpharm Chemical Regent Co.,Ltd (Shanghai, China). Bovine serum albumin(BSA, =98%) was purchased from Shanghai Jinsui Bio-Technology Co., Ltd (Shanghai, China). Triton X-100 were purchased from BBI Life Sciences Co., Ltd. (Shanghai, China). The solvents were purchased from Maclean. All of the aqueous solutions were prepared using deionized distilled water. 2.2. Characterization The surface functionalities of supports and immobilized enzyme were recorded by Fourier transform infrared (FTIR) spectroscopy using a FTIR spectrometer (Thermo Fisher Scientific, Nicolet-460, USA) in the range of 4000–500 cm1. The elemental mapping of the products was investigated by field emission scanning electron microscopy (99FESEM) instrument (ZEISS, SUPRA 40, Germany). The activity of the immobilized enzyme and the concentration of enzyme solution were assayed spectrophotometrically using UV–Vis spectrophotometer (AEO, A560, China). The elemental analysis of GO was provided by X-ray photoelectron spectroscopy ((Thermo Fisher Scientific, Escalab 250Xi, USA). Zeta potential analyzer (Malvern, ZS990, U.K) was used for determination of the Zeta potential of the carriers. 2.3. Preparation of functional carriers GO was synthesized by modified Hummers method from graphite power as described in our previous work [9]. A suitable amount of GO powder was taken, and pure water was added to prepare the solution of 0.5 mg/mL coarse GO dispersion. After centrifugation at 3000 rmp for 5 min, the supernatant was taken as stable graphene oxide dispersion. 40 mg of the stabilized GO dispersion were added to 40 mg of the amino acid and reacted at 25 °C for 12 h. The mixture was centrifuged for 15 min at 10000 rpm and washed three times with phosphate buffer saline (PBS) to remove the excess of amino acids. The resulting sample was freeze-dried as our carrier. 2.4. Preparation of immobilized enzymes In a typical immobilization experiment, after 10 min sonication of a mixture containing support(5 mg) and 5 mL PBS(50 mM, pH 7.0), 2.5 mL TL Lipase were added to this dispersed mixture, shaken at 25 °C for 3 h . The suspension was then centrifuged for 15 min and washed three times with PBS in order to remove all nonimmobilized enzyme. Before and after enzyme immobilization, the amount of TL Lipase in the supernatant was measured by the Bradford method using bovine serum albumin as the standard [37,38]. The loading capacity was defined as the amount of immo-

213

AL ¼

Cinitial  Csupernatant msupport

ð1Þ

2.5. Assay of enzymatic activity The catalytic activity of the enzyme was measured in PBS by the ability to hydrolyze pNPP. The substrate solution was prepared from 10 mL of a pNPP isopropanol solution, adjusted to a volume of 500 mL with a PBS (50 mM, pH 7.0) containing 0.4 wt% of X100. After the substrate solution was incubated in a water bath at 50 °C for 2 min, the enzyme solution was added and maintained in a 50 °C water bath for 20 min. Finally, 1.0 mL of the reaction solution was diluted 5 times with pure water, and the ultraviolet absorption wavelength was measured at 410 nm. One unit of lipase activity (U) is defined as the amount of enzyme that catalyzes the hydrolysis of pNPP per minute to produce 1 lmol of pNP under the above conditions. 2.6. Determination of immobilization parameters Immobilized protein loading (qe, g/g of support) was calculated according to Eq. (2) [39]:

qe ¼

V  ðC 0  C e Þ m

ð2Þ

where V is the volume of enzyme solution (mL), C0 is the initial protein concentration (mg/mL), Ce is the protein concentration after immobilization (mg/mL) and m is the mass of support (g). The relative activity and the activity recovery are two terms to determine the success of enzyme immobilization [40]. The relative activity is the percentage of total immobilized enzyme activity from the free enzyme that was measured. The activity recovery (AR) was calculated by Eq. (3):

ARð%Þ ¼

OA  100% IA

ð3Þ

where OA and IA are the observed activity after enzyme immobilization and initial activity before enzyme immobilization(U/g of protein), respectively. Relative activity (RA) was calculated as Eq. (4):

RAð%Þ ¼

IE  100% FE

ð4Þ

where FE and IE are the enzymatic activity before and after immobilization, respectively ((U). 2.7. Effect of pH To assay the pH stability of free and immobilized TL Lipase at room temperature, the residual activity of TL Lipase was measured after 1 h incubation at a specified pH ranging from 5 to 10 at the temperature of 25 °C. 2.8. Effect of temperature The thermal stability of free and immobilized TL Lipase in PBS was assessed by incubating the lipase solution at any specified temperature ranging from 25 °C to 70 °C for 1 h at pH 7.0. The residual activity of TL Lipase was then assayed at room temperature after the heat stress, and taken as an index of thermal stability.

214

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

Intensity(a.u.)

(a) A

E

B

F

C

G

D 0

200

400

600

800

1200 0

1000

200

400

Binding Energy (eV)

600

800

1000

1200

Binding Energy (eV)

(b) C-O

Intensity(a.u.)

A

C-C/C=C

C=O

E

B

F

C

G

D

280

285

290

295

280

285

Binding Energy (eV)

290

295

Binding Energy (eV)

Fig. 1. The high resolution XPS spectra (a) and C 1s (b) peak for GO and RGO (A: GO, B: Cys-GO, C: Arg-GO, D: Lys-GO, E: Glu-GO, F: Phe-GO, G: Trp-GO).

Table 1 The relative content of each C-containing structure and the carbon-oxygen ratio of GO and RGO. Carriers

CAC/C@C (%)

CAO (%)

C@O (%)

C/O

GO Cys-GO Arg-GO Lys-GO Glu-GO Phe-GO Trp-GO

43.18 45.59 53.85 55.94 51.86 46.86 45.8

46.67 46.11 40.72 39.17 42.75 46.25 47.1

10.16 8.3 5.42 4.9 5.39 6.9 7.1

1.87093 1.98779 2.51688 2.54441 2.31597 2.11459 2.07298

2.9. Adsorption isotherms The two most commonly used adsorption models [41] were used to fit the adsorption process of lipase on functionalized GO. The Langmuir model is commonly used to describe the physical adsorption of monolayers [42,43], and it is assumed that the surface properties of the adsorbent are uniform and there is no interaction between adsorbed molecules. The Freundlich model has no assumptions and is an empirical equation [44]. The Freundlich model can also be used to describe multimolecular adsorption [45,46]. The equal ratio of solid-liquid ratio was used, and the carrier concentration was 1 mg/mL. Langmuir equation:

10.5 A D G

10.0

B E

C F

qe ¼

qmax K L C e 1 þ K LCe

ð5Þ

Freundlich equation:

pH

9.5

a s

9.0

1

qe ¼ K F C ne

where qe and Ce represent the same parameters as in equation (1), qmax the theoretically calculated maximum adsorption capacity (mg/g GO), KL is the adsorption equilibrium constant related to the affinity between the adsorbent and adsorbate, KF the Freundlich constant, an indicator of adsorption capacity, and 1/n an empirical constant related to the magnitude of the adsorption driving force

3.5 3.0 2.5 0

ð6Þ

2

4

6

8

10

12

Time (h) Fig. 2. pH change trend of GO and RGO (A: GO, B: Cys-GO, C: Arg-GO, D: Lys-GO, E: Glu-GO, F: Phe-GO, G: Trp-GO).

2.10. Thermodynamic studies Thermodynamic parameters in terms of free energy change (DG), enthalpy change (DH) and entropy change (DS) have been calculated respectively.

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

Gibbs free energy was determined according to Equation (7) [37]:

DG ¼ nRT

ð7Þ

where DG is the Gibbs free energy (kJ/mol), R is the universal gas constant (8.314  103 kJ/molK), T is the absolute temperature (298.15 K), and n is the exponential constant of Freundlich equation. The enthalpy change (DG) and entropy change (DS) as a function of temperature are expressed by Eqs. (8) and (9), respectively [47]. The results of DH and DS are obtained from the slope and the intercept of the plot of ln K0 against 1/T.

In

1 DH ¼ InK 0  Ce RT

ð8Þ

DH  DG T

ð9Þ

DS ¼

where R is the universal gas constant (8.314 J
mol1K1) and T is the solution temperature in Kelvin. K0 is the thermodynamic equilibrium constant for the adsorption process. The K0 was determined by plotting ln(Ce/qe) versus Ce and extrapolating to zero Ce as suggested by Khan and Singh (1987) [48]. 3. Results and discussion The XPS survey spectra of GO and RGO are shown in Fig. 1a. It can be seen from the Fig. 1a that the amino acid reduction changes the carbon-to-oxygen ratio, and induce N element. Among them, Lys and Arg reduced GO have higher carbon-oxygen ratio compared to other amino acids RGO. The presence of N in RGO indicates the adsorption of amino acid. This suggests that Lys and

215

Arg reduced GO (Lys-GO, Arg-GO) may have a strong hydrophobicity due to their high carbon to oxygen ratio. Cys-GO has low carbon to oxygen ratio and strong hydrophilicity, indicating that Cys has weaker reduction effect on GO. The C1s peak fittings of GO and RGO are shown in Fig. 1b. Detailed carbon species are summarized in Table 1. After the reaction with amino acids for 12 h, the functionalized groups of GO changed dramatically. The C@O bond of GO reduced by Lys, Arg and Glu was obvious, indicating that these three amino acids mainly act on the C@O bond of GO. Surface of GO enriches with oxygen-containing functional groups such as hydroxyl, carboxyl and epoxy groups. The dissociation of hydrogen ions on the hydroxyl and carboxyl groups renders GO acidic in aqueous solution. By adding a certain amount of amino acids, the amino acid itself will affect the pH value of the aqueous solution of GO. At the same time, the reduction of amino acids will consume the oxygen-containing functional groups on the surface of GO and change the pH of the solution. The pH change of the GO solution after the addition of the amino acid was monitored, and the results were shown in Fig. 2. After GO was dispersed in water, the pH was 3.17, and there was no significant change. It can be seen that adding Lys and Arg into GO solutions exhibit pH larger than 10, because they contain more amino groups. Then the pH sharply decreased after they reacted with GO. The pH decrease of the GO dispersion with the other amino acid addition is small, indicating that the interaction between them and GO is weak. Arg and Lys have higher isoelectric points, so the GO dispersion with Arg and Lys added has the highest initial pH. Glu has the lowest isoelectric point, so the initial pH is the lowest. The stability of the GO dispersion depends mainly on the surface electromotive potential. When the motor potential of GO surface does not produce a sufficiently large repulsive force, the

Fig. 3. SEM images of different resolutions of GO (a,b), Glu-GO (c,d) and Lys-GO (e,f).

216

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

suspension becomes unstable and causes aggregation. The surface of GO contains hydrophilic functional groups such as carboxyl group or hydroxyl group. When GO is dispersed in an aqueous solution, the carboxyl group ionizes hydrogen ion and the hydroxyl group also forms hydrated anion with the OH in an alkaline environment, both causing GO surface to be negatively charged. As shown in Fig. 4a, the absolute value of the zeta potential of amino acid functionalized GO is lower than that of the original GO, which is attributed to the reduction of oxygen-containing functional

-50

(a)

Zeta Potential (mV)

-40

-30

-20

-10

0

GO

(b)

O

s-G

Cy

O O O O O s-G e-G g-G p-G u-G Ly Ph Gl Tr Ar

80

Contact Angle (°)

60

40

20

0

GO

O O O O O O s-G s-G e-G g-G p-G u-G Ly Cy Gl Ph Ar Tr

Fig. 4. Zeta potential (a) and contact angle (b) of GO and RGO.

GO

transmittance (%)

A

OH

C=O

C-O

groups. Since Arg-GO and Lys-GO are in an alkaline environment, their absolute value of Zeta is larger than that of other amino acids. The morphologies of GO, Glu-GO, and Lys-GO are shown in Fig. 3. It indicates that the GO surface has a certain degree of wrinkles, and amino acid modification has little effect on its wrinkles. The pleated structure of GO is the result of the stacking of sheets, and the increase in pleats reduces the accessible specific surface area in contact with the protein and reduces the protein loading. As shown in Fig. 4b, for Glu-GO, Phe-GO, Trp-GO, and Cys-GO, the increment of contact angle can be observed, indicating the increase of GO surface hydrophobicity. Moreover, its contact angle increases as the ability to reduce amino acids increases. In contrast, the contact angles of Arg-GO and Lys-GO are reduced, suggesting the increase of surface hydrophilicity. As confirmed by XPS data (Fig. 1), the increase of carbon-oxygen ratio in Arg-GO and LysGO also proves the increased hydrophilicity, which may be due to amino acid adsorption. The FTIR spectra of amino-modified GO-LIP and RGO-LIP are depicted in Fig. 5. After amino acid treatment, the C@O peak and the CAO peak of GO are weakened, and the peaks of C@O and CAOAC of Arg-GO and Lys-GO are almost disappeared [25,49]. In addition, studies by Tran [24] and Chen [25] et al. have also shown that amino acids mainly react with epoxy groups, hydroxyl groups, carboxyl groups or the like on GO via an amino group or an R group, and form a double bond on GO. When the lipase is bound to the carrier by physical adsorption, electrostatic adsorption and hydrophobic interaction are the main forces, which are beneficial to the maintenance of the protein structure. Fig. 5 displayes the FITR spectra of lipase adsorbed GO. The characteristic peaks of lipase at 1646 cm1 and 1536 cm1 are assigned to the amide I band and the amide II band, respectively. When the lipase is adsorbed on GO or RGO, the peak intensity of the amide II band is obviously enhanced. Because our test uses reflective infrared, it only detects the surface of the material. For GO-LIP or RGO-LIP, there is a large amount of lipase adsorption on the surface, so the C@O and CAO bonds on the surface of the carrier are not obvious in the infrared spectrum of the figure below. On the other hand, after lipase adsorption, the hydroxyl peak is shifted from 3423 cm1 to 3376 cm1, suggesting the formation of hydrogen bonds [50]. The spectrum of lipase and GO is significantly different at the range of 800 cm1-1200 cm1, which is an absorption peak of the peptide group of the protein [51]. Both the hydrophobicity and the charge properties of the carrier are critical to the catalytic performance of the immobilized lipase. The effect of the carrier on the adsorption and catalytic properties of free lipase was also examined. As shown in Fig. 6, the pristine GO protein loading reached 124 mg/g at an initial protein concentra-

E C-N

OH

F

B

G

C

H

D II band I band

C-N

4000

3500

3000

2500

2000

1500 -1

Wavenumber (cm )

1000

500 4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Fig. 5. FTIR spectra of GO-LIP and RGO-LIP(A: GO-LIP, B: Cys-GO-LIP, C: Arg-GO-LIP, D: Lys-GO-LIP, E: Glu-GO-LIP, F: Phe-GO-LIP, G: Trp-GO-LIP, H:Free lipase).

217

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

tion of 5 mg/mL. The amount of lipase adsorbed on GO is mainly affected by the hydrophobicity and charge properties of the carrier. The enzyme loading of Arg-GO and Lys-GO is low (<94 mg/g), probably due to the extremely hydrophilic external surface of the carriers. However, the extremely strong hydrophilicity easily leads to the formation of a thicker water layer between the medium and the enzyme[52], and weakens the interaction between the enzyme and the medium. On the other hand, the more hydrophobic the carrier, the more difficult it is for the lipase to contact the carrier 200

300

1.0

200

150

150 100 100 50

50

°C

(a)

A B C D E F G

0.8

0.6

qe (g/g)

a s

Protein Loading (mg/g)

250

Relative Activity (%)

[14]. The enzymatic activity of the immobilized lipase is related to hydrophobic and charge properties of the carrier. Glu-GO immobilized lipase has the highest relative enzymatic activity. Although Arg and Lys have a stronger reduction effect on GO, Glu-GO is more suitable for the immobilization of TL lipase. Compared to other amino acid functionalized GOs, the surface properties of Glu-GO are better matched to the surface properties of TL lipase. The Arg-GO and Lys-GO with hydrophilic surface display low lipase activity, mainly attributed to the low enzyme loading. Meanwhile,

0.4

0.2

0 IP IP IP IP IP IP IP IM TL GO-L GO-L GO-L GO-L GO-L GO-L GO-L s s e g p u l Ly Ph G Cy Tr Ar

--- Langmuir — Freundlich

0.0 0.0

0.2

Fig. 6. Enzyme loading and relative activity of GO and RGO.

0.4

0.6

1.0 200

B-LIP D-LIP F-LIP H

(b)

A B C D E F G

°C

0.8

150

0.6

qe (g/g)

Relative Acitivity (%)

(a)

A-LIP C-LIP E-LIP G-LIP

100

0.4

0.2

50

--- Langmuir — Freundlich

0.0 0.0

0 5

6

7

8

9

0.2

0.6

250 A-LIP C-LIP E-LIP G-LIP

200

B-LIP D-LIP F-LIP H

(c)

150

100

1.0

0.6 0.4

--- Langmuir — Freundlich

0.2

50

0 30

40

50

60

0.8

A B C D E F G

°C

0.8

qe (g/g)

Relative Activity (%)

0.4

Ce (g/l)

10

pH

(b)

0.8

Ce (g/l)

70

Temperature (°C) Fig. 7. pH stability (a) and thermal stability (b) of GO and RGO(A: GO-LIP, B: CysGO-LIP, C: Arg-GO-LIP, D: Lys-GO-LIP, E: Glu-GO-LIP, F: Phe-GO-LIP, G: Trp-GO-LIP, H: Free lipase).

0.0 0.0

0.2

0.4

0.6

0.8

Ce (g/l) Fig. 8. Adsorption isotherms for RGO and GO at 25 °C (a), 35 °C (b), and 50 °C (c), respectively (A: GO, B: Cys-GO, C: Arg-GO, D: Lys-GO, E: Glu-GO, F: Phe-GO, G: TrpGO).

218

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

after the lipase is immobilized on the carrier, excessive hydrophilicity may cause the lid above the lipase active center to be difficult to open. Among them, the enzyme loading of Cys-GO is higher, but the catalytic activity of Cys-GO-LIP is lower. The possible reason is that Cys has a small amount of adsorption on GO, and the sulfhydryl group has an inhibitory effect on the catalytic activity of lipase [53]. pH is an important factor that determines the catalytic performance of immobilized lipase because the change in pH can affect the degree of ionization and charge of GO surface functionality. The pH stability of GO and RGO immobilized lipases are shown in Fig. 7a. As seen in the Figure, the optimum pH value of free lipase is 8 which is consistent with the literature [54], and that of the immobilized lipase is 6 or 7. The optimum pH of the immobilized lipase shifts to the left due to the effects of the proton state of the three amino acid residues on the active site [55], as well as the interaction between non-catalytic residues [56] after exposure to the lipase active center. Arg-GO-LIP and Lys-GO-LIP, in which amino acid adsorption is present, are relatively stable at different pH values. This suggests that amino acids may increase the stability of lipase, and other reagents such as betaine [57], detergents [58], etc., have also been shown to increase the stability of TL lipase. (See Fig. 8.). The thermal stability of enzymes is of great importance in practical operational conditions. As described in Section 2.8, the thermal stability detection range of free lipase and immobilized lipase ranged 25  70 °C, and the results are shown in Fig. 7a. Arg-GO-LIP and Lys-GO-LIP have lower relative enzymatic activities, but better thermal resistance. When the temperature is higher than 50 °C, the relative enzymatic activities of five immobilized lipases, such as that of Glu-GO-Lipase (Glu-GO-LIP), are still higher than 100%. Compared to GO immobilized lipase, the amino acidsfunctionalized GO immobilized lipase exhibits higher relative enzymatic activity and slightly poor thermal resistance. A series of experiments were carried out to investigate the effects of the initial enzyme concentration and adsorption temperature on the adsorption of free lipase on GO and RGO. The adsorption curves were plotted with qe and Ce as axes, and the adsorption process was fitted using the Langmuir isotherm model and Freundlich isotherm model. The Langmuir isotherm model is suitable for the ideal molecular layer adsorption, and the Freundlich isotherm model is an empirical equation. The values of the isotherm

parameter, such as the maximum adsorption capacity, and the correlation coefficient (R2) are described in Table 2. It can be observed from the correlation coefficient that the Freundlich isotherm model is more suitable for experimental data. As the temperature increases, the increment in adsorption capacity indicates that the adsorption process is an endothermic process. The 1/n obtained by Freundlich fitting is<1, indicating that the adsorption of lipase on GO and RGO is preferential adsorption. As can be seen, the amount of lipase adsorbed on GO and RGO is as follows: Phe-GO > Trp-GO > Cys-GO > Glu-GO > GO > Lys-GO > Arg-GO

The langmuir adsorption model predicts the maximum amount of adsorption, and this is basically consistent with the previously calculated order of protein loading. Part of the inconsistency may be due to differences in protein concentration and solid-liquid ratio. GO has a strong hydrophilicity and a large amount of negative charge, which is not favorable to the adsorption of lipase. On the other hand, the hydrophobicity of Arg-GO and Lys-GO is too weak, making it also detrimental to the adsorption of lipase. Thermodynamic calculations are applied to determine if the process is spontaneous. It can be seen from Table 3 that the adsorption Gibbs free energy of lipase on both RGO and GO is negative, indicating that the adsorption process is spontaneous. The result of DGdecreases as the temperature increases, indicating that the temperature rise is favorable for adsorption. The positive value of DSmeans that the adsorption of lipase on RGO and GO is a process of the disorder increasing. In general, when the enzyme molecule is adsorbed on the carrier, the movement is hindered, and the degree of disorder is reduced. However, high temperature operation tends to change the structure of the enzyme molecules, resulting in increased disorder. For the adsorption of TL lipase on GO, the entropy produced by adsorption is smaller than the entropy change caused by the change of enzyme molecules, so the overall process shows an increase in disorder. The positive DH indicates that the adsorption of lipase on RGO and GO is an endothermic process. The adsorption enthalpy of lipase on Arg-GO and Trp-GO is more than 40 kJ/mol, suggesting that the chemical adsorption of the enzyme on the two supports. The entropy of adsorption increases to other carriers, indicating that chemistry adsorption has a great influence on the conformation of lipase.

Table 2 Fitting data of adsorption isotherm for RGO and GO. Temperature

25 °C

35 °C

50 °C

Carrier

GO Cys-GO Arg-GO Lys-GO Glu-GO Phe-GO Trp-GO GO Cys-GO Arg-GO Lys-GO Glu-GO Phe-GO Trp-GO GO Cys-GO Arg-GO Lys-GO Glu-GO Phe-GO Trp-GO

Langmuir

Freundlich

qmax

KL

R2

KF

n

R2

0.50458 0.7216 0.39399 0.47364 0.52427 0.96162 0.68808 0.53498 0.79773 0.53824 0.58988 0.56478 1.13707 0.70598 0.60929 0.89875 0.71832 0.69931 0.56555 1.2239 0.95827

22.28262 13.66808 9.97125 4.86984 25.69847 6.08636 4.35689 28.74482 16.58228 9.06731 4.36336 33.65139 6.1734 12.18671 25.83941 26.49424 9.14516 5.51891 48.60547 7.14893 14.20073

0.97406 0.9543 0.95409 0.97032 0.92447 0.98548 0.99244 0.88202 0.94462 0.9526 0.97089 0.88886 0.98973 0.97133 0.88545 0.93961 0.92088 0.97334 0.8579 0.98397 0.96435

0.59617 0.8357 0.44064 0.46081 0.64379 1.05715 0.64325 0.65268 0.92733 0.58808 0.5377 0.67905 1.22782 0.86284 0.73259 1.15394 0.78249 0.6921 0.71992 1.38409 1.10848

3.89241 3.20858 2.55853 2.24762 3.92455 2.12776 2.20658 3.90731 3.26024 2.56297 2.25681 3.97185 2.15849 2.68198 3.92043 3.29937 2.63857 2.28857 4.21323 2.18424 3.00875

0.99327 0.9951 0.99021 0.97126 0.98211 0.98981 0.98689 0.98112 0.99029 0.99363 0.97658 0.96627 0.98599 0.98254 0.98364 0.99538 0.97681 0.98806 0.98772 0.98903 0.96501

219

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220 Table 3 Thermodynamic data for the TL lipase adsorption on RGO and GO. Carrier

qe (mg/g)

GO

200 400 600 200 400 600 200 400 600 200 400 600 200 400 600 200 400 600 200 400 600

Cys-GO

Arg-GO

Lys-GO

Glu-GO

Phe-GO

Trp-GO

4G(KJ/mol)

4S(J/mol
K)

4H

298.15 K

313.15 K

328.15 K

298.15 K

313.15 K

328.15 K

(KJ/mol)

9.6437

10.0055

10.5280

7.9495

8.3485

8.8602

6.3389

6.5630

7.0857

5.5686

5.7790

6.1458

9.7234

10.1708

11.3143

5.2717

5.5273

5.8656

5.4670

6.8678

8.0798

122.2138 120.1523 118.8912 155.9614 149.3216 145.4408 189.8958 183.7045 180.0636 122.7136 119.7941 117.9748 120.2854 98.2092 85.3133 90.0056 85.8622 83.4350 291.8690 233.1106 199.1240

119.4204 117.4258 116.2057 152.1932 145.7691 142.0143 184.4579 178.4676 174.9449 119.4125 116.5878 114.8276 117.8327 96.4732 83.9960 87.9132 83.9043 81.5559 286.9408 230.0901 197.2070

115.4923 113.5903 112.4269 146.7096 140.5837 137.0033 177.5098 171.7977 168.4386 115.0024 112.3089 110.6304 115.9009 95.5334 83.6356 84.8780 81.0553 78.8159 277.3677 223.1572 191.8011

26.7761 26.1617 25.7859 38.5271 36.5483 35.3919 50.2498 48.4049 47.3258 31.0584 30.1308 29.5879 26.1218 19.5429 15.6935 21.5515 20.3152 19.5919 81.5138 64.0436 53.8719

The character of small amount of adsorption on RGO/GO and the favorable thermal stability are also consistent with chemisorption. The physical adsorption of lipase is evidenced by the adsorption enthalpy of the lipase on the support (<40 kJ/mol). The adsorption enthalpy decreases with the increase of the equilibrium adsorption amount. It is speculated that the heterogeneity of the GO surface enables the enzyme molecule to occupy the site with small binding energy. As the amount of adsorption increases, the site with small binding energy is gradually filled, then the remaining enzyme molecules can only be fixed with sites with higher binding energy, that is, the enthalpy increases. 4. Conclusions This study combines the interaction of surface functionalized amino acids with GO and immobilized enzymes. In the process of enzyme immobilization, the surface properties of GO are regulated by using a green reducing agent, amino acid, thereby regulating the catalytic performance of the enzyme. The surface charge and hydrophobic properties of GO can be regulated by using different kinds of amino acids. The negative charge of amino acidmodified GO is significantly reduced, and the hydrophobicity increased under control. Among all amino acids, Arg and Lys have both strong reducibility and adsorption on GO. In contrast, Cys has the weakest reduction effect on GO. Due to the interfacial activation phenomenon of TL lipase, by changing the charge properties and surface hydrophobicity of the carrier, the TL lipase can be immobilized on the carrier in the form of the lid opening. GluGO-Lipase has the highest relative enzymatic activity among other immobilized samples. Through adsorption isotherm and thermodynamic studies, it was found that the adsorption of lipase on RGO and GO is consistent with the Freundlich model, and PheGO and Cys-GO have the largest enzyme load. Except for partially chemisorbed carriers, the adsorption of lipase on RGO and GO mainly belongs to the physical adsorption process, and the adsorption capacity increases with heating up to 70 °C. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No.: 21878142, 21636003),

the Program for Changjiang Scholars and Innovative Research Team in University (IRT_14R28), the Major Research Plan of the National Natural Science Foundation of China (21390204), the Jiangsu Synergetic Innovation Center for Advanced BioManufacture, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] Y. Deng, X.R. Huang, X.L. Wu, Review on graphene oxide composites, Mater. Rev. 26 (2012) 84–87. [2] W. Gao, L.B. Alemany, L.J. Ci, P.M. Ajayan, New insights into the structure and reduction of graphite oxide, Nat. Chem. 1 (2009) 403–408. [3] N.C.D. Nath, I.Y. Jeon, M.J. Ju, S.A. Ansari, J.B. Baek, J.J. Lee, Edge-carboxylated graphene nanoplatelets as efficient electrode materials for electrochemical supercapacitors, Carbon 142 (2019) 89–98. [4] N. Parveen, S.A. Ansari, M.O. Ansari, M.H. Cho, Manganese dioxide nanorods intercalated reduced graphene oxide nanocomposite toward high performance electrochemical supercapacitive electrode materials, J. Coll. Interf. Sci. 506 (2017) 613–619. [5] N. Parveen, S.A. Ansari, S.G. Ansari, H. Fouad, M.H. Cho, Intercalated reduced graphene oxide and its content effect on the supercapacitance performance of the three dimensional flower-like b-Ni(OH)2 architecture, New J. Chem. 41 (2017) 10467–10475. [6] Y. Liu, Q. Li, Y.Y. Feng, G.S. Ji, T.C. Li, J. Tu, X.D. Gu, Immobilisation of acid pectinase on graphene oxide nanosheets, Chem. Pap. 68 (2014) 732–738. [7] X. Gu, J. Gao, X. Li, Y. Wang, Immobilization of papain onto graphene oxide nanosheets, J. Nanosci. Nanotechno. 18 (2018) 3543–3547. ˇ a, Z. Marie, B.A. Daniel, P. Martin, S. Zdeneˇk, Graphene oxide [8] H. Son immobilized enzymes show high thermal and solvent stability, Nanoscale 7 (2015) 5852–5858. [9] W. Zhuang, L.J. He, J.H. Zhu, J.W. Zheng, X.J. Liu, Y.H. Dong, J.L. Wu, J.W. Zhou, Y. Chen, H.J. Ying, Efficient nanobiocatalytic systems of nuclease P1 immobilized on PEG-NH2 modified graphene oxide: effects of interface property heterogeneity, Coll. Surf. B. Biointerf. 145 (2016) 785–794. [10] V. Mehnati-Najafabadi, A. Taheri-Kafrani, A.K. Bordbar, Xylanase immobilization on modified superparamagnetic graphene oxide nanocomposite: effect of PEGylation on activity and stability, Int. J. Biolog. Macromol. 107 (2018). [11] M. Mathesh, B. Luan, T.O. Akanbi, J.K. Weber, Opening lids- modulation of lipase Immobilization by graphene oxides, ACS Catal. 6 (2016) 4760–4768. [12] S. Stankovich, D.A. Dikin, R.D. Piner, K.A. Kohlhaas, A. Kleinhammes, Y.Y. Jia, Y. Wu, S.B.T. Nguyen, R.S. Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558–1565. [13] P.Y. Zhu, M. Shen, S.H. Xiao, D. Zhang, Experimental study on the reducibility of graphene oxide by hydrazine hydrate, Physcia B 406 (2011) 498–502. [14] X.F. Gao, J. Jang, S. Nagase, Hydrazine and thermal reduction of graphene oxide: reaction mechanisms, product structures, and reaction design, J. Phy. Chem. C 114 (2010) 832–842. [15] S. Park, J. An, J.R. Potts, A. Velamakanni, S. Murali, R.S. Ruoff, Hydrazinereduction of graphite- and graphene oxide, Carbon 49 (2011) 3019–3023.

220

W. Zhou et al. / Journal of Colloid and Interface Science 546 (2019) 211–220

[16] C.K. Chua, M. Pumera, Chemical reduction of graphene oxide: a synthetic chemistry viewpoint, Chem. Soc. Rev. 43 (2013) 291–312. [17] H.J. Shin, K.K. Kim, A. Benayad, S.M. Yoon, H.K. Park, I.S. Jung, M.H. Jin, H.K. Jeong, J.M. Kim, J.Y. Choi, Efficient reduction of graphite oxide by sodium borohydride and Its effect on electrical conductance, Adv. Funct. Mater. 19 (2010) 1987–1992. [18] Y. Liu, D.S. Yu, C. Zeng, Z.C. Miao, L.M. Dai, Biocompatible graphene oxidebased glucose biosensors, Langmuir 26 (2010) 6158–6160. [19] A. Ambrosi, C.K. Chua, A. Bonanni, M. Pumera, Lithium aluminum hydride as reducing agent for chemically reduced graphene oxides, Chem. Mater. 24 (2012) 2292–2298. [20] S. Pandit, M. De, Interaction of amino acids and graphene oxide: trends in thermodynamic properties, J. Phys. Chem. C 121 (2017) 600–608. [21] L. Shanghao, A.N. Aphale, I.G. Macwan, P.K. Patra, W.G. Gonzalez, M. Jaroslava, R.M. Leblanc, Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins, ACS Appl. Mater. Inter. 4 (2012) 7069–7074. [22] H. Vovusha, S. Sanyal, B. Sanyal, Interaction of nucleobases and aromatic amino acids with graphene oxide and graphene flakes, J. Phys. Chem. Lett. 4 (2013) 3710–3718. [23] S.S. Mallineni, J. Shannahan, A.J. Raghavendra, A.M. Rao, J.M. Brown, R. Podila, Biomolecular interactions and biological responses of emerging twodimensional materials and aromatic amino acid complexes, ACS Appl. Mater. Inter. 8 (2016) 16604–16611. [24] D.N.H. Tran, S. Kabiri, D. Losic, A green approach for the reduction of graphene oxide nanosheets using non-aromatic amino acids, Carbon 76 (2014) 193–202. [25] D.Z. Chen, L.D. Li, L. Guo, An environment-friendly preparation of reduced graphene oxide nanosheets via amino acid, Nanotechnology 22 (2011) 325601. [26] J. Gao, F. Liu, Y.L. Liu, N. Ma, Z.Q. Wang, X. Zhang, Environment-friendly method to produce graphene that employs vitamin C and amino acid, Chem. Mater. 22 (2010) 2213–2218. [27] P. Adlercreutz, Immobilisation and application of lipases in organic media, Chem. Soc. Rev. 42 (2013) 6406–6436. [28] A. Zaks, A.M. Klibanov, Enzyme-catalyzed processes in organic solvents, Ann. N.Y. Acad. Sci. 501 (2010). 129 129. [29] Z.Y. Wang, N. Li, M.H. Zong, A simple procedure for the synthesis of potential 6-azauridine prodrugs by thermomyces lanuginosus lipase, J. Mol. Catal. B: Enzym. 59 (2009) 212–219. [30] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst, J. Mol. Catal. B: Enzym. 62 (2010) 197– 212. [31] H.P. Dong, J.F. Li, Y.M. Li, L.J. Hu, D.P. Luo, Improvement of catalytic activity and stability of lipase by immobilization on organobentonite, Chem. Eng. J. 181– 182 (2012) 590–596. [32] J.L. Kaar, A.M. Jesionowski, J.A. Berberich, M. Roger, A.J. Russell, Impact of ionic liquid physical properties on lipase activity and stability, J. Am. Chem. Soc. 125 (2003) 4125–4131. [33] M.D. Alves, F.M. Aracri, E.C. Cren, A.A. Mendes, Isotherm, kinetic, mechanism and thermodynamic studies of adsorption of a microbial lipase on a mesoporous and hydrophobic resin, Chem. Eng. J. 311 (2016) 1–12. [34] E.T. Hwang, M.B. Gu, Enzyme stabilization by nano/microsized hybrid materials, Eng. Life Sci. 13 (2013) 49–61. [35] S. Tadepalli, Z.Y. Wang, K.K. Liu, Q.S. Jiang, J. Slocik, Influence of surface charge of the nanostructures on the biocatalytic activity, Langmuir 33 (2017) 6611– 6619. [36] D.H. Zhang, L.X. Yuwen, Y.L. Xie, W. Li, X.B. Li, Improving immobilization of lipase onto magnetic microspheres with moderate hydrophobicity/ hydrophilicity, Coll. Surf. B. Biointerf. 89 (2012) 73–78. [37] W.Q. Song, Y.Y. Liu, L.W. Qian, L.Y. Niu, L.Q. Xiao, H. Yu, W. Yan, X.D. Fan, Hyperbranched polymeric ionic liquid with imidazolium backbones for highly efficient removal of anionic dyes, Chem. Eng. J. 287 (2016) 482–491.

[38] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [39] F.A. Lage, J.J. Bassi, M.C. Corradini, L.M. Todero, J.H. Luiz, A.A. Mendes, Preparation of a biocatalyst via physical adsorption of lipase from thermomyces lanuginosus on hydrophobic support to catalyze biolubricant synthesis by esterification reaction in a solvent-free system, Enzyme Microb. Technol. 84 (2016) 56–67. [40] R.A. Sheldon, S.V. Pelt, Enzyme immobilisation in biocatalysis: why, what and how, Chem. Soc. Rev. 42 (2013) 6223–6235. [41] K.Y. Foo, B.H. Hameed, Insights into the modeling of adsorption isotherm systems, Chem. Eng. J. 156 (2010) 2–10. [42] A. Chakraborty, B. Sun, An adsorption isotherm equation for multi-types adsorption with thermodynamic correctness, Appl. Therm. Eng. 72 (2014) 190–199. [43] Y.Y. Zhao, X.Y. Dong, L.L. Yu, L. Yang, S. Yan, Implications from protein adsorption onto anion- and cation-exchangers derivatized by modification of poly(ethylenimine)-Sepharose FF with succinic anhydride, Biochem. Eng. J. 132 (2018) 78–79. [44] I. Slavik, W. Uhl, H. Börnick, E. Worch, Assessment of SOC adsorption prediction in activated carbon filtration based on Freundlich coefficients calculated from compound properties, RSC Adv. 6 (2016) 19587–19604. [45] L.H. Terrell, Theory of physical adsorption, Adv. Catal. 4 (1952) 211–258. [46] C. Duran, D. Ozdes, A. Gundogdu, H.B. Senturk, Kinetics and isotherm analysis of basic dyes adsorption onto almond shell (prunus dulcis) as a low cost adsorbent, J. Chem. Eng. Data 56 (2011) 2136–2147. [47] W.S.W. Ngah, M.A.K.M. Hanafiah, Adsorption of copper on rubber (Hevea brasiliensis) leaf powder: kinetic, equilibrium and thermodynamic studies, Biochem. Eng. J. 39 (2016) 521–530. [48] A.A. Khan, R.P. Singh, Adsorption thermodynamics of carbofuran on Sn (IV) arsenosilicate in H+, Na+ and Ca2+ forms, Colloids Surf. 24 (1987) 33–42. [49] S. Bose, T. Kuila, Dual role of glycine as a chemical functionalizer and a reducing agent in the preparation of graphene: an environmentally friendly method, J. Mater. Chem. 22 (2012) 9696–9703. [50] N.Zˇ. Prlainovic´, D.I. Bezbradica, Z.D. Knezˇevic´-Jugovic´, S.I. Stevanovic´, M.L.A. Ivic´, P.S. Uskokovic´, D.Zˇ. Mijin, Adsorption of lipase from Candida rugosa on multi walled carbon nanotubes, J. Ind. Eng. Chem. 19 (2013) 279–285. [51] K. Ramani, P. Saranya, S.C. Jain, G. Sekaran, Lipase from marine strain using cooked sunflower oil waste: production optimization and application for hydrolysis and thermodynamic studies, Bioprocess Biosyst. Eng. 36 (2013) 301–315. [52] D.H. Zhao, C.W. Peng, J. Zhou, Lipase adsorption on different nanomaterials: a multi-scale simulation study, PCCP 17 (2014) 840–850. [53] K. Chakraborty, R. Paulraj, Purification and biochemical characterization of an extracellular lipase from pseudomonas fluorescens MTCC 2421, J. Agric. Food. Chem. 57 (2009) 3869–13866. [54] R. Fernandez-Lafuente, Lipase from Thermomyces lanuginosus : Uses and prospects as an industrial biocatalyst, J. Mol Catal. B: Enzym 62 (2010) 197– 212. [55] D. Thakshila, J.M. Swails, M.E. Harris, A.E. Roitberg, D.M. York, Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations, Biochemistry 54 (2015) 1307–1313. [56] O.N. Alvaro, B.A. Andrews, J.A. Asenjo, A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity, Biotechnol. Bioeng. 86 (2010) 573–586. [57] T. Soderlund, K. Zhu, J. Arimatti, P. Kinnunen, Effects of betaine on the structural dynamics of thermomyces (Humicola) lanuginosa lipase, Coll. Surf. B. Biointerf. 26 (2002) 75–83. [58] G. Fernandez-Lorente, J.M. Palomo, J.M. Guisan, R. Fernandez-Lafuente, Effect of the immobilization protocol in the activity, stability, and enantioslectivity of Lecitase Ò, Ultra. J. Mol. Catal. B: Enzym. 47 (2007) 99–104.