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Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase: An Efficient and Stable Biocatalyst for Asymmetric Synthesis of (R)-3-Quinuclidinol with Regeneration of Coenzymes In Situ Yuhan Chen 1 , Qihua Jiang 1 , Lili Sun 1 , Qiang Li 2 , Liping Zhou 1 , Qian Chen 1 , Shanshan Li 1 , Mingan Yu 1 and Wei Li 1,3, * 1

2

3

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Department of Medicinal Chemistry, School of Pharmacy, Chongqing Medical University, Chongqing 400016, China; [email protected] (Y.C.); [email protected] (Q.J.); [email protected] (L.S.); [email protected] (L.Z.); [email protected] (Q.C.); [email protected] (S.L.); [email protected] (M.Y.) Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological & Chemical engineering, Chongqing University of Education, Chongqing 400067, China; [email protected] Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, China Correspondence: [email protected]; Tel.: +86-23-6848-5161

Received: 14 July 2018; Accepted: 30 July 2018; Published: 15 August 2018







Abstract: Enzymes are biocatalysts. In this study, a novel biocatalyst consisting of magnetic combined cross-linked enzyme aggregates (combi-CLEAs) of 3-quinuclidinone reductase (QNR) and glucose dehydrogenase (GDH) for enantioselective synthesis of (R)-3-quinuclidinolwith regeneration of cofactors in situ was developed. The magnetic combi-CLEAs were fabricated with the use of ammonium sulfate as a precipitant and glutaraldehyde as a cross-linker for direct immobilization of QNR and GDH from E. coli BL(21) cell lysates onto amino-functionalized Fe3 O4 nanoparticles. The physicochemical properties of the magnetic combi-CLEAs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and magnetic measurements. Field emission scanning electron microscope (FE-SEM) images revealed a spherical structure with numerous pores which facilitate the movement of the substrates and coenzymes. Moreover, the magnetic combi-CLEAs exhibited improved operational and thermal stability, enhanced catalytic performance for transformation of 3-quinuclidinone (33 g/L) into (R)-3-quinuclidinol in 100% conversion yield and 100% enantiomeric excess (ee) after 3 h of reaction. The activity of the biocatalysts was preserved about 80% after 70 days storage and retained more than 40% of its initial activity after ten cycles. These results demonstrated that the magnetic combi-CLEAs, as cost-effective and environmentally friendly biocatalysts, were suitable for application in synthesis of (R)-3-quinuclidinol essential for the production of solifenacin and aclidinium with better performance than those currently available. Keywords: enzyme immobilization; magnetic nanoparticles; combined cross-linked enzyme aggregates; reuse; (R)-3-quinuclidinol

Catalysts 2018, 8, 334; doi:10.3390/catal8080334

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1. Introduction Enantiomerically pure or enriched alcohols with excellent enantiomeric excess (ee) are highly valuable chiral building blocks for the production of wide range fine chemicals, agrochemicals and particularly pharmaceuticals [1–3]. For instance, (R)-3-quinuclidinol is a key chiral intermediate of many drugs such as aclidinium bromide [4], solifenacin succinate [5] and revatropate [6]. Aclidinium and revatropate are anticholinergic for long-term management of chronic obstructive pulmonary disease (COPD) which leads to more than 3 million deaths worldwide every year [7] and is also the third leading cause of death in United States following cancer and heart disease. Solifenacin, a competitive muscarinic receptor antagonist which attenuates bladder contraction, was approved in 2004 to treat overactive bladder (OAB) in the USA and the EU [8]. Recently published studies showed that when clinical benefits and risks are considered, aclidinium and tiotropium generate similar value to COPD patients, but when considering convenience criteria, aclidinium may be preferred [9]. (R)-3-Quinuclidinol can be synthesized by resolution of racemic mixtures of (dl)-3-quinuclidinone or by chemical, biocatalytic asymmetric synthesis. Chemical synthesis is frequently used to synthesize (R)-3-quinculidinol. For instance, asymmetric hydrogenation of 3-quinuclidinone using RuBr2[(S,S)-xylskewphos](pica) as chiral catalysts in a base containing ethanol has given (R)-3-quinuclidinol in 88–90% ee [10]. After that, a much better result, (R)-3-quinuclidinol in 97–98% ee, was obtained under the synergistic catalysis of RuCl2 [(S)-binap][(R)-iphan] and t-C4 H9 OK [11]. However, these methods suffer from several limitations. The metal catalysts are expensive, toxic, and the transition metal may contaminate the product. An alternative access to (R)-3-quinuclidinol in 42% overall yield and 96% ee is Aspergillus melleus protease-catalyzed kinetic resolution of (dl)-3-quinuclidinol esters [12]. Nevertheless, no chemical or resolution of racemic compounds can compare with the biocatalytic asymmetric reduction of 3-quinuclidinone, in which biocatalysts produce the products in 100% theoretical conversion yield and exceptionally high ee value. Furthermore, biocatalysts can operate under milder and more environmentally friendly conditions than the traditional chemical synthesis methods, thus avoiding using more severe conditions which can lead to problems with isomerization, racemization and rearrangement [13,14]. The biocatalyst can be a newly isolated Nocardia sp. and Rhodococcus erythropolis [15] or a screened fungal system belonging to Mucor piriformis [16], or a recombinant ketoreductase [17,18]. Nevertheless, the real breakthrough in biocatalysts came from the development of recombinant DNA technology [19,20]. However, more notable is that enzyme immobilization contributed to facilitating the progress of practical industrial applications of biocatalysts and recycling for long periods of time [20]. Unfortunately, most enzyme immobilization techniques have required a purified enzyme [21–23], but such processes are often too costly and low-yielding [24–26]. In particular, it is difficult to purify the low concentration of extracellular enzymes, which is often produced in cell culture medium [27]. Currently, coupling the purification and immobilization steps by nickel column may be a strategy to design a selective immobilization process for his-tagged protein [28–30], but the nickel column is expensive and needs to be regenerated after running about three times, and the purity of his-tagged protein was low [28]. Hence, there is a need to develop a simple and cost-effective immobilization technique for the biosynthesis of (R)-3-quinuclidinol in high yields and high optical purity and in situ recycling of cofactors. A substantial effort has been applied to the development of effective immobilization strategies to improve the long-term operational stability of enzyme and to facilitate its efficient recovery and recycling [31,32]. Cross-linked enzyme aggregates (CLEAs) may be a potential method for enzyme immobilization due to clear advantages: high volumetric productivity, superior storage stability, amenability for easy scaling-up and excellent recyclability, especially the method does not require purified enzyme and can be employed for the direct immobilization of an enzyme from crude cell lysate [31–33]. CLEAs can be simply prepared by precipitating enzyme solution at the optimum pH by addition of organic solvents or inorganic salts, and then cross-linked by a bifunctional cross-linking agent, such as glutaraldehyde (GA). As a result, the cross-linking

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increases the chance that any multi-subunit enzyme was cross-linked to another component of the biocatalyst, avoiding enzyme dissociation and contributing to stabilizing the quaternary structure of enzyme [34]. So far, several enzymes including penicillin G amidase, alcalase, hydrolases, oxidoreductases, lyases, esterases, etc. have been successfully immobilized as CLEAs with high specific activity and activity recovery [31,32,35]. Interestingly, combined cross-linked enzyme aggregates (combi-CLEAs) are a novel prospect for co-immobilization of two or more enzymes, which can catalyze various cascade and non-cascade bioconversion processes [36]. For example, acombi-CLEAs of glucose dehydrogenase (GDH) and ketoreductase were developed and employed repeatedly in the reduction of ethyl 4-chloro-3-oxobutanoate to syntheses the key atorvastatin intermediate ethyl 4-chloro-3-hydroxybutyrate [37]. A significant advantage of combi-CLEAs is a robust regeneration system for cofactors, the improved pH and thermal stability, high concentration of substrate tolerance, long-term operational stability and reusability, allowing its uses at relatively extreme conditions than the soluble counterparts. The authors believed that the strategy could be widely applied to produce various chiral alcohols with high efficiency and low cost [37]. However, it is difficult to deal with and fully recover the combi-CLEAs particles due to it forming clumps leading to internal mass-transfer limitations during conventional centrifugation or filtration treatments [38–40]. In a further elaboration, the smart magnetic CLEAs were prepared by cross-linking of enzyme aggregates onto magnetic nanoparticles (MNPs) [40,41]. Due to the magnetic nature, the magnetic biocatalysts can be easily separated from reaction mixture by an external magnet without the need for a filtration or centrifugation step, thus overcoming the disadvantage of squeezing of the CLEAs, and can be employed in a magnetically stabilized fluidized bed reactor, giving novel combinations of biotransformations and downstream processing [31,32]. However, the application of MNPs still has some disadvantages including aggregation, stability, leaching, and toxicity [42,43]. Usually, these problems can be circumvented by coating a layer of inert silica onto the surface of MNPs. Moreover, the surface of silica coated MNPs can be further functionalized with 3-aminopropyl trimethoxysilane (APTES) [44,45]. To our knowledge, application of amine functionalized silica coated MNPs for the preparation of combi-CLEAs remain scarcely investigated. Therefore, the aim of this study is to develop the magnetic combi-CLEAs of 3-quinuclidinone reductases (QNR) and glucose dehydrogenase (GDH) for enantioselective synthesis of (R)-3-quinuclidinol with regeneration of coenzymes in situ. The magnetic combi-CLEAs were prepared by co-precipitation of amine functionalized-MNPs, QNR and GDH from E. coli BL(21) cell lysates by agents such as (NH4 )2 SO4 to form aggregates, followed by cross-linking the aggregates using bifunctional agents such as GA. The physicochemical properties of the resulting biocatalyst were carried out by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), transmission electron microscopy (TEM) and field emission scanning electron microscope (FE-SEM). To permit retention of significant levels of enzyme activity, the effects of precipitant agents, cross-linker concentration, pH and temperature on the enzyme activity were optimized. The storage stability, reactivity, reusability and characteristic morphological changes of during recycling of the biocatalysts were also tested. 2. Results 2.1. Preparation and Characterization of the Magnetic Combi-CLEAs of QNR and GDH The fragment of the qnr gene encoding QNR and gdh gene encoding GDH with endogenous promoter and ribosomal recognition sequences has been inserted into pET28a to give pET28a-QNR and pET28a-GDH. Optimized cultivation conditions including a low expression temperature of 25 ◦ C for 48 h and the presence of 0.2 mM isopropyl β-D-Thiogalactoside (IPTG) afforded a similar cell stability of the cells expressing the QNR and GDH compared to the host strain E. coli BL(21) (Figure 1). A very significant band appeared at 25.70 kDa and 28.25 kDa in accordance with the calculated molecular weight derived from the amino acid sequence of the full QNR and GDH.

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Figure 1. 12% SDS-PAGE gel of protein samples. Lane M, protein molecular weight markers; Lane 1 Figure 1. 12% SDS-PAGE gel of protein samples. Lane M, protein molecular weight markers; Lane 1 to to Lane 4, Lane 5 to Lane 8 for the E. coli (His-QNR), E. coli (His-GDH) before induction, after Lane 4, Lane 5 toSDS-PAGE Lane 8 forgel theofE.protein coli (His-QNR), E. coliM,(His-GDH) before induction, after induction, Figure 1. 12% samples. Lane protein molecular weight markers; Lane 1 induction, the cell lysate supernatant and the cell lysate precipitate, respectively. thetocell lysate supernatant and8 the Lane 4, Lane 5 to Lane for cell the lysate E. coliprecipitate, (His-QNR),respectively. E. coli (His-GDH) before induction, after induction, the cell lysate supernatant and the cell lysate precipitate, respectively.

Magnetic combi-CLEAs of QNR-GDH biocatalyst preparation involves a sequence of two Magnetic combi-CLEAs QNR-GDH biocatalystofpreparation involves sequence of two physic-chemical steps startingof with co-precipitation QNR, GDH and Fea3O 4@SiO2-NH2 Magnetic steps combi-CLEAs of QNR-GDH biocatalyst preparation involves a -NH sequence of two physic-chemical starting with co-precipitation of QNR, GDH and Fe O @SiO nanoparticles 3 4 and2 NH22- attached nanoparticles followed by cross-linking the enzyme aggregates with themselves physic-chemical steps starting with co-precipitation of QNR, GDH and Fe3O4@SiO2-NH2 followed by cross-linking the enzyme aggregates with themselves NH22--NH attached the surfaces on the surfaces of Fe3O4@SiO 2-NH 2 nanoparticles (Scheme 1), where Feand 3O4@SiO 2 were on employed nanoparticles followed by cross-linking the enzyme aggregates with themselves and NH2- attached of (Scheme Fe3 Owe as Fe support for2 -NH enzyme immobilization. For 1), thiswhere purpose, first2 -NH synthesized magnetic as Fesupport 3O4 3 O4 @SiO 2 nanoparticles 4 @SiO 2 were employed on the surfaces of Fe3O4@SiO2-NH2 nanoparticles (Scheme 1), where Fe3O4@SiO2-NH2 were employed for enzyme immobilization. For this purpose, we first synthesized magnetic Fe O nanoparticles. nanoparticles. The nanoparticles were then coated with tetraethylorthosilicate (TEOS) followed by 4 as support for enzyme immobilization. For this purpose, we first synthesized 3magnetic Fe3O4 The nanoparticles were then coated with conditions, tetraethylorthosilicate (TEOS) followed by functionalizing functionalizing with APTES in alkaline thus preventing these nanoparticles from nanoparticles. The nanoparticles were then coated with tetraethylorthosilicate (TEOS) followed by clustering and their chemical Thethese resultant Fe3O4@SiOfrom 2-NH 2 nanoparticles were with APTES inimproving alkaline conditions, thus stability. preventing nanoparticles clustering and improving functionalizing with APTES in alkaline conditions, thus preventing these nanoparticles from mixedchemical with cellstability. lysates containing QNRFe and GDH. After precipitation and cross-linking under the their The resultant O @SiO -NH nanoparticles were mixed with cell lysates 4 2The resultant 2 clustering and improving their chemical3stability. Fe3O4@SiO2-NH2 nanoparticles were optimized conditions such as precipitant agents, cross-linker concentration and the cross-linking containing QNR GDH. After precipitation and cross-linking under the conditions such mixed with celland lysates containing QNR and GDH. After precipitation andoptimized cross-linking under the time, the magnetic combi-CLEAs were obtained. Totheensure successful preparation of magnetic as precipitant agents, cross-linker concentration and cross-linking time, the magnetic combi-CLEAs optimized conditions such as precipitant agents, cross-linker concentration and the cross-linking combi-CLEAs, FTIR, XRD and FE-SEMpreparation were employed to confirm these results. FTIR, XRD and FE-SEM were obtained. To ensure successful of magnetic time, the magnetic combi-CLEAs were obtained. To ensurecombi-CLEAs, successful preparation of magnetic were employed toFTIR, confirm results. were employed to confirm these results. combi-CLEAs, XRDthese and FE-SEM

Scheme 1. The diagram of the preparation of the magnetic combi-CLEAs biocatalysts. Scheme 1. The diagram of the preparation of the magnetic combi-CLEAs biocatalysts.

1. The diagram of the preparation of the magneticgroups combi-CLEAs Firstly, Scheme FTIR analyses was carried out to detect the functional of Fe3O4biocatalysts. , Fe3O4@SiO2-NH2, free enzyme and magnetic combi-CLEAs. As can be seen form Figure 2A(a), the absorption peak at Firstly, FTIR analyses was carried out to detect the functional groups of Fe3O4, Fe3O4@SiO2-NH2, −1 was 578 cm attributed towas the carried Fe-O bond stretching the characteristic peak also Firstly, FTIR analyses out to detect thevibration, functionaland groups of Fe3 O4 , Fe3 O , 4 @SiO 2 -NH free enzyme and magnetic combi-CLEAs. As can be seen form Figure 2A(a), the absorption peak at 2 appeared in and Figure 2A(b,d),combi-CLEAs. indicative of As no can changes in form the structure of Fe3the O4 after chemical free enzyme magnetic be seen Figure 2A(a), absorption 578 cm−1 was attributed to the Fe-O bond stretching vibration, and the characteristic peakpeak also at −1 and 1627 cm−1 were associated with-OH groups available −1 was modification. The peaks about at 3410 cmbond 578 cm attributed to the Fe-O stretching vibration, and the characteristic peak also appeared in Figure 2A(b,d), indicative of no changes in the structure of Fe3O4 after chemical −1 from the surface of Fe32A(b,d), O4. In theindicative Fe3O4@SiO2−1 -NH 2 spectrum, the peaks at 465, 790, 940 and 1092 cm appeared in Figure of no changes in the structure of Fe O after chemical 4 modification. The peaks about at 3410 cm and 1627 cm−1 were associated with-OH3 groups available −1 Si-O-Si −1 were were assigned to thepeaks O-Si-O bending vibration, symmetric, Si-Oassociated symmetric and Si-O-Si modification. The at 3O 3410 cm 1627 cm from the surface of Fe3O4. about In the Fe 4@SiO 2-NHand 2 spectrum, the peaks at 465, 790, 940with-OH and 1092 groups cm−1 asymmetric stretching vibration of O the. coated silica shell, respectively. The broad band atat3400–3500 available from the surface of Fe In the Fe O @SiO -NH spectrum, the peaks 465, 790, were assigned to the O-Si-O bending vibration, Si-O symmetric and Si-O-Si940 3 4 3 4 Si-O-Si 2 symmetric, 2 −1 can be ascribed to the characteristic peaks of NH2− groups of APTES which were overlain by cm − 1 and 1092 cm stretching were assigned to the O-Si-O vibration, Si-O-Si symmetric, symmetric asymmetric vibration of the coatedbending silica shell, respectively. The broad bandSi-O at 3400–3500 hydroxyl groups stretching vibrations. The peaks at 2930 −cm−1 corresponds to-CH2 bond stretching −1 can and asymmetric vibration of the coated silica of shell, respectively. The broad band cmSi-O-Si be ascribed to stretching the characteristic peaks of NH 2 groups APTES which were overlain by vibrations of aminopropyl from APTES [44]. The results demonstrated that Fe3O4@SiO2 nanoparticles hydroxyl groups stretching vibrations. peaks at 2930 cm−1ofcorresponds to-CH 2 bond stretching at 3400–3500 cm−1 can be ascribed to theThe characteristic peaks NH2 − groups of APTES which were were successfully modified by APTES (Figure 2A(b)). FTIR spectrum of−QNR and GDH before vibrations of aminopropyl APTESvibrations. [44]. The results demonstrated that1Fe 3O4@SiO2 nanoparticles overlain by hydroxyl groupsfrom stretching The peaks at 2930 cm corresponds to-CH2 bond (Figure 2A(c)) and after immobilization (Figure 2A(d)) presented changes in the characteristic peaks were successfully by APTES (Figure 2A(b)). FTIRresults spectrum of QNR and GDH before stretching vibrationsmodified of aminopropyl from APTES [44]. The demonstrated that Fe O 4 @SiO2 of functional groups. Especially decreasing of intensity at 1500 and 1550 cm−1 correspond to3N-H (Figure 2A(c)) andsuccessfully after immobilization 2A(d)) presented changes in the characteristic peaks nanoparticles were modified(Figure by APTES (Figure 2A(b)). FTIR spectrum of QNR and GDH vibration of free enzymes indicated that the NH2− group of QNR and GDH reacted with the NH2− −1 correspond of functional groups. Especially decreasing of intensity at 1500 and 1550 cm to N-H before (Figure 2A(c)) and after immobilization (Figure 2A(d)) presented changes in the characteristic vibration of free enzymes indicated that the NH2− group of QNR and GDH reacted with the NH2−

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Catalystsof 2018, 8, x FOR PEER REVIEW 5 of to 16 peaks functional groups. Especially decreasing of intensity at 1500 and 1550 cm−1 correspond Catalysts 2018, 8, x FOR PEER REVIEW 5 of 16 N-H vibration of free enzymes indicated that the NH2 − group of QNR and GDH reacted with the − attached group ononFeFe 3O 4@SiO 2 by GA cross-linking. XRD wasused usedto toidentify identify thecrystalline crystalline structure NH attached Fe3 O @SiO by GA cross-linking. was used to thestructure crystalline 2 group group attached 3on O4@SiO 24by GA2cross-linking. XRD was XRD theidentify of all samples. As can be seen in Figure 2B, the bare Fe 3 O 4 exhibited characteristic crystalline peaks structure of all samples. Asseen can in beFigure seen in2B, Figure 2B,Fe the Fe3 O4 characteristic exhibited characteristic crystalline of all samples. As can be the bare 3Obare 4 exhibited crystalline peaks at at ◦ ,43.60°, ◦ ,53.70°, ◦ ,57.20° ◦ and which around 35.80°, 62.70°, consistent withthe the standard peaks at30.40°, around 30.40 35.80 43.60 53.70◦and ,and 57.20 62.70◦are ,are which are consistent with the standard around 30.40°, 35.80°, 43.60°, 53.70°, 57.20° 62.70°, which consistent with standard Fe3Fe O43O4 XRD data (JCPDS, card 89-0950) [46]. In addition, it was found (Figure 2B(c)) that the coating Fe3XRD O4 XRD (JCPDS, 89-0950) In addition, it was found (Figure 2B(c)) that coating datadata (JCPDS, cardcard 89-0950) [46].[46]. In addition, it was found (Figure 2B(c)) that thethe coating procedure did not affect the phase change of Fe333O O444 nanoparticles nanoparticlesbecause becausethe theSiO SiO 2 and and APTES procedure didnot notaffect affectthe thephase phasechange changeof of Fe Fe O nanoparticles 2 and APTES procedure did because the SiO APTES 2 coatings are amorphous. Therefore, new peaks attributable to these groups could not be detected coatings are amorphous. Therefore, new peaks attributable to these groups could not be detected byby coatings are amorphous. Therefore, new peaks attributable to these groups could not be detected by XRD analysis. For free enzyme (Figure 2B(a)), their trends were similar to the XRD of the magnetic XRD analysis.For Forfree freeenzyme enzyme(Figure (Figure 2B(a)), 2B(a)), their trends the magnetic XRD analysis. trendswere weresimilar similartotothe theXRD XRDofof the magnetic combi-CLEAs (Figure 2B(d)). Nevertheless, crystallinity ofthe the samples was clearly decreased combi-CLEAs (Figure 2B(d)). Nevertheless, crystallinity samples was clearly decreased combi-CLEAs (Figure 2B(d)). Nevertheless, thethe crystallinity of of the samples was clearly decreased after after the coating or/and immobilization process (Figure 2B(c,d)), mainly due to the partial destruction after the coating or/and immobilization process (Figure 2B(c,d)), mainly due to the partial destruction the coating or/and immobilization process (Figure 2B(c,d)), mainly due to the partial destruction of the regularity ofthe theFe Fe3O 3O 4crystal structure structure after and of of thetheregularity 4crystal after coating coating with with aasilica silicashell shelland andAPTES APTES and regularity of the Feof 3 O4 crystal structure after coating with a silica shell and APTES and subsequently subsequently subjected to the GA cross-linking procedure. subsequently subjected to the GAprocedure. cross-linking procedure. subjected to the GA cross-linking

Figure 2. (A) FTIR spectra of (a) Fe3O4; (b) Fe3O4@SiO2-NH2; (c) free enzyme; (d) magnetic combiFigure 2. (A) (A)FTIR FTIRspectra spectra of O(b) ; Fe (b)3OFe free enzyme; (d) magnetic 43O 3 O4 @SiO 2 ; (c)enzyme; Figure 2. (a)(a) Fe3Fe O43;Fe 4@SiO 2-NH2;-NH (c) free (d) magnetic combiCLEAs. (B) XRD of (a) freeof enzyme; (b) 4; (c) Fe3O4@SiO2-NH2; (d) magnetic combi-CLEAs. combi-CLEAs. (B) XRD of (a) free enzyme; (b) Fe O ; (c) Fe O @SiO -NH ; (d) magnetic combi-CLEAs. 3 Fe 4 3O4@SiO 3 42-NH2;2 (d) magnetic 2 CLEAs. (B) XRD of (a) free enzyme; (b) Fe3O4; (c) combi-CLEAs.

The magnetic behaviors were confirmed by the magnetization curves (Figure 3a), which were The magnetic behaviorswere were confirmed by the magnetization curves (Figure 3a), which were typical plot of magnetization against applied magnetic field (M−H loop) at room and The magnetic behaviors confirmed by the magnetization curves (Figure 3a),temperature, which were typical typical plot of magnetization against applied magnetic field (M−H loop) at room temperature, and crossed the zero point, inductive of the superparamagnetic The saturation magnetization plot of magnetization against applied magnetic field (M−Hproperties. loop) at room temperature, and crossed value of Fe 3O 4@SiO andsuperparamagnetic magnetic combi-CLEAs was about 66.79 and 40.88 emu/g, crossed zero point,2-NH inductive of the superparamagnetic properties. Theemu/g saturation magnetization the zero the point, inductive of2 the properties. The saturation magnetization value of respectively. The decreased superparamagnetic behaviors of the magnetic combi-CLEAs could be value of Fe 3 O 4 @SiO 2 -NH 2 and magnetic combi-CLEAs was about 66.79 emu/g and 40.88 emu/g, Fe3 O4 @SiO2 -NH2 and magnetic combi-CLEAs was about 66.79 emu/g and 40.88 emu/g, respectively. explained due to cross-linking of proteins on the surface of Fe 3 O 4 @SiO 2 -NH 2 nanoparticles quenching respectively. decreased superparamagnetic behaviors ofcombi-CLEAs the magnetic could combi-CLEAs coulddue be The decreasedThe superparamagnetic behaviors of the magnetic be explained the magnetic moment. Although the superparamagnetic behaviors of2-NH the 2magnetic combi-CLEAs explained due toof cross-linking of proteins onFe the surface of Fe 3O 4@SiO nanoparticles quenching to cross-linking proteins on the surface of O @SiO -NH nanoparticles quenching the magnetic 3 4 2 2 decreasing manner, they well dispersed phosphate buffered saline (PBS) (Figure thedisplayed magnetic moment. Although thewere superparamagnetic behaviors of the magnetic combi-CLEAs moment. Although the superparamagnetic behaviors of the in magnetic combi-CLEAs displayed decreasing 3b) and the superparamagnetic properties were high enough to make easy separation from displayed decreasing were well dispersed phosphate saline (PBS)liquid (Figure manner, they were well manner, dispersedthey in phosphate buffered salinein(PBS) (Figure buffered 3b) and the superparamagnetic media (about 10 s) in the present of an external magnetic field (Figure 3c), suggesting the liquid as3b) and the superparamagnetic properties were high enough to make easy separation from properties were high enough to make easy separation from liquid media (about 10 s) in the present of synthesized magnetic combi-CLEAs could be efficiently applied in asymmetric synthesis of (R)-3media (about 10 s) in the(Figure present an external field magnetic (Figure 3c), suggestingcould the asan external magnetic field 3c),ofsuggesting the magnetic as-synthesized combi-CLEAs be quinuclidinol. synthesized magnetic combi-CLEAs could be efficiently applied in asymmetric synthesis of (R)-3efficiently applied in asymmetric synthesis of (R)-3-quinuclidinol.

quinuclidinol.

Figure 3. (a) Magnetic hysteresis curves of Fe3 O4 @SiO2 -NH2 and magnetic combi-CLEAs, Figure 3. (a) Magnetic hysteresis curves of Fe3O4@SiO2-NH2 and magnetic combi-CLEAs, (b) PBS(b) PBS-dispersivity of the magnetic combi-CLEAs and (c) the collection of the magnetic combi-CLEAs dispersivity of the magnetic combi-CLEAs and (c) the collection of the magnetic combi-CLEAs by an byexternal an external magnetic field. magnetic field. Figure 3. (a) Magnetic hysteresis curves of Fe3O4@SiO2-NH2 and magnetic combi-CLEAs, (b) PBSdispersivity of the magnetic combi-CLEAs and (c) the collection of the magnetic combi-CLEAs by an external magnetic field.

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TEM images (Figure 4a) suggested that nearly monodisperse Fe3O4@SiO2-NH2 nanoparticles TEM images (Figure 4a) nanoparticles TEM 4a) suggested suggestedthat thatnearly nearlymonodisperse monodisperseFeFe 3O 4@SiO22-NH -NH22 nanoparticles 3O 4 @SiO were successfully prepared and the average size was about 50 nm. A relatively smooth surface was were successfully successfully prepared and the average size was about 50 nm. A relatively smooth smooth surface surface was obtained because of silica coating. Compared with these bare particles, it was obviously observed obtained because because of of silica coating. Compared Compared with with these these bare bare particles, particles, it it was obviously observed observed obtained that combi-CLEAs were inlayed within magnetic nanoparticles (The combi-CLEAs embedded in the that combi-CLEAs combi-CLEAs were inlayed within magnetic magnetic nanoparticles nanoparticles (The (The combi-CLEAs combi-CLEAs embedded in the that nanoparticles was indicated by the red arrow), clearly showing a well-defined spherical appearance nanoparticles was was indicated indicated by by the the red red arrow), clearly showing a well-defined spherical appearance appearance nanoparticles (Figure 4b). Based on morphology, the magnetic combi-CLEAs are classified as type 1 which permit Based on on morphology, morphology, the magnetic combi-CLEAs combi-CLEAs are classified as type 1 which permit (Figure 4b). Based better mass transfer. Furthermore, the surface of the magnetic combi-CLEAs showed relatively high better mass mass transfer. transfer. Furthermore, Furthermore, the surface surface of the magnetic combi-CLEAs combi-CLEAs showed showed relatively high better numbers of perforations that might facilitate the substrates/cofactors transfer, leading to superior numbers of perforations that might facilitate the substrates/cofactors transfer, leading leading to to superior superior numbers substrates/cofactors transfer, catalytic efficiency. catalytic efficiency. catalytic

Figure 4. (a) TEM images of the Fe3O4@SiO2-NH2 and (b) FE-SEM images of the magnetic combiFigure 4. (a) (a) TEM TEMimages images O4@SiO 2-NH2 and (b) FE-SEM images of the magnetic combiFigure 4. of of thethe Fe3Fe O43@SiO CLEAs. 2 -NH2 and (b) FE-SEM images of the magnetic combi- CLEAs. CLEAs.

2.2. Screening Screening of of Precipitant Precipitant Agents Agents for for Magnetic Magnetic Combi-CLEAs Preparation 2.2. 2.2. Screening of Precipitant Agents for Magnetic Combi-CLEAs Preparation Activityrecovery recoveryofofthe theimmobilized immobilizedenzyme enzyme a key cost determining parameter of CLEAs. Activity is is a key cost determining parameter of CLEAs. To Activity recovery of the immobilized enzyme is a key cost determining parameter of CLEAs. To To preserve the maximum enzyme activity in magnetic combi-CLEAs, it is essential to optimize preserve the maximum enzyme activity in magnetic combi-CLEAs, it is essential to optimize different preserve the maximum enzyme activity in magnetic combi-CLEAs, it is essential to optimize different different parameters for the preparation of magnetic combi-CLEAs. an efficient precipitation of parameters for the preparation of magnetic combi-CLEAs. Firstly,Firstly, an efficient precipitation of all parameters for the preparation of magnetic combi-CLEAs. Firstly, an efficient precipitation of all all enzymes in active its active form is of significant importance the preparationofofmagnetic magneticcombi-CLEAs. combi-CLEAs. enzymes in its form is of significant importance inin the preparation enzymes in its active form is of significant importance in the preparation of magnetic combi-CLEAs. Therefore, five precipitants including ammonium sulfate, sulfate, acetone, acetone, ethanol, ethanol, isopropanol, isopropanol, DMSO DMSO were were Therefore, Therefore, five precipitants including ammonium sulfate, acetone, ethanol, isopropanol, DMSO were tested to to evaluate evaluate their their abilities abilities of of precipitating precipitating QNR QNR and and GDH. GDH. After After the the formation formation of of physical physical tested tested to evaluate their abilities of precipitating QNR and GDH. After the formation of physical aggregates, the the precipitate precipitate was was redissolved, redissolved, then then the the remaining remaining activities activities were were measured. measured. Figure Figure 55 aggregates, aggregates, the precipitate was redissolved, then the remaining activities were measured. Figure 5 showed ammonium sulfate acting as the best precipitating agent compared to four organic solvents, showed ammonium sulfate acting as the best precipitating agent compared to four organic solvents, showed ammonium sulfate acting as the best precipitating agent compared to four organic solvents, and 97.2% and activity waswas respectively achieved for QNR and GDH, which which agrees and and98.5% 98.5%ofofrecovered recovered activity respectively achieved for QNR and GDH, and 97.2% and 98.5% of recovered activity was respectively achieved for QNR and GDH, which with previous studies studies [47,48]. [47,48]. Ammonium sulfate was the was best the option it can usually agrees with previous Ammonium sulfate bestbecause option because it canpreserve usually agrees with previous studies [47,48]. Ammonium sulfate was the best option because it can usually the enzyme of the biocatalyst While[47,48]. ethanol,While isopropanol, DMSO and acetone exhibited preserve theactivity enzyme activity of the[47,48]. biocatalyst ethanol, isopropanol, DMSO and preserve the enzyme activity of the biocatalyst [47,48]. While ethanol, isopropanol, DMSO and relatively lower therelatively recoveredlower activity: and 27.6%, 58.7% and 36.1%, 54.8% andand 57.9%, and54.8% 85.6% acetone exhibited the 35.3% recovered activity: 35.3% and 27.6%, 58.7% 36.1%, acetone exhibited relatively lower the recovered activity: 35.3% and 27.6%, 58.7% and 36.1%, 54.8% and 57.9%, 76.2% for and Therefore, ammoniumTherefore, sulfate was chosen assulfate precipitant and andQNR 85.6% andGDH, 76.2%respectively. for QNR and GDH, respectively. ammonium was and 57.9%, and 85.6% and 76.2% for QNR and GDH, respectively. Therefore, ammonium sulfate was agent for magnetic preparation. chosen as further precipitant agentcombi-CLEAs for further magnetic combi-CLEAs preparation. chosen as precipitant agent for further magnetic combi-CLEAs preparation.

Figure Figure 5. 5. Effect Effectof ofprecipitants precipitantson onactivity activityrecovery recovery of ofthe themagnetic magneticcombi-CLEAs. combi-CLEAs. The Themeasurements measurements Figure 5. Effect of precipitants on activity recovery of the magnetic combi-CLEAs. The measurements were performed in triplicate. were performed in triplicate. were performed in triplicate.

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2.3. Effect of Cross-Linker Concentration and Cross-linking Time on Activity Recovery of Magnetic CombiCLEAs 2.3. Effect of Cross-Linker Concentration and Cross-linking Time on Activity Recovery of Magnetic Combi-CLEAs GA is a bialdehyde functional molecule commonly used as a cross-linking reagent to prepare GAHowever, is a bialdehyde functional used as a cross-linking reagent to prepare CLEAs. it is important to molecule note that commonly if too little cross-linker and cross-linking time are not CLEAs. to However, it is important note thatof if enzyme too littlemolecules, cross-linker and too cross-linking time areand not enough form CLEAs that leads to leaching while much cross-linker enough to form CLEAs that leads to leaching of enzyme molecules, while too much cross-linker cross-linking time can cause the destruction of enzyme’s flexibility which is essential for its activity and cross-linking time maximum can cause enzyme the destruction flexibility which is forthe its [39,49,50]. To preserve activity of in enzyme’s the biocatalysts, therefore, weessential optimized activity [39,49,50]. To preserve maximum enzyme activity in the biocatalysts, therefore, we optimized concentration of cross-linker and cross-linking time. As shown in Figure 6, the activity recovery of the concentration cross-linker and cross-linking As shown Figure 6, recovery of QNR and GDH inofthe magnetic combi-CLEAs wastime. increased within increase in the GAactivity concentration and QNR and GDH in and the magnetic wasfor increased increase GA concentration cross-linking time over 93%combi-CLEAs activity recovery the twowith enzymes wasinobtained at 20 mM and GA cross-linking time and over 93%Therefore, activity recovery the two enzymes wasofobtained at 20 mM GA and and 3 h cross-linking time. in the for further optimization magnetic combi-CLEAs 3 h cross-linking time.20 Therefore, the further optimizationtime of magnetic combi-CLEAs preparation preparation process, mM GAinand 3 h cross-linking were determined as the optimal process, 20 mM GA and 3 h cross-linking time were determined as the optimal conditions. conditions.

Figure 6. Effect Effect of of GA GAconcentration concentration(a) (a)and andcross-linking cross-linking time activity recovery of QNR Figure 6. time (b)(b) on on thethe activity recovery of QNR and and GDH in the magnetic combi-CLEAs. The measurements were performed in triplicate. GDH in the magnetic combi-CLEAs. The measurements were performed in triplicate.

2.4. for Magnetic Magnetic Combi-CLEAs Combi-CLEAs Activity Activity Recovery Recovery 2.4. Optimal Optimal pH pH and and Temperature Temperature for The ◦ C. The influence influence of of pH pH on on enzyme enzyme activity activity was was investigated investigated in in the the range range from from 5.0 5.0 to to 10.0 10.0 at at 25 25 °C. As can be seen from Figure 7a, for the free enzyme the optimized pH was at 7.0, which shifted to pH As can be seen from Figure 7a, for the free enzyme the optimized pH was at 7.0, which shifted to pH 8.0 8.0 for the magnetic combi-CLEAs. incould pH could betodue the change in acidic andamino basic for the magnetic combi-CLEAs. The The shiftshift in pH be due theto change in acidic and basic amino acid sideionization chain ionization in the microenvironment around the active and the interaction acid side chain in the microenvironment around the active site, andsite, the interaction between between the enzyme and cross-linking agent [51]. In addition, the relative activity of magnetic combithe enzyme and cross-linking agent [51]. In addition, the relative activity of magnetic combi-CLEAs was CLEAs was always kept those higherofthan of at free enzyme various pH value. Nevertheless, pH always kept higher than free those enzyme various pHatvalue. Nevertheless, at pH 9.0–10.0atboth 9.0–10.0 both theand freethe enzyme and combi-CLEAs the magnetic combi-CLEAs activity, the free enzyme magnetic had significanthad losssignificant of activity,loss butofthe activitybut of the the activity of the latter decreases slower than the former. The influence of temperature on the relative latter decreases slower than the former. The influence of temperature on the relative activity of the free activity the free enzymecombi-CLEAs and the magnetic combi-CLEAs was monitored byactivity measuring enzyme enzyme of and the magnetic was monitored by measuring enzyme as a function activity as a function of temperature between 15 and 50°C after 4 h incubation. The optimum ◦ of temperature between 15 and 50 C after 4 h incubation. The optimum temperature of the former temperature of the former wasshowed 25 °C, whereas the latter highest activity at 30 °C (Figure 7b). was 25 ◦ C, whereas the latter highest activity atshowed 30 ◦ C (Figure 7b). Nevertheless, the relative Nevertheless, activity the free enzyme and magnetic combi-CLEAs was activity of boththe the relative free enzyme and of theboth magnetic combi-CLEAs wasthe gradually decreased with further gradually decreased with further increase in temperature, but the activity of the latter still decreases increase in temperature, but the activity of the latter still decreases slower than the former. The data slower than thethe former. Thecombi-CLEAs data suggested that the magnetic had contributed the suggested that magnetic had contributed to thecombi-CLEAs resilience of the enzyme againsttohigh resilience of the enzyme against highresults temperature, agreed with This the results obtained byto XRD temperature, which agreed with the obtainedwhich by XRD analysis. was probably due the analysis. This was probably due to the fact that the Fe 3O4@SiO2-NH2 nanoparticles could retarded fact that the Fe3 O4 @SiO2 -NH2 nanoparticles could retarded heat transfer and collapse of the CLEAs heat transfer and collapse of the structure test temperatures, andenhanced the rigidity of the active structure at test temperatures, andCLEAs the rigidity of theat active conformation was by cross-linking conformation was enhanced byaggregates cross-linking the formation of enzyme [52]. In during the formation of enzyme [52].during In addition, good storage stability aggregates is another essential addition, good storage stability is another essential requirement for a biocatalyst to be employed in requirement for a biocatalyst to be employed in industrial applications. The free enzyme and the industrial applications. The free enzyme andatthe were stored in PBS at 4free °C magnetic combi-CLEAs were stored in PBS 4 ◦magnetic C for a 70combi-CLEAs days period. Storage stability of the for a 70 days period. Storage stability of the free enzyme and magnetic combi-CLEAs was presented enzyme and magnetic combi-CLEAs was presented in Figure 7c. The magnetic combi-CLEAs still in Figureabout 7c. The magnetic combi-CLEAs still70retained about 80% its initial activity after 70 28.6% days. retained 80% of its initial activity after days. However, theoffree enzyme retained only However, free enzyme retained only and 28.6% and respectively. 30.5% of its initial activity for QNR stability and GDH, and 30.5%the of its initial activity for QNR GDH, The improved storage of respectively. The improved storage stability of magnetic combi-CLEAs might be due to the covalent

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magnetic combi-CLEAs might be due to the covalent cross-linking between the enzyme aggregates by GA, which avoided enzyme leaching, denaturation andwhich autodigestion, and limited the conformational cross-linking between the enzyme aggregates by GA, avoided enzyme leaching, denaturation changes of the enzyme. and autodigestion, and limited the conformational changes of the enzyme.

Figure 7. Effect Effect of of pH pHand andtemperature temperatureononthe the free enzyme and magnetic combi-CLEAs activity. (a) Figure 7. free enzyme and magnetic combi-CLEAs activity. (a) pH, pH, (b) temperature and (c) storage stability of the free enzyme and magnetic combi-CLEAs. Activity (b) temperature and (c) storage stability of the free enzyme and magnetic combi-CLEAs. Activity was was assayed °C standard in the standard at pHThe 7.5–8.0. The measurements were assayed at 25 ◦atC 25 in the reactionreaction mediummedium at pH 7.5–8.0. measurements were performed performed in triplicate.in triplicate.

2.5. Time-Course of Biosynthesis of (R)-3-quinuclidinol 2.5. Time-Course of Biosynthesis of (R)-3-quinuclidinol The ability of the biocatalysts to catalyze the synthesis of (R)-3-quinuclidinol with substrateThe ability of the biocatalysts to catalyze the synthesis of (R)-3-quinuclidinol with substrate-coupled coupled cofactor regeneration was tested (Scheme 2). The biotransformation of 3-quinuclidinone into cofactor regeneration was tested (Scheme 2). The biotransformation of 3-quinuclidinone into (R)-3-quinuclidinol was carried out by the free enzyme at 25 °C, pH 7.0 or by the magnetic combi(R)-3-quinuclidinol was carried out by the free enzyme at 25 ◦ C, pH 7.0 or by the magnetic CLEAs at 30 °C, pH◦ 8.0, respectively, and glucose was used as co-substrate. The samples were combi-CLEAs at 30 C, pH 8.0, respectively, and glucose was used as co-substrate. The samples withdrawn at appropriate intervals from reaction system and analyzed by gas chromatography (GC). were withdrawn at appropriate intervals from reaction system and analyzed by gas chromatography The results showed (Figure 8a) that the magnetic combi-CLEAs completely transformed 3(GC). The results showed (Figure 8a) that the magnetic combi-CLEAs completely transformed quinuclidinone into the corresponding (R)-3-quinuclidinol with 100% ee and 100% conversion yield 3-quinuclidinone into the corresponding (R)-3-quinuclidinol with 100% ee and 100% conversion in 3 h, which is about two times faster than that of free enzyme. The conversion yield and ee value yield in 3 h, which is about two times faster than that of free enzyme. The conversion yield and ee agreed with the pervious reported results [18,53]. Three of the employed enzymes, QNR in this study value agreed with the pervious reported results [18,53]. Three of the employed enzymes, QNR in from Microbacterium luteolum JCM9174, ArQR from Agrobacterium radiobacter ECU2556 [18] and RrQR this study from Microbacterium luteolum JCM9174, ArQR from Agrobacterium radiobacter ECU2556 [18] from Rhodotorula rubra [53], were novel enzymes belonging to the short-chain alcohol and RrQR from Rhodotorula rubra [53], were novel enzymes belonging to the short-chain alcohol dehydrogenase/reductase superfamily due to their low amino acid identity between each other. The dehydrogenase/reductase superfamily due to their low amino acid identity between each other. difference in reaction velocity and reaction time may be due to the difference in the biocatalysts The difference in reaction velocity and reaction time may be due to the difference in the biocatalysts loading, cofactor concentration and enzyme intrinsic property. The intermediate in samples could be loading, cofactor concentration and enzyme intrinsic property. The intermediate in samples could also displayed by thin layer chromatography (TLC) and stained by iodine, suggesting that the be also displayed by thin layer chromatography (TLC) and stained by iodine, suggesting that the versatile semi-quantitative method can be used for determining the changes in the concentration of versatile semi-quantitative method can be used for determining the changes in the concentration of substrate and products in the reaction progress with time(Figure 8b for free enzyme, and c for the substrate and products in the reaction progress with time(Figure 8b for free enzyme, and c for the magnetic combi-CLEAs). Compared to the ever reports [18,53], The shorter period should be magnetic combi-CLEAs). Compared to the ever reports [18,53], The shorter period should be attributed attributed to the fast diffusion of 3-quinuclidinone between QNR and GDH due to the short distance to the fast diffusion of 3-quinuclidinone between QNR and GDH due to the short distance between between QNR and GDH in the magnetic combi-CLEAs. Hence, co-immobilizing QNR and GDH in magnetic combi-CLEAs is an effective strategy for asymmetric synthesis (R)-3-quinuclidinol.

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QNR and GDH in the magnetic combi-CLEAs. Hence, co-immobilizing QNR and GDH in magnetic combi-CLEAs anPEER effective strategy for asymmetric synthesis (R)-3-quinuclidinol. Catalysts 2018, 8, x is FOR REVIEW 9 of 16 Catalysts 2018, 8, x FOR PEER REVIEW

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Scheme 2. The magnetic combi-CLEAs biocatalysts for the production of (R)-3-quinuclidinol with Scheme 2.2. The combi-CLEAs Schemeregeneration The magnetic magnetic combi-CLEAs biocatalysts biocatalysts for for the the production production of of(R)-3-quinuclidinol (R)-3-quinuclidinol with with cofactor in situ. cofactor regeneration in situ. cofactor regeneration in situ.

Figure 8. Reaction time-course for the bioreduction of 3-quinuclidinone into (R)-3-quinuclidinol Figure 8.8. by Reaction time-course for the bioreduction of 3-quinuclidinone 3-quinuclidinone into (R)-3-quinuclidinol (R)-3-quinuclidinol Figure Reaction bioreduction of catalyzed the freetime-course enzyme or for the the magnetic combi-CLEAs, respectively. into Samples (0.5 mL) were catalyzed by the free enzyme or the magnetic combi-CLEAs, respectively. Samples (0.5 mL) mL) were were catalyzed by enzymeintervals or the magnetic combi-CLEAs, Samples (0.5 withdrawn at the the free appropriate and analyzed by GC (a) respectively. or by TLC (b,c). withdrawnat atthe theappropriate appropriateintervals intervalsand andanalyzed analyzedby byGC GC(a) (a)or orby byTLC TLC(b,c). (b,c). withdrawn

For applications of ketoreductase, recycling is of significant importance. The main advantage of For applications of ketoreductase, recycling is of significant importance. The mainresulted advantage of the magnetic combi-CLEAs is its easyrecycling separation the reaction mixtures. in of a For applications of ketoreductase, is offrom significant importance. The This main advantage the magnetic combi-CLEAs is its easy separation from the reaction mixtures. This resulted in a complete recovery of the immobilized enzymes and cost-effective industrial applications [51]. In our the magnetic combi-CLEAs is its easy separation from the reaction mixtures. This resulted in a complete complete recovery of the immobilized enzymes and cost-effective industrial applications [51]. In our experience, theimmobilized magnetic combi-CLEAs eminently recyclable. After each cycle, theexperience, magnetic recovery of the enzymes and are cost-effective industrial applications [51]. In our experience, the magnetic combi-CLEAs are eminently recyclable. After each cycle, the magnetic combi-CLEAs could be successfully separated from After the reaction medium using a combi-CLEAs magnet, and the magnetic combi-CLEAs are eminently recyclable. each cycle, the magnetic combi-CLEAs could be successfully separated from the reaction medium using biocatalyst a magnet, was and employed again in the next cycle. As shown in Figure 9a, the magnetic combi-CLEAs could be successfully separated from the reaction medium using a magnet, and employed again in employed again the nextand cycle. shown in Figure 9a,time the magnetic combi-CLEAs biocatalyst able to cycle upAs toin10 runs, theAs complete conversion for each run was 3,was 3, 3.5, 4, 4,was 5, the next cycle. shown in Figure 9a, the magnetic combi-CLEAs biocatalyst able3.5, to 4, cycle up able to cycle up to 10 runs, and the complete conversion time for each run was 3, 3, 3.5, 3.5, 4, 4, 4,3-5, 5.5 and 7 h, respectively. After the successive 7th cycle, reduction of 0.33 g (2.0 mmol) to 10 runs, and the complete conversion time for each run was 3, 3, 3.5, 3.5, 4, 4, 4, 5, 5.5 and 7 h, 5.5 and 7 h, respectively. the successive 7th cycle, of 30.33 g of (2.0 mmol) 3quinuclidinone with mg ofAfter the7th magnetic combi-CLEAs thereduction presence of mmol glucose respectively. After the50 successive cycle, reduction of 0.33in g (2.0 mmol) 3-quinuclidinone with 50and mg quinuclidinone with 50 mg ofadenine the magnetic combi-CLEAs in the presence of 3 conversion mmol of glucose and +) afforded 6.6 mg reduced nicotinamide dinucleotide (NAD over 100% efficiency of the magnetic combi-CLEAs in the presence of 3 mmol of+ glucose and 6.6 mg reduced nicotinamide 6.6 mg reduced nicotinamide adenine dinucleotide (NADat) 10th afforded over 100% conversion efficiency + ) afforded and (R)-3-quinuclidinol in 100% ee at 300 min.100% However, cycle, it took min to reach 100% adenine dinucleotide (NAD over conversion efficiency and 420 (R)-3-quinuclidinol in and (R)-3-quinuclidinol in 100% ee at 300 min. However, at 10th cycle, it took 420 min to reach 100% yield, 180 min longer than the 7th cycle. The residual activity of the magnetic combi-CLEAs during 100% ee at 300 min. However, at 10th cycle, it took 420 min to reach 100% yield, 180 min longer than yield, 180 min longer the 7th cycle. The residual activity ofthat the the magnetic combi-CLEAs during the wasthan alsoactivity presented in Figure 9b.combi-CLEAs It was found activity of magnetic combithe reaction 7th cycle.cycles The residual of the magnetic during the reaction cycles was also the reaction cycles was also presented in Figure 9b. It was found that the activity of magnetic combiCLEAs was to the 7th andofdecreased approximately 54.3% and presented inwell-maintained Figure 9b. It wasup found that thebatch activity magnetic to combi-CLEAs was59.1%, well-maintained CLEAs well-maintained upQNR, to theand 7th batch decreased to approximately 54.3% and 43.5% ofwas its activity for 60.9%,and 58.3% 45.8% GDH at original 8,59.1%, 9 andactivity 10 cycles, up to the 7thoriginal batch and decreased to approximately 59.1%,and 54.3% and for 43.5% of its for 43.5% of its original activity for QNR, and 60.9%, 58.3% and 45.8% for GDH at 8, 9 and 10 cycles, respectively. The obvious decrease of the catalytic after the 7th cycle, especially in the 9th and QNR, and 60.9%, 58.3% and 45.8% for GDH at 8, 9 activity and 10 cycles, respectively. The obvious decrease of respectively. Thebe obvious decrease of the catalytic activity after the 7thofcycle, especiallycombi-CLEAs in the 9th and 10th runs, could due the denaturation, collapse the magnetic the catalytic activity afterto the 7thenzyme cycle, especially in thepartial 9th and 10th runs, could be due to the enzyme 10thsubstantial runs, could be duefrom to the enzyme denaturation, partial collapse the magnetic combi-CLEAs and leakage support intocombi-CLEAs the reaction batch duringofrepeated use. denaturation, partial collapse the of the magnetic and substantial leakage from the support and substantial leakage from the support into the reaction batch during repeated use. into the reaction batch during repeated use.

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Figure 9.9.Reusability Reusability of of magnetic combi-CLEAs for the the bioreduction of 3-quinuclidinone 3-quinuclidinone into (R)-3(R)-3Figure of magnetic combi-CLEAs for of into Figure 9. Reusability magnetic combi-CLEAs for bioreduction the bioreduction of 3-quinuclidinone into quinuclidinol, (a) conversion yield of 3-quinuclidinone and ee of (R)-3-quinuclidinol; (b) relative quinuclidinol, (a) conversion yield of 3-quinuclidinone and ee of (R)-3-quinuclidinol; (b) relative (R)-3-quinuclidinol, (a) conversion yield of 3-quinuclidinone and ee of (R)-3-quinuclidinol; (b) relative activity of QNR and GDH in magnetic magnetic combi-CLEAs. activity of of QNR QNR and and GDH GDH in magnetic combi-CLEAs. activity

In addition, addition, we also also utilized utilized the the FE-SEM FE-SEM to to further investigate investigate the the change change of of the the magnetic magnetic combicombiIn In addition,we we also utilized the FE-SEMfurther to further investigate the change of the magnetic CLEAs surface surface structure structure during during the the cycles cycles of of repeated repeated use. use. The results results revealed revealed that that almost almost no no CLEAs combi-CLEAs surface structure during the cycles of repeated The use. The results revealed that almost obvious morphologic change was observed, and the magnetic biocatalyst presented a spherical or obvious morphologic change was observed, and the no obvious morphologic change was observed, and themagnetic magneticbiocatalyst biocatalystpresented presentedaaspherical spherical or or nearly spherical structure even after the seventh cycle (Figure 10a–g), indicating that the morphologic nearly nearly spherical spherical structure structure even even after after the the seventh seventh cycle cycle (Figure (Figure 10a–g), 10a–g), indicating indicating that that the the morphologic morphologic integrity of of the magnetic magnetic combi-CLEAs was was obviously in in close agreement agreement with its its enzyme activity activity integrity integrity of the the magnetic combi-CLEAs combi-CLEAs was obviously obviously in close close agreement with with its enzyme enzyme activity (Figure 9b). As can be seen from Figure 10h–j, the eighth, ninth and tenth cycles experienced partial (Figure (Figure 9b). 9b). As As can can be be seen seen from from Figure Figure 10h–j, 10h–j, the the eighth, eighth, ninth ninth and and tenth tenth cycles cycles experienced experienced partial partial collapse of of spherical structure, structure, the disappearance disappearance of small small hole present present on the the surface of of the magnetic magnetic collapse collapse of spherical spherical structure, the the disappearance of of small hole hole present on on the surface surface of the the magnetic combi-CLEAs and and then aggregation aggregation into aa bigger bigger size of of the the combi-CLEAs, combi-CLEAs, respectively. respectively. Because Because combi-CLEAs combi-CLEAs and then then aggregation into into a bigger size size of the combi-CLEAs, respectively. Because particle size of the combi-CLEAs can affect mass transfer of the substrates and prevent substrates particle particle size size of of the the combi-CLEAs combi-CLEAs can can affect affect mass mass transfer transfer of of the the substrates substrates and and prevent prevent substrates substrates contacting with with the the inner inner enzymes enzymes of of the the combi-CLEAs, combi-CLEAs, the the inner inner enzymes enzymes present present in the the bigger bigger size size contacting contacting with the inner enzymes of the combi-CLEAs, the inner enzymes present inin the bigger size of of the magnetic combi-CLEAs were inaccessible and could not form complexes with the substrates of magnetic combi-CLEAs inaccessible and could not complexes form complexes with the substrates thethe magnetic combi-CLEAs werewere inaccessible and could not form with the substrates [48,54], [48,54], hence, hence, leading leading to to the the loss loss of of enzyme enzyme activity activity of of the the magnetic magnetic combi-CLEAs. combi-CLEAs. From From another [48,54], hence, leading to the loss of enzyme activity of the magnetic combi-CLEAs. From another pointanother of view, point of of view, view, the the change change in in surface surface structure structure of of magnetic magnetic combi-CLEAs combi-CLEAs supports supports the the value value of of using using point the change in surface structure of magnetic combi-CLEAs supports the value of using combi-CLEAs in combi-CLEAs in combination with the Fe 3 O 4 @SiO 2 -NH 2 to yield magnetic combi-CLEAs with combi-CLEAs in combination with the Fe3O4@SiO2-NH2 to yield magnetic combi-CLEAs with combination with the Fe3 O4 @SiO 2 -NH2 to yield magnetic combi-CLEAs with excellent resistance to excellent resistance resistance to to mechanical mechanical stress, stress, such such as as shake shake or or agitate agitate and and wash, wash, allowing allowing high high recovery recovery excellent mechanical stress, such as shake or agitate and wash, allowing high recovery of the biocatalyst after of the biocatalyst after many cycles and providing (R)-3-quinuclidinol in high ee and yield. of the biocatalyst after many cycles and providing (R)-3-quinuclidinol in high ee and yield. many cycles and providing (R)-3-quinuclidinol in high ee and yield.

Figure 10. Cont.

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Figure Figure 10. 10. The The morphology morphology change change of of image image of of the themagnetic magnetic combi-CLEAs combi-CLEAs during during 10 10 cycles cycles observed observed by by FE-SEM. FE-SEM.

3. Materials and Methods 3. Materials and Methods 3.1. 3.1. Materials Materials Recombinant Recombinant plasmid plasmid pET28a-QNR pET28a-QNRand and pET28a-GDH pET28a-GDHwas wasprovided providedby byTaihe Taihe (Beijing, (Beijing,China). China). Competent cells E. coli BL(21) (DE3) was from Tiangen (Beijing, China). Page Ruler Prestained Protein Competent cells E. coli BL(21) (DE3) was from Tiangen (Beijing, China). Page Ruler Prestained Ladder was form Fisher Scientific (Waltham, MA, Protein Ladder was Thermo form Thermo Fisher Scientific (Waltham, MA,USA). USA). Isopropy-l-βIsopropy-l-β-DD-thiogalacto-thiogalactopyranoside (IPTG), 3-aminopropyltriethoxysilane (APTES), Tetraethylorthosilicate all pyranoside (IPTG), 3-aminopropyltriethoxysilane (APTES), Tetraethylorthosilicate (TEOS),(TEOS), all reagents reagents for sodium polyacrylamide gel electrophoresis (SDS-PAGE), 3-quinuclidone for sodium dodecyldodecyl sulfate sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 3-quinuclidone and and standard (R)-3-quinuclidinol were obtained from Sigma-Aldrich (Shanghai, China). Other standard (R)-3-quinuclidinol were obtained from Sigma-Aldrich (Shanghai, China). Other chemicals chemicals andwere reagents were the highest purityThe available. Thewater deionized water was ultrapure and reagents the highest purity available. deionized was ultrapure produced by produced by Millipore system (Bedford, MA, USA). Millipore system (Bedford, MA, USA). 3.2. Expression Expression of of Recombinant Recombinant Enzyme QNR and GDH Recombinant plasmid of pET28a-QNR and pET28a-GDH was transformed into competent E. coli BL(21)(DE3), and the resulting cells were respectively denoted as E. coli (QNR) and E. coli (GDH) (GDH) and and cultivated in inLuria LuriaBroth Broth medium supplemented 100 μg/mL of kanamycin 37overnight °C. The medium supplemented with with 100 µg/mL of kanamycin at 37 ◦ C.atThe overnight culture was inoculated andatgrown 37 °C. Expression of the target enzyme was induced culture was inoculated and grown 37 ◦ C.atExpression of the target enzyme was induced until until OD600 reached by adding to aconcentration final concentration 0.2Cells mM. growth Cells growth OD600 reached aboutabout 0.6 by0.6 adding IPTG IPTG up to up a final of 0.2 of mM. along ◦ C.hThe along with enzyme expression continued for2548 at 25 Thethen cells were then pelleted by with enzyme expression continued for 48 h at cells°C. were pelleted by centrifugation ◦ centrifugation (12,000× g for 5 min) at 4 °C, washed using ice PBS, 7.4) and resuspended in mLgram PBS (12,000× g for 5 min) at 4 C, washed using ice PBS, 7.4) and resuspended in 50 mL PBS50per per cells. cells were lysed using XC-CD2500 Ultrasonic Oscillator (Ningbo, wetgram cells. wet Then theThen cells the were lysed using XC-CD2500 Ultrasonic Oscillator (Ningbo, China)China) on ice. ◦ on ice. The supernatant was collected by centrifugation (15,000× g for 20 min) at 4 °C. After filtrated The supernatant was collected by centrifugation (15,000× g for 20 min) at 4 C. After filtrated by by filter membrane, supernatant analyzed by SDS-PAGE concentration of 0.220.22 µmμm filter membrane, thethe supernatant waswas analyzed by SDS-PAGE andand the the concentration of the the proteins determined by Bradford protein assay proteins waswas determined by Bradford protein assay [55].[55]. Enzyme Activity Activity Assay Assay 3.3. Enzyme Enzyme activity activity was wasassayed assayedspectrophotometrically spectrophotometricallyatat2525°C◦ C monitoring absorbance Enzyme byby monitoring thethe absorbance at at 340 corresponding thechange changeininoxidized oxidizednicotinamide nicotinamide adenine adenine dinucleotide dinucleotide (NADH) (NADH) 340 nmnm corresponding to tothe was consisted of PBS, 3 µmol 3-quinuclidinone and 0.3 µmol concentration [17]. [17]. 11mL mLofofassay assaymixture mixture was consisted of PBS, 3 μmol 3-quinuclidinone and 0.3 + for GDH), NADH for QNR 10 µmol and 1 µmol and an and appropriate amountamount of QNR μmol NADH for (or QNR (or 10glucose μmol glucose and 1NAD μmol NAD+ for GDH), an appropriate of QNR or GDH. In the case of the magnetic combi-CLEAs, the magnetic biocatalysts were separated using a magnet from the reaction mixture and then the appearance of NADH was measured. One

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or GDH. In the case of the magnetic combi-CLEAs, the magnetic biocatalysts were separated using a magnet from the reaction mixture and then the appearance of NADH was measured. One unit of QNR or GDH activity was defined as the amount of enzyme that catalyzed 1 µmol NADH or NAD+ per minute. All experiments were performed in triplicates. 3.4. Immobilization of QNR and GDH 3.4.1. Preparation of NH2 -Functionalized Fe3 O4 Nanoparticles (Fe3 O4 @SiO2 -NH2 ) Magnetic Fe3 O4 nanoparticles were prepared according to the pervious reported method [56]. In brief, 0.27 g FeCl3 ·6H2 O and 0.72 g NaAc dissolved in 10 mL of ethylene glycol under stirring. Then the resulting yellow solution was further treated at 200 ◦ C for 10 h in the Teflon-sealed autoclaves. The final products were washed with ethanol and water several times, used freshly or dried by vacuum freeze-drying. To functionalize the Fe3 O4 nanoparticles with -NH2 groups, 100 mg of freshly prepared Fe3 O4 nanoparticles were homogeneously dispersed in 100 mL mixture solution containing ethanol and water in a volume ratio of 4:1 and 1 mL of concentrated NH3 ·H2 O. 100 µL of TEOS was added and the mixture was allowed to react with vigorous stirring for 6.0 h at 50 ◦ C. The resulting Fe3 O4 @SiO2 nanoparticles were separated by a magnetic field, washed and redispersed in a mixed solution containing ethanol and water in a volume ratio of 3:4 and 1 mL concentrated NH3 ·H2 O, followed by the drop-wise addition of 300 µL APTES with vigorous stirring. After 6 h, the precipitate was washed and denoted as Fe3 O4 @SiO2 -NH2 nanoparticles. 3.4.2. Preparation of Magnetic Combi-CLEAs of QNR and GDH Magnetic combi-CLEAs of QNR and GDH were prepared according the pervious reported method [37]. Typically, 10 mg Fe3 O4 @SiO2 -NH2 nanoparticles were incubated with 2 mL of the cell lysate containing same units of QNR and GDH (total protein concentration was 4.5 mg/mL) in the presence of precipitant agents with continuous shaking at 4 ◦ C for 30 min to complete precipitate enzymes. Then chilled GA was added up to the appropriate concentration and the stirring continued for 3 h at 4 ◦ C. The pellets were collected using magnetic field. Unreacted GA and unbound enzymes were removed by washing with PBS. The final magnetic combi-CLEAs obtained were kept in PBS at 4 ◦ C. The effects of precipitants ((NH4 )2 SO4 , isopropanol, ethanol, DMSO and acetone), GA concentration and cross-linking time on the activity recovery were tested. The activity recovery of QNR and GDH in the magnetic combi-CLEAs was determined by following equation [40,47]. Activity recovery(%) =

Total activity o f each enzyme in magnetic combi −CLEAs(U ) Total starting activity o f each enzyme used f or magnetic combi −CLEAs(U )

× 100

Subsequently, the influence of pH and temperature on magnetic combi-CLEAs activities was also studied. 3.5. Application of Magnetic Combi-CLEAs for Biosynthesis (R)-3-quinuclidinol To test the ability of the biocatalysts to biotransform 3-quinuclidone to (R)-3-quinuclidinol, the reaction was carried out as follows: 50 mg magnetic combi-CLEAs, 0.33 g 3-quinuclidinone (2.0 mmol), 0.54 g glucose (3.0 mmol), 6.6 mg NAD+ were mixed in 10 mL of PBS. The reaction system was incubated at 25 ◦ C with continuous stirring at 100 rpm and the pH was maintained at 7.5–8.0 by titrating 5 M NaOH. Aliquots (0.5 mL) were withdrawn at 0.5, 1, 2, 3, 4, 5 and 6 h, respectively, the conversion of 3-quinuclidone was monitored TLC analysis. Silica gel coated plates were activated at 110 ◦ C for 30 min and 1 µL samples were applied. The mixtures of CH2 Cl2 and CH3 OH (9:1, v/v) was selected as the eluting solution. The resulting chromatography plates were air dried, and stained with iodine. The ee value and conversion yield of 3-quinuclidone was determined by GC according to the previous report [57]. After the reaction completed, the magnetic combi-CLEAs were collected using magnet and washed with PBS.

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3.6. Reusability of Enzymes in Magnetic Combi-CLEAs To estimate the reusability of magnetic combi-CLEAs to produce (R)-3-quinuclidinol under the same reaction conditions as described above, the biocatalysts were recovered with a magnet after each run, washed twice with PBS, and reused again for the next assay cycle. The retained enzyme activity was compared according to that of initial magnetic combi-CLEAs, which was defined as 100%. 3.7. Characterization The morphology and size of the Fe3 O4 @SiO2 -NH2 nanoparticles and the magnetic combi-CLEAs were determined by TEM (JEM-1400 plus Electron Microscope, JEOL, Tokyo, Japan) and FE-SEM (JSM-6700F, Hitachi High-Technologies, JEOL, Tokyo, Japan), respectively. FTIR analysis ofFe3 O4 , Fe3 O4 @SiO2 -NH2 , free enzyme and magnetic combi-CLEAs were carried out on a Thermo Nicolet 6700 FTIR spectrometer (Madison, WI, USA) at a resolution of 4 cm−1 over the wavelength range 4000–400 cm−1 under transmission mode. XRD measurements were conducted on a DX-1000 X-ray diffractometer (Dandong Fanyuan Instrument Co. LTD, Liaoning, China) to investigate the crystal structure by use of copper Kα (λ = 0.154056 nm) as the source of radiation. The sample were scanned in the 2θ range between 3◦ and 80◦ at 2◦ /min with a scan step of 0.02◦ and a filament voltage of 40 kV at room temperature. The magnetic properties were determined under a varying magnetic field by VSM using a physical property measurement system DynaCool9 (Quantum, Denver, CO, USA). 4. Conclusions In summary, we successfully fabricated the magnetic combi-CLEAs by co-precipitation followed by cross-linking QNR, GDH and Fe3 O4 @SiO2 -NH2 nanoparticles. Various preparation conditions were optimized, and the physicochemical properties were characterized. The resulting biocatalysts exhibited high activity retention, improved operational stability and excellent catalytic property for the bioreduction of 3-quinuclidinone to (R)-3-quinuclidinol. Moreover, the biocatalysts maintain the superparamagnetic behavior, allowing for easy separation from the reaction medium and reusability for 10 cycles with 100% ee and 100 conversion yield. Such high catalytic efficiency and excellent magnetic recyclability might make economically viable the application of cost enzymes and thus opens a new horizon for biosynthesis of (R)-3-quinuclidinol. Author Contributions: Y.C., Q.J., Q.C. and W.L. designed the experiments; Y.C., Q.J., L.S., L.Z. and S.L. performed the experiments; L.S., Q.L., L.Z., S.L., M.Y. and W.L. analyzed the data; Y.C., Q.J., Q.C., M.Y. and W.L. wrote the paper. Funding: This research was funded by Municipal Science and Technology Committee of Chongqing: CSTC2014jcyjA0019. Acknowledgments: This work was supported by School of Pharmacy, Chongqing Medical University. Conflicts of Interest: The authors declare no conflict of interest.

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