Improved Catalytic Properties of Thermomyces Lanuginosus Lipase Immobilized Onto Newly Fabricated Polydopamine-Functionalized Magnetic Fe3o4 Nanoparticles

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Article Improved Catalytic Properties of Thermomyces lanuginosus Lipase Immobilized onto Newly Fabricated Polydopamine-Functionalized Magnetic Fe3O4 Nanoparticles

Yanhong Bi 1,2, Zhaoyu Wang 2, Rui Zhang 2, Yihan Diao 2, Yaoqi Tian 1 and Zhengyu Jin 1,*

1 State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; [email protected] (Y.B.); [email protected] (Y.T.) 2 School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an 223003, China; [email protected] (Z.W.); [email protected] (R.Z.); [email protected] (Y.D.) * Correspondence: [email protected]

Received: 11 April 2020; Accepted: 18 May 2020; Published: 24 May 2020

Abstract: In this study, magnetic Fe3O4 nanoparticles coated with polydopamine possessing abundant amino groups (Fe3O4@PDA) were conveniently prepared, detailed, and characterized, and then firstly used as a supporting matrix for immobilizing Thermomyces lanuginosus lipase (Fe3O4@PDA@TLL). The effects of some crucial factors on the immobilization efficiency were investigated and the optimal protein loading and activity recovery were found to be 156.4 mg/g and 90.9%, respectively. Characterization studies revealed that Fe3O4@PDA@TLL displayed a broader pH and temperature adaptability as compared to the free TLL, which allows its use at wider ranges of reaction conditions. With regard to the stabilities, the immobilized TLL clearly displayed improved pH, thermal, and solvent tolerance stabilities compared to the free enzyme, suggesting that the biocompatible Fe3O4@PDA might be an outstanding material for immobilizing TLL and acting as alternative support for different enzymes.

Keywords: magnetic nanoparticle; immobilization; Thermomyces lanuginosus lipase; stability

1. Introduction Enzyme immobilization has been described as a facile and powerful tool for improving the properties of enzymes. Accordingly, it has been applied extensively in the areas of the food, pharmaceutical, and cosmetic industries [1,2]. Physical adsorption, entrapment/encapsulation, cross-linking, and covalent binding are among the typical immobilization approaches for the coupling of the enzyme with carrier material [1], which is often crucial to optimizing enzyme behavior in industrial processes. Though the immobilized enzymes have been well known for over four decades, they are still largely restricted to industrial-scale applications, particularly for use in nonaqueous enzymatic catalysis systems because of their inadequate reusability, operational stability, recovery, and shelf-life [3,4]. Recent advances in modern nanoscience allow for a variety of choices in exploring novel immobilization strategies to overcome the aforementioned drawbacks of the enzymes, which may offer new possibilities for establishing sustainable, highly efficient, and overall cost-effective industrial processes [5–7]. Among the available nano-immobilization protocols, surface-modified magnetic nanoparticles (MNPs) possessing excellent characteristics of biocompatibility, chemical stability, and dispersity are receiving considerable attention as robust immobilization supports [3]. Modified materials—including polydopamine (PDA), chitosan, and cellulose—have proved to be

Processes 2020, 8, 629; doi:10.3390/pr8050629 www.mdpi.com/journal/processes Processes 2020, 8, 629 2 of 15 eminent modified agents for masking the magnetic nanoparticles because of their outstanding advantages derived from the existence of numerous functional groups such as amino, hydroxyl, and hydroxymethyl groups, which can be coupled easily with the enzyme protein [8–10]. For example, Wang et al. investigated pullulanase immobilization using Fe3O4 MNPs modified with functional groups and found that ionic adsorption of pullulanase on Fe3O4@PDA-polyethyleneimine- glycidyltrimethylammonium gave a high performance and a durable catalyst [11]. Based on the surface available functional or functionalized entities, Lou et al. explored a series of convenient and efficient nanoparticle-based catalytic supports like Fe3O4@polydopamine, Fe3O4@cellulose, and Fe3O4@chitosan@cellulose to immobilize lipase and papain by physical adsorption or covalent coupling method [3,5]. Their further characterization indicated that the catalytic features such as activity, organic solvent tolerance, and reusability were substantially improved in their respective catalytic systems. Thermomyces lanuginosus lipase (TLL) is a versatile biocatalyst and is widely used in the oleochemical industry, biodiesel production, organic synthesis, and environmental protection [12]. However, like most of the industrial enzymes, TLL also suffers from leaching from the carrier support, easy denaturation by organic solvent, and poor recyclability, inevitably resulting in loss of their catalytic activities and extremely high cost [13]. With the development of the nanoparticle-based immobilization strategy, magnetic nano-Fe3O4 has emerged as a desirable alternative to traditional materials for assembling immobilized TLL with markedly improved performances [14–16]. Therefore, in this study, we report the development of a novel nanobiocatalyst by using a PDA biomimetic coating to combine TLL with magnetic Fe3O4 nanoparticle (Fe3O4@PDA@TLL). The structural features, catalytic activity, and stability of the free and immobilized enzymes were investigated in detail to characterize their desirable properties. The findings might offer primary guidelines for the enzyme nano-immobilization techniques and are of interest in industrial biotechnology.

2. Materials and Methods

2.1. Catalysts and Chemicals T.lanuginosus lipase (TLL) was purchased from Novozymes Co., Ltd., China. Dopamine hydrochloride, 2-methyltetrahydrofuran (MeTHF), p-nitrophenyl palmitate (p-NPP), and p-nitrophenol were procured from Aladdin Reagent Co. Ltd. (Shanghai). Choline chloride/glycerol (ChCl/GL, 1:2), choline chloride/ ethylene glycol (ChCl/EG, 1:2), choline chloride/urea (ChCl/UR, 1:3), and choline chloride/acetic acid (ChCl/AA, 1:2) were kindly donated by Professor Zhangqun Duan (Academy of State Administration of Grain, China). All other chemicals were of the highest reagent grade commercially available and used as received.

2.2. Preparation of Fe3O4@PDA

The magnetic Fe3O4 nanoparticles were prepared by a chemical coprecipitation method, as reported earlier, with slight modiﬁcations [8]. According to this method, 0.87 g FeCl 6H O and 0.31 g FeCl 4H O 3· 2 2· 2 were dissolved in 25 mL deionized water under the nitrogen atmosphere. The mixture was then placed in a 75 ◦C water bath with vigorous stirring. Afterwards, 1.5 mol/L NaOH solution was added dropwise and the pH of the solution was maintained at 9.0–10 under continuous stirring for 1.0 h. The precipitated particles were washed with deionized water and dried in a vacuum for 24 h. To prepare PDA-coated Fe3O4 (Fe3O4@PDA), dopamine hydrochloride and Fe3O4 nanoparticles, which were dispersed in deionized water for 10 min under sonication, with the molar ratio of 1:1, were mixed and stirred vigorously. The pH of the mixture was adjusted to 8.5 by adding the above NaOH solution. After stirring for 24 h at room temperature, the Fe3O4@PDA was formed, collected with an external magnet and rinsed with deionized water repeatedly. Processes 2020, 8, 629 3 of 15

2.3. Preparation of Immobilized Lipase

In a typical experiment, 0.4 g of Fe3O4@PDA was added to 12 mL of phosphate buﬀer (50 mM, pH 8.0) at 25 ◦C, and 2.4 mL of TLL solution (260 mg/mL) was then added to the sample. The mixture was stirred at 250 rpm for 4.0 h. After the stipulated time, the immobilized sample was recovered and continuously washed until no protein was detected. The enzyme-loaded Fe3O4@PDA was designated as Fe3O4@PDA-TLL. The concentration of the residual TLL in the supernatant and washings was subsequently determined using Bradford’s method [17]. The immobilization yield of TLL was calculated using the following equation:

Y = [(m m m )/m ] 100 (1) 0 − 1 − 2 0 × where m0 is the mass of TLL protein initially added to the phosphate buﬀer, m1 and m2 are the protein content of the supernatant and wash solution after immobilization. All data in this formula are averages of duplicate experiments.

2.4. Assay of Enzyme Activity The activity of free and immobilized TLL was assayed according to the p-nitrophenyl palmitate (p-NPP) method with slight modifications [18]. The activity assay was conducted in 1.0 mL Tris-HCl buffer (50 mM, pH 8.0) containing 0.1 mL p-NPP solution (a quantity of 30 mg p-NPP was dissolved in 10 mL isopropanol). The reaction was initiated with the addition of either 0.1 g immobilized enzyme or 0.1 mL TLL solution and incubated at 37 ◦C and 250 rpm for 10 min. After a predetermined time, the reaction was stopped by adding 5.0 mL of 95% ethanol. The absorbance of the reaction mixture was determined at 410 nm and the activity was calculated. One unit of activity (U) was defined as the amount of enzyme required to produce 1.0 µmol p-nitrophenol (p-NP) in 1.0 min under the above conditions. The specific activities of the free TLL and Fe3O4@PDA-TLL were 3921 U/mL and 8022 U/g, respectively.

2.5. Surface Characterization of the Immobilized Lipase The morphologies of various pristine and lipase-couple nanoparticles were envisaged using a transmission electron microscopy (TEM, Tecnai G2 20) with an accelerating voltage of 200 kV.The Fourier Transform infrared spectroscopy (FT-IR spectra) was obtained with a Nicolet 670 spectrometer using KBr pellets. A PANalytical X’Pert Pro instrument using Cu KR (k) 0.154 nm radiation carried out small-angle X-ray diﬀraction (XRD) measurements. The surface composition was performed on X-ray photoelectron spectroscopy (XPS) with an ESCALAB–250 electron spectrometer with 13 kV high pressure and 20 mA electric current using Mg Kα as radiation.

2.6. Effect of pH and Temperature on the Free and Immobilized Lipase The optimum reaction pH and temperature of the free and immobilized lipases were determined as the relative activity in Tris-HCl buffer with a variety of pH (from 6.5 to 9.0) and temperature (from 10 ◦C to 55 ◦C), following the same procedure as in Section 2.4. The highest activity of the lipase incubated under different conditions was defined as 100%. All the experiments were repeated three times and the standard deviation was calculated.

2.7. pH Stability Studies of Free and Immobilized Lipase The pH stabilities of free and immobilized lipase were carried out by incubating them in Tris-HCl buffer (50 mM) adjusted to various pH values (pH = 6.0–10) at 25 ◦C for 12 h. Then the residual activity was determined, as described in Section 2.4, and the activity of the lipase without incubation was defined as 100% (the method for measuring and defining residual enzyme activity was the same with thermal and solvent tolerance stability studies). Processes 2020, 8, 629 4 of 15

2.8. Thermal Stability Studies of Free and Immobilized Lipase The thermal stabilities were examined by incubating the free or immobilized TLL in Tris-HCl buﬀer (50 mM, pH = 8.0) at diﬀerent temperatures (25–80 ◦C) for 12 h.

2.9. Solvent Tolerance Stability Studies of Free and Immobilized Lipase Solvent tolerance stabilities were measured after the lipases were incubated in seven typical organic solvents, such as acetonitrile, ethanol, ChCl/GL (1:2), ChCl/EG (1:2), ChCl/UR (1:3), and ChCl/AA (1:2) for 12 h at 25 ◦C.

2.10. Operational Stability Studies of Free and Immobilized Lipase The operational stabilities of immobilized TLL during the batch hydrolysis of p-NPP were investigated. When the maximum p-NP yield was achieved, the Fe3O4@PDA-TLL was separated by filtration and washed three times. Then, the reused enzyme was added into the fresh reaction mixture at 25 ◦C, followed by the assay of its activity and maximum product yield. The initial activity obtained in the first batch was defined as 100 %.

3. Results and Discussion

3.1. Preparation and Characterization of Fe3O4@PDA@TLL Several characterization techniques were used to identify the topological/chemical structures and other physicochemical properties of the bare and lipase-coupled nanoparticles. The morphology and size of bare Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@TLL were first analyzed by TEM. The TEM images are portrayed in Figure1. The Fe 3O4 nanoparticles were mostly regular spherical or ellipsoidal shape with an average diameter of 18.7 nm measured using a dynamic laser scattering particle size (DLS) analyzer (Brookhaven, NanoBrook 90Plus, USA), as shown in Figures1A and2A. After surface modification with a polymeric shell by self-assembly of PDA in alkaline aqueous solution, it was seen that the magnetic microspheres were wrapped with a thin PDA layer and showed a tendency to aggregate due to the interaction between PDA and magnetic nanoparticles), conforming to the formation of the uniform PDA shell, see Figure1B. Further, the introduction of enzyme protein to PDA-coated Fe3O4 nanoparticles through the Schiff base reaction between amine groups shows no distinct changes in the morphology of the Fe3O4@PDA and Fe3O4@PDA@TLL, as seen in Figure1C, but a relatively aggregative appearance was observed compared to the precursor Fe3O4 particles. This is in good agreement with the previous results [19,20]. Moreover, based on the analysis of the size distribution, the mean diameters of aggregated Fe3O4@PDA and Fe3O4@PDA@TLL were estimated to be 24.4 and 29.8 nm, as shown in Figure2B and 2C, respectively, which were obviously larger than that of the MNPs and consistent with those recorded in the TEM images. XPS was applied to analyze the chemical compositions of all types of the bare and enzyme-attached nanoparticles as seen in Figure3A. Wide scan XPS spectra indicated that the characteristic peaks of Fe 2p (Fe 2p1/2 723.97 eV, Fe 2p3/2 709.97 eV) and O1s (529.97 eV) were detected in all the examined samples, which were in consonance with the previous reports [21]. The additional binding energy peaks of C 1s of 283.97 eV and N 1s of 400.97 eV appeared in Fe3O4@PDA and a significant enhancement in Fe3O4@PDA@TLL nanocomposites corroborated the successful grafting of PDA and enzyme protein onto the nano-Fe3O4 particles [8]. Processes 2020, 8, x FOR PEER REVIEW 5 of 15

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Figure 1. TEM images of Fe3O4 (A), Fe3O4 nanoparticles coated with polydopamine possessing abundant amino groups (Fe3O4@PDA) (B), and the aforementioned used as a supporting matrix for

Figureimmobilizing 1. TEM Thermomyces images of Fe 3lanuginosusO4 (A), Fe3 Olipase4 nanoparticles (Fe3O4@PDA@TLL) coated with (C). polydopamine possessing Figureabundant 1. aminoTEM images groups of (Fe Fe3O3O4@PDA)4 (A), Fe (B3),O4 and nanoparticles the aforementioned coated with used polydopamine as a supporting possessing matrix for abundantimmobilizing amino Thermomyces groups (Fe3O lanuginosus4@PDA) (B), lipase and the (Fe3 aforementionedO4@PDA@TLL) used (C). as a supporting matrix for immobilizing Thermomyces lanuginosus lipase (Fe3O4@PDA@TLL) (C).

100 ACB

80 100 ACB

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0 20 1 10 100 0 Size (nm)

Figure 2. Size distribution1 of Fe3O4 (A), Fe 103O4@PDA (B), and Fe3O4@PDA@TLL 100 (C). Figure 2. Size distribution of Fe3O4 (A), Fe3O4@PDA (B), and Fe3O4@PDA@TLL (C). Size (nm) XPS was applied to analyze the chemical compositions of all types of the bare and enzyme- attached nanoparticlesFigure 2. Size as distribution seen in Figure of Fe3 O3A.4 (A Wide), Fe3O scan4@PDA XPS (B ),spectra and Fe 3indicatedO4@PDA@TLL that ( Cthe). characteristic peaks of Fe 2p (Fe 2p1/2 723.97 eV, Fe 2p3/2 709.97 eV) and O1s (529.97 eV) were detected in all the examinedXPS was samples, applied which to analyze were in the consonance chemical withcompos theitions previous of all reports types [21]. of the The bare additional and enzyme- binding attached nanoparticles as seen in Figure 3A. Wide scan XPS spectra indicated that the characteristic peaks of Fe 2p (Fe 2p1/2 723.97 eV, Fe 2p3/2 709.97 eV) and O1s (529.97 eV) were detected in all the examined samples, which were in consonance with the previous reports [21]. The additional binding

Processes 2020, 8, x FOR PEER REVIEW 6 of 15 energy peaks of C 1s of 283.97 eV and N 1s of 400.97 eV appeared in Fe3O4@PDA and a significant enhancement in Fe3O4@PDA@TLL nanocomposites corroborated the successful grafting of PDA and enzyme protein onto the nano-Fe3O4 particles [8]. Processes 2020, 8, 629 6 of 15

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100 200 300 400 500 600 700 800 Binding energy (eV)

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Figure 3. XPS spectra (A), FT-IR spectra (B), and XRD patterns (C) of Fe3O4 (1), Fe3O4@PDA (2) and Figure 3. XPS spectra (A), FT-IR spectra (B), and XRD patterns (C) of Fe3O4 (1), Fe3O4@PDA (2) and Fe3O4@PDA@TLL (3). Fe3O4@PDA@TLL (3).

FT-IR spectra were also obtained to evaluate the immobilized enzyme on the surface of Fe3O4@PDA, 1 as seenFT-IR in spectra Figure3 B.were The also appearance obtained of to a strongevaluate absorption the immobilized band of Feenzyme3O4 spectra on the atsurface 573 cm of− Ferepresents3O4@PDA, the as vibration seen in Figure of the 3B. Fe-O The function appearance group of [8 a]. strong According absorption to the recent band reportof Fe3O by4 spectra Sureshkumar at 573 −1 According to1 the recent report by cmet al. represents [22], dopamine the vibration showed some of the peak Fe-O absorptions function betweengroup [8]. 1000–1600 cm− . After the successful Sureshkumar et al. , dopamine showed some peak absorptions between 1000 1600 cm−1. 1 functionalization of[22] MNPs with PDA, the stretching vibrations of the carbonyl– group at 1640After cm the− , 1 1 successfulC=C bending functionalization group at 1510 of cm MNPs− , and with a phenolic PDA, the hydroxyl stretching group vibrations at 1270 of cm the− incarbonyl the Fe3 Ogroup4@PDA at 1640samples cm−1 are, C=C clearly bending seen ingroup curves at 21510 and cm 3, Figure−1, and3 B,a phenolic proving thathydroxyl PDA shellgroup had at been1270 successfullycm−1 in the 1 1 Fecoated3O4@PDA on the samples Fe3O4 surfaceare clearly [8,21 seen]. Furthermore, in curves 2 and the 3, peaks Figure at 10403B, proving cm− and that 1510 PDA cm shell− assigned had been to −1 −1 successfullythe C-N and coated C=C bending on the Fe vibrations,3O4 surface respectively, [8,21]. Furthermore, were broader the peaks than those at 1040 of Fecm3O 4and@PDA, 1510 which cm assignedindicated to the the immobilization C-N and C=C of thebending enzyme vibrations, onto the support.respectively, The resultswere ofbroader XRD experimentsthan those areof Feshown3O4@PDA, in Figure which3C. indicated Regarding the theimmobilization Fe 3O4 nanoparticles, of the enzyme the sixonto characteristic the support. peaksThe results at 2θ of= 30.2XRD◦ , experiments35.5◦, 43.1◦, 53.5are ◦shown, 57.0◦ ,in and Figure 62.7◦ 3C.corresponding Regarding the to theFe3O lattice4 nanoparticles, planes of (220), the six (311), characteristic (400), (422), peaks (511), atand 2θ(440), = 30.2°, respectively, 35.5°, 43.1°, which 53.5°, are 57.0°, typical and characteristics62.7° corresponding of Fe3O to4 [the8]. lattice After coatingplanes of with (220), PDA (311), and 3 4 (400),immobilization (422), (511), of and TLL, (440), the XRD respectively, data of Fe which3O4@ are PDA typical and Fe characteristics3O4@ PDA@TLL of Fe seenO [8]. in curves After coating 2 and 3, 3 4 3 4 withFigure PDA3C, and reveal immobilization that all the main of TLL, peaks the wereXRD overlappeddata of Fe O with@ PDA the and di ff ractionFe O @ PDA@TLL peak of the seen Fe3 Oin4 , curvesas seen 2 inand curve 3, Figure 1, Figure 3C, 3revealC. This that fully all indicatesthe main thatpeaks the were Fe 3 overlappedO4 cores maintained with the diffraction their crystalline peak ofstructure the Fe3O in4, theas seen immobilized in curve complex.1, Figure 3C. This fully indicates that the Fe3O4 cores maintained their crystalline structure in the immobilized complex. 3.2. Immobilization of TLL onto Fe3O4@PDA 3.2. Immobilization of TLL onto Fe3O4@PDA It is well documented that the immobilization efficiency and activity of the immobilized enzyme are stronglyIt is well dependent documented on that the immobilization the immobilization parameters efficiency such and as activity the enzyme-support of the immobilized ratio, pH enzyme of the aresolution, strongly or thedependent working on temperature. the immobilization For example, parameters the loading such amount as the enzyme-support of enzyme protein ratio, on Fe pH3O 4of@ thePDA solution, was increased or the working from 128.5 temperature. mg/g to 189.3 For mg exam/g asple, the enzyme-supportthe loading amount ratio of increased enzyme fromprotein 0.52 on to Fe2.6,3O as4@ shownPDA was in Figure increased4A. Thefrom enzyme 128.5 mg/g activity to recovery189.3 mg/g was as firstthe increasedenzyme-support and then ratio a decreasing increased fromtendency 0.52 to was 2.6, noticed, as shown and in the Figure best ratio4A. The of enzyme enzyme to activity support recovery proved towas be first about increased 1.6 with and an activitythen a decreasingrecovery and tendency a protein was loading noticed, of 85.5%and the and best 178.4 ratio mg of/ g,enzyme respectively. to support Surprisingly, proved to the be protein-loadingabout 1.6 with ancapacity activity of recovery the Fe3O and4@ PDAa protein is obviously loading of greater 85.5% than and those178.4 reportedmg/g, respectively. in most literature Surprisingly, [14,15 ,the23]. protein-loadingThe higher concentration capacity ofof the the initial Fe3O TLL4@ PDA protein is usedobviously for immobilization greater than may those be responsiblereported in for most this literaturephenomenon. [14,15,23]. However, The higher no obvious concentration correlation of the was initial observed TLL protein between used the enzymefor immobilization protein loading may beand responsible catalytic activity for this of phenomenon. the immobilized However, TLL. This no isobvious similar correlation to the immobilization was observed oflaccase between on the the enzymemagnetic protein Fe3O4 nanoparticlesloading and [24catalytic]. Although activity the activityof the immobilized recovery of the TLL. immobilized This is similar enzyme to would the

Processes 2020, 8, x FOR PEER REVIEW 8 of 15 immobilization of laccase on the magnetic Fe3O4 nanoparticles [24]. Although the activity recovery of theProcesses immobilized2020, 8, 629 enzyme would be enhanced with the increase in initial enzyme concentration,8 of 15 excessive enzyme protein absorption on the surface of Fe3O4@ PDA forms intensive intermolecular steric hindrance, thus leading to the diffusional restrictions of substrates and products and be enhanced with the increase in initial enzyme concentration, excessive enzyme protein absorption deactivation of the immobilized enzyme [24]. on the surface of Fe3O4@ PDA forms intensive intermolecular steric hindrance, thus leading to the diﬀusional restrictions of substrates and products and deactivation of the immobilized enzyme [24].

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Relative activity (%) (2) Protein loading (mg/g) 50 120

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Figure 4. Cont.

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50 Protein loading (mg/g) (2) 120

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Figure 4. Effect of immobilization conditions on activity recovery (1) and protein loading (2). (A)Effect Figure 4. Effect of immobilization conditions on activity recovery (1) and protein loading (2). (A) of enzyme-support ratio (12 mL phosphate buffer (50 mM, pH 8.5) containing 0.4 g Fe3O4@PDA, Effect of enzyme-support ratio (12 mL phosphate buffer (50 mM, pH 8.5) containing 0.4 g Fe3O4@PDA, different amount of TLL, 25 ◦C, 10 h); (B)Effect of immobilization time (12 mL phosphate buffer (50 mM, different amount of TLL, 25 °C, 10 h); (B) Effect of immobilization time (12 mL phosphate buffer (50 pH 8.5) containing 0.4 g Fe3O4@PDA, 2.4 mL TLL, 25 ◦C, different immobilization time); (C)Effect of mM, pH 8.5) containing 0.4 g Fe3O4@PDA, 2.4 mL TLL, 25 °C, different immobilization time); (C) immobilization pH (12 mL phosphate buffer (50 mM, pH 6.5–9.0) containing 0.4 g Fe3O4@PDA, 2.4 mL Effect of immobilization pH (12 mL phosphate buffer (50 mM, pH 6.5–9.0) containing 0.4 g TLL, 25 ◦C, 4.0 h); (D)Effect of immobilization temperature (12 mL phosphate buffer (50 mM, pH 8.0) Fe3O4@PDA, 2.4 mL TLL, 25 °C, 4.0 h); (D) Effect of immobilization temperature (12 mL phosphate containing 0.4 g Fe3O4@PDA, 2.4 mL TLL, 10–55 ◦C, 4.0 h). buffer (50 mM, pH 8.0) containing 0.4 g Fe3O4@PDA, 2.4 mL TLL, 10–55 °C, 4.0 h). The effect of coupling time on the immobilization process was also studied and exemplified in FigureThe 4effectB. The of protein coupling loading time on on the the immobilization immobilization process material was increased also studied with prolongingand exemplified coupling in Figuretime 4B. up toThe 12 protein h, while loading a reduction on the in immobilizati the proportionon ofmaterial residual increased activity waswith observedprolonging after coupling reaching timethe up plateau to 12 h, coupling while a reactionreduction time in the of 4.0proportion h, with of enzyme residual immobilization activity was observed and activity after retention reaching of the161.5 plateau mg/ gcoupling and 88.4%, reaction respectively. time of 4.0 In h, consideration with enzyme of immobilization these observations, and activity the optimal retention coupling of 161.5time mg/g used and for 88.4%, TLL lipase respectively. conjugation In consideration on Fe3O4@PDA of these was observations, 4.0 h. Of the the tested optimal pH coupling values ranging time used for TLL lipase conjugation on Fe3O4@PDA was 4.0 h. Of the tested pH values ranging from 6.5

Processes 2020, 8, x FOR PEER REVIEW 10 of 15 Processes 2020, 8, 629 10 of 15 to 9.0 as depicted in Figure 4C, the alkaline immobilization conditions such as pH 7.5–8.5 seem to be fromfavorable 6.5 to 9.0 for as TLL depicted immobilization in Figure 4onC, Fe the3O alkaline4@PDA, immobilizationretaining about conditions90.9% of its such maximum as pH activity 7.5–8.5 at pH 8.0. Interestingly, the enzyme loadings displayed a gradually increasing trend with the seem to be favorable for TLL immobilization on Fe3O4@PDA, retaining about 90.9% of its maximum activityenhancement at pH 8.0. of Interestingly,pH, indicating the that enzyme the high loadings pH might displayed be beneficial a gradually for protein increasing deposition trend withon the thesurface enhancement of Fe3O4 of@PDA pH, [21]. indicating Temperature that the also high plays pH mightan import be beneficialant role in forthe proteinimmobilization deposition process, on influencing not only the activity recovery but also the enzyme loading. As shown in Figure 4D, the surface of Fe3O4@PDA [21]. Temperature also plays an important role in the immobilization process,increasing influencing the temperature not only the from activity 10 °C recovery to 25 °C but brought also the about enzyme a gradual loading. improvement As shown in Figure in both4D, the above crucial parameters. The optimum activity recovery and enzyme loading were 90.9% and 156.4 increasing the temperature from 10 ◦C to 25 ◦C brought about a gradual improvement in both the above crucialmg/g, parameters. respectively. The A optimum further increase activity recoveryin temperature and enzyme resulted loading in a were drastic 90.9% drop and in 156.4 the mgactivity/g, respectively.recovery, presumably A further increasedue to the in partial temperature inactivation resulted of the in aTLL drastic at high drop temperatures. in the activity recovery, presumably due to the partial inactivation of the TLL at high temperatures. 3.3. Effect of the pH and Temperature on Free and Immobilized TLL 3.3. Effect of the pH and Temperature on Free and Immobilized TLL For a better understanding of the property of the immobilized Fe3O4@PDA@TLL, the effects of

pHFor and a bettertemperature understanding were studied of the on property the basis of of the determining immobilized the Fe hydrolysis3O4@PDA@TLL, of p-NPP, the as eff shownects of in pHFigure and temperature 5. For example, were the studied influence on the of basis pH ranging of determining from 6.5 the to hydrolysis9.0 was first of pexamined-NPP, as shownat 25 °C in to Figureinvestigate5. For example,the optimum the influencepH wherein of pHthe rangingenzymes from are most 6.5 to active. 9.0 was Results first examinedshowed that at the 25 optimal◦C to investigatepH for the the free optimum enzyme pH was wherein 7.5, while the enzymes after immobilization, are most active. the Results optimal showed pH thatfor Fe the3O optimal4@PDA@TLL pH forshifted the free to enzyme 8.0, as was seen 7.5, in while Figure after 5A. immobilization, Unquestionably, the optimalFe3O4@PDA@TLL pH for Fe3O displayed4@PDA@TLL broader shifted pH toadaptability 8.0, as seen inand Figure higher5A. residual Unquestionably, activity than Fe that3O4 @PDA@TLLof the free counterpart. displayed Temperature broader pH adaptabilityinvestigations andrevealed higher residualthat although activity the than immobilized that of the and free free counterpart. TLL presented Temperature the same investigations temperature optima, revealed the thatimmobilized although the nanobiocatalyst immobilized and exhibited free TLL enhanced presented theheat same resistance temperature to a wider optima, temperature the immobilized range, nanobiocatalystretaining more exhibited than 53.3% enhanced of the heatmaximum resistance activity to a among wider temperature the tested temperatures, range, retaining as presented more than in 53.3%Figure of the 5B. maximum These positive activity changes among themight tested be temperatures, associated with as presented the charge in Figureof the5 B.various These positivefunctional changesgroups might including be associated amino groups with the in chargeimmobilization of the various carriers functional [25], which groups interact including with aminoenzyme groups protein inby immobilization covalent bond carriers formation [25 and], which ensure interact the conformational with enzyme integrity protein and by covalentadjustability, bond thus formation reducing andthe ensure sensitivity the conformational of pH and temper integrityature. and Previous adjustability, studies thus reported reducing a similar the sensitivity phenomenon of pH andof the temperature.behavior of Previousthe immobilized studies enzyme reported on a similarnanomaterials phenomenon [14,26]. of the behavior of the immobilized enzyme on nanomaterials [14,26].

A 110 Fe3O4@PDA@TLL Free TLL 100

70 Relative activity (%)activity Relative 60

40 6.5 7.0 7.5 8.0 8.5 9.0 pH

Figure 5. Cont.

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B Fe O @PDA@TLL 100 3 4 Free TLL 90

Relative activity (%) 50

30 10 20 30 40 50 60 Temperature (°C)

Figure 5. Effect of pH (A) and temperature (B) on the activity of free TLL and Fe3O4@PDA@TLL. Figure 5. Effect of pH (A) and temperature (B) on the activity of free TLL and Fe3O4@PDA@TLL. 3.4. Stability of Free and Immobilized TLL 3.4. Stability of Free and Immobilized TLL In addition to facilitating recovery for reuse, the increase in volume-specific enzyme loading, and simplificationIn addition to of facilitating downstream recovery processing, for reuse, another the increase significant in volume-specific driving force enzyme for exploring loading, noveland simplification immobilization of techniquesdownstream to processing, immobilize another biocatalyst significant is to achieve driving higher force for stability exploring towards novel environmentalimmobilization perturbations. techniques Thisto isimmobilize of both commercial biocatalyst and is scientific to achieve interest higher [4,13]. stability The pH stabilitytowards profileenvironmental illustrates perturbations. that the residual This catalytic is of both activity commercial of Fe3O and4@PDA@TLL scientific interest was apparently [4,13]. The higher pH stability than theprofile free form illustrates after incubation that the residual for 12 h catalytic at the corresponding activity of Fe pH3O from4@PDA@TLL 6.0–10, especially was apparently at the optimal higher pHthan ofthe 8.0, free with form a 20.3% after improvement incubation for of 12 the hrelative at the corresponding activity, see Figure pH from6A. This 6.0–10, confirms especially previous at the findings optimal ofpH pH of eff 8.0,ect onwith the a behavior20.3% improvement of the immobilized of the relative enzyme. activity, Of the examinedsee Figure temperatures 6A. This confirms ranging previous from 25findings◦C to 80 of◦C pH depicted effect on in Figurethe behavior6B, although of the theimmobilized residual activities enzyme. of Of Fe the3O 4examined@PDA@TLL temperatures and free TLLranging exhibited from decreasing 25 °C tendency,to 80 °C theirdepicted retained in activitiesFigure in6B, the although whole temperature the residual range activities are rather of diFefferent.3O4@PDA@TLL For free TLL, and thefree relative TLL exhibited activity decreasing drastically reducestendency, from their 100% retained to 17.5%, activities indicating in the poorwhole stabilitytemperature to temperature. range are On rather the otherdifferent. hand, For the free immobilized TLL, the Ferelative3O4@PDA@TLL activity drastically exerted a considerablyreduces from enhanced100% to stability17.5%, inindicating the same temperaturepoor stability range, to temperature. and retained 35.1%On the activity other even hand, at 80 the◦C. immobilized The reason forFe these3O4@PDA@TLL phenomena exerted might bea considerably that the immobilization enhanced stability effectively in protectsthe same the temperature protein subunit range, from and unfoldingretained and35.1% limits activity the conformation even at 80 transition °C. The of re theason enzyme for protein,these phenomena thus strengthening might thebe rigiditythat the ofimmobilization the spatial conformation effectively of protects the enzyme the protein andsubunit leading from to unfold improvementing and of limits the heat the stabilityconformation [27]. transitionSatisfactory of the solvent enzyme tolerance protein, of thus the biocatalystsstrengthening has the been rigidity proven of tothe be spatial an essential conformation characteristic of the andenzyme provides protein the non-aqueousand leading to enzymatic improvement catalysis of the industry heat stability with a potentially [27]. wide application [4,28]. As is evidentSatisfactory from solvent the results tolerance in Figure of the6C biocatalysts that Fe 3O has4@PDA@TLL been proven displayed to be an essential substantially characteristic higher catalyticand provides activity the as non-aqueous compared to enzymatic the free enzyme catalysis in industry all the examined with a potentially solvents. wide For instance,application[4,28]. after a 12As h–incubation is evident periodfrom the in theresults strong-polar in Figure organic 6C that DMSO Fe3O which4@PDA@TLL can easily displayed result in enzymesubstantially inactivation higher bycatalytic stripping activity the essential as compared water layer to the o fffreethe enzyme enzyme in molecules all the examined [29], the immobilizedsolvents. ForTLL instance, still achieved after a 12 2.0h–incubation times higher period activity in recovery the strong-polar (61.3% versus organic 30.1%) DMSO compared which can to thateasily of theresult free in enzyme. enzyme Ininactivation contrast toby traditional stripping solvents, the essential the four water eco-friendly layer off ChCl-based the enzyme DESs, molecules which have[29], provedthe immobilized to be a sustainable TLL still andachieved promising 2.0 times alternative higher activity to conventional recovery organic(61.3% versus media 30.1%) [30], showed compared good to that to excellent of the free capacity enzyme. toIn maintain contrast enzymeto traditional activity, solvents, especially the four for eco-fr the immobilizediendly ChCl-based Fe3O4 @[email protected], which have In ChCl proved/GL to (1:2), be a forsustainable example, theand recovered promising activity alternative of the to immobilized convention TLLal organic was 88.5%, media which [30], isshowed higher good than thatto excellent of the freecapacity enzyme. to maintain Nearly all enzyme of the activity activity, of especially the free TLL for was the lostimmobilized after incubation Fe3O4@PDA@TLL. in ChCl/AA, In while ChCl/GL the immobilized(1:2), for example, biocatalyst the recovered still exhibited activity a notable of the activity immobilized (34.4% TLL of initial was 88.5%, activity). which Moreover, is higher as evidentthan that fromof the the free data enzyme. depicted Nearly in Figure all6 D,of thethe immobilizedactivity of the lipase freestill TLL retained was lost more after than incubation 70.2% of in its ChCl/AA, original while the immobilized biocatalyst still exhibited a notable activity (34.4% of initial activity).

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ProcessesMoreover,2020, 8 ,as 629 evident from the data depicted in Figure 6D, the immobilized lipase still retained12 ofmore 15 than 70.2% of its original activity even after being repeatedly employed for eight batches, suggesting that Fe3O4@PDA@TLL had satisfactory operational stability in the examined system. activity even after being repeatedly employed for eight batches, suggesting that Fe3O4@PDA@TLL had satisfactory operational stability in the examined system.

A Fe O @PDA@TLL 90 3 4 Free TLL 80

Relative activity (%) activity Relative 50

30 6.07.08.09.010.0 pH

B 100 Fe O @PDA@TLL 3 4 90 Free TLL

Relative activity (%) 40

20 20 30 40 50 60 70 80 Temperature (°C)

Figure 6. Cont.

Processes 2020, 8, x FOR PEER REVIEW 13 of 15 Processes 2020, 8, 629 13 of 15

C 100 Fe O @PDA@TLL 3 4 Free TLL 80

40 Relative activity (%)

0 le ol O ) ) ) ) itri an S (1:2 (1:2 (1:3 (1:2 ton Eth DM GL EG UR AA ce Cl/ Cl/ Cl/ Cl/ A Ch Ch Ch Ch

D 100 Fe O @PDA@TLL 3 4

40 Relative activity (%) activity Relative

0 12345678 Batch

Figure 6. pH (A), thermal (B), solvent tolerance (C), and operational (D) stabilities of the enzyme. Figure 6. pH (A), thermal (B), solvent tolerance (C), and operational (D) stabilities of the enzyme. 4. Conclusions 4. Conclusions In conclusion, T. lanuginosus lipase was successfully immobilized on Fe3O4@PDA, which was preparedIn conclusion, as a biocompatible T. lanuginosus matrix lipase and characterizedwas successfully by TEM,immobilized FT-IR, XRD,on Fe WPS,3O4@PDA, and DLSwhich with was regardprepared to their as a morphology, biocompatible composition, matrix and and characterized structure characteristics. by TEM, FT-IR, The XRD, hydrolysis WPS, and of p DLS-NPP with in phosphateregard to butheirﬀer morphology, served as a model composition, reaction and toexamine structure the characteristics. catalytic properties The hydrolysis of the immobilized of p-NPP in phosphate buffer served as a model reaction to examine the catalytic properties of the immobilized enzyme. The results reported in this paper clearly show that Fe3O4@PDA@TLL exhibited much better resistanceenzyme. The against results pH, reported thermal, in and this solventpaper clearly denaturation show that and Fe3 satisfactoryO4@PDA@TLL recyclability exhibited much for p-NPP better hydrolysis.resistance against Taken together,pH, thermal, these and ﬁndings solvent demonstrated denaturation a promisingand satisfactory application recyclability potential for ofp-NPP the compositehydrolysis. particles Taken fortogether, enzyme these immobilization. findings demonstrated a promising application potential of the composite particles for enzyme immobilization. Author Contributions: Conceptualization, Z.W., Z.J.; Investigation, Y.B., R.Z.; Methodology, Y.B., Z.W.; ProjectAuthor administration, Contributions: Z.W.; Conceptualization, Supervision, Z.J.; Z.W., Writing—original Z.J.; Investigation, draft, Y.B., Y.B., R.Z.; Y.D.; Methodology, Writing—review Y.B., andZ.W.; editing, Project Y.T.administration, All authors have Z.W.; read Supervision, and agreed Z.J.; to the Writing—original published version draft, of the Y.B., manuscript. Y.D.; Writing—review and editing, Y.T. All authors have read and agreed to the published version of the manuscript.

Processes 2020, 8, 629 14 of 15

Funding: This research received no external funding. Acknowledgments: This research was sponsored by the National Natural Science Foundation of China (Nos. 21676114 and 21706088), Qing Lan Project of Jiangsu Province of China, Six Talent Peaks Project in Jiangsu Province (No. SWYY–011; YY–061), and Graduate Research and Innovation Projects of Jiangsu Province (No. SJCX20_0898) for financial support. Conflicts of Interest: The authors declare no conflict of interest.

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