Magnetic Combined Cross-Linked Enzyme Aggregates of Ketoreductase and Alcohol Dehydrogenase

catalysts

Article 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 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.) 2 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] 3 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 Fe3O4 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 www.mdpi.com/journal/catalysts Catalysts 2018, 8, 334 2 of 16

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-C4H9OK [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 Catalysts 2018, 8, 334 3 of 16 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)2SO4 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) (Figure1). 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. Catalysts 2018, 8, 334 4 of 16 Catalysts 2018, 8, x FOR PEER REVIEW 4 of 16

Catalysts 2018, 8, x FOR PEER REVIEW 4 of 16

Figure 1. 1. 12%12% SDS-PAGE SDS-PAGE gel gel of of protein protein samples. samples. Lane Lane M, M, protein protein molecular molecular weight weight markers; markers; Lane Lane 1 1 to to Lane 4, Lane 5 to Lane 8 for the E. coli (His-QNR), E. coli (His-GDH) before induction, after LaneFigure 4, Lane 1. 12% 5 to SDS-PAGE Lane 8 for gel the ofE. protein coli (His-QNR), samples. LaneE. coli M,(His-GDH) protein molecular before induction,weight markers; after induction, Lane 1 induction, the cell lysate supernatant and the cell lysate precipitate, respectively. theto cell Lane lysate 4, Lane supernatant 5 to Lane and 8 thefor cellthe lysateE. coli precipitate,(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 of QNR-GDH biocatalyst preparation involves a sequence of two physic-chemical steps starting with co-precipitation of QNR, GDH and Fe3O4@SiO2-NH2 Magnetic combi-CLEAs of QNR-GDH biocatalyst preparation involves a sequence of two physic-chemicalnanoparticles followed steps starting by cross-linking with co-precipitation the enzyme aggregates of QNR, GDH with and themselves Fe3O4@SiO and2 -NHNH22- nanoparticlesattached physic-chemical steps starting with co-precipitation of QNR, GDH and Fe3O4@SiO2-NH2 followedon the surfaces by cross-linking of Fe3O4@SiO the2-NH enzyme2 nanoparticles aggregates (Scheme with themselves1), where Fe and3O4@SiO NH22--NH attached2 were onemployed the surfaces nanoparticles followed by cross-linking the enzyme aggregates with themselves and NH2- attached ofas Fesupport3O4@SiO for2 -NHenzyme2 nanoparticles immobilization. (Scheme For 1this), where purpose, Fe 3 Owe4@SiO first2 -NHsynthesized2 were employed magnetic asFe support3O4 on the surfaces of Fe3O4@SiO2-NH2 nanoparticles (Scheme 1), where Fe3O4@SiO2-NH2 were employed fornanoparticles. enzyme immobilization. The nanoparticles For we thisre then purpose, coated we with ﬁrst tetraethylorthosilicate synthesized magnetic (TEOS) Fe3O followed4 nanoparticles. by as support for enzyme immobilization. For this purpose, we first synthesized magnetic Fe3O4 Thefunctionalizing nanoparticles with were APTES then coatedin alkaline with conditio tetraethylorthosilicatens, thus preventing (TEOS) these followed nanoparticles by functionalizing from nanoparticles. The nanoparticles were then coated with tetraethylorthosilicate (TEOS) followed by withclustering APTES and in improving alkaline conditions, their chemical thus stability. preventing The these resultant nanoparticles Fe3O4@SiO from2-NH clustering2 nanoparticles andimproving were functionalizing with APTES in alkaline conditions, thus preventing these nanoparticles from theirmixed chemical with cell stability.lysates containing The resultant QNR Fe andO GDH.@SiO After-NH precipitationnanoparticles and were cross-linking mixed with under cell the lysates clustering and improving their chemical3 stability.4 2The resultant2 Fe3O4@SiO2-NH2 nanoparticles were optimized conditions such as precipitant agents, cross-linker concentration and the cross-linking containingmixed with QNR cell and lysates GDH. containing After precipitation QNR and GDH. and cross-linking After precipitation under theand optimized cross-linking conditions under the such time, the magnetic combi-CLEAs were obtained. To ensure successful preparation of magnetic asoptimized precipitant conditions agents, cross-linker such as precipitant concentration agents, and cross-linker the cross-linking concentration time, the and magnetic the cross-linking combi-CLEAs combi-CLEAs, FTIR, XRD and FE-SEM were employed to confirm these results. weretime, obtained. the magnetic To ensure combi-CLEAs successful preparationwere obtained. of magneticTo ensure combi-CLEAs, successful preparation FTIR, XRD ofand magnetic FE-SEM werecombi-CLEAs, employed to FTIR, conﬁrm XRD these and FE-SEM results. were employed to confirm these results.

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. Firstly, SchemeFTIR analyses 1. The diagramwas carried of the out preparation to detect the of thefunctional magnetic groups combi-CLEAs of Fe3O4 biocatalysts., 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, 578 cmFirstly,−1 was FTIR attributed analyses to wasthe carriedFe-O bond out tostretching detect the vibration, functional and groups the characteristic of Fe O , Fe Opeak@SiO also-NH , free enzyme and magnetic combi-CLEAs. As can be seen form Figure 2A(a), the3 absorption4 3 4 peak2 at 2 appeared in Figure 2A(b,d), indicative of no changes in the structure of Fe3O4 after chemical free578 enzyme cm−1 was and attributed magnetic to combi-CLEAs. the Fe-O bond As stretching can be seen vibration, form Figure and 2theA(a), characteristic the absorption peak peak also at 578modification. cm−1 was The attributed peaks about to theat 3410 Fe-O cm bond−1 and stretching1627 cm−1 were vibration, associated and with-OH the characteristic groups available peak also appeared in Figure 2A(b,d), indicative of no changes in the structure of Fe3O4 after chemical from the surface of Fe3O4. In the Fe3O4@SiO2-NH2 spectrum, the peaks at 465, 790, 940 and 1092 cm−1 appearedmodification. in Figure The peaks2A(b,d), about indicative at 3410 cm of−1 and no changes1627 cm−1 inwere the associated structure with-OH of Fe 3 Ogroups4 after available chemical were assigned to the O-Si-O bending vibration,−1 Si-O-Si symmetric,−1 Si-O symmetric and Si-O-Si modiﬁcation.from the surface The of peaks Fe3O4. aboutIn the Fe at3O 34104@SiO cm2-NHand2 spectrum, 1627 cmthe peakswere at associated465, 790, 940 with-OH and 1092 groupscm−1 asymmetric stretching vibration of the coated silica shell, respectively. The broad band at 3400–3500 availablewere assigned from the to surfacethe O-Si-O of Fe bending3O4. In thevibration, Fe3O4 @SiOSi-O-Si2-NH symmetric,2 spectrum, Si-O thesymmetric peaks at and 465, Si-O-Si 790, 940 cm−1 can be ascribed− to the characteristic peaks of NH2− groups of APTES which were overlain by andasymmetric1092 cm 1stretchingwere assigned vibration to theof the O-Si-O coated bending silica shell, vibration, respectively. Si-O-Si The symmetric, broad band Si-O at 3400–3500 symmetric hydroxyl groups stretching vibrations. The peaks at 2930 cm−1 corresponds to-CH2 bond stretching andcm Si-O-Si−1 can be asymmetric ascribed to stretchingthe characteristic vibration peaks of theof NH coated2− groups silica of shell, APTES respectively. which were The overlain broad bandby vibrations of aminopropyl−1 from APTES [44]. The results demonstrated−1 that− Fe3O4@SiO2 nanoparticles at 3400–3500hydroxyl groups cm canstretching be ascribed vibrations. to the The characteristic peaks at 2930 peaks cm of corresponds NH2 groups to-CH of APTES2 bond stretching which were were successfully modified by APTES (Figure 2A(b)). FTIR spectrum of− QNR1 and GDH before overlainvibrations by hydroxyl of aminopropyl groups from stretching APTES vibrations. [44]. The results The peaks demonstrated at 2930 cm that Fecorresponds3O4@SiO2 nanoparticles to-CH bond (Figure 2A(c)) and after immobilization (Figure 2A(d)) presented changes in the characteristic peaks2 stretchingwere successfully vibrations modified of aminopropyl by APTES from (Figure APTES 2A(b)). [44]. TheFTIR results spectrum demonstrated of QNR and that GDH Fe Obefore@SiO of functional groups. Especially decreasing of intensity at 1500 and 1550 cm−1 correspond to3 N-H4 2 nanoparticles(Figure 2A(c)) were and successfully after immobilization modiﬁed (Figure by APTES 2A(d (Figure)) presented2A(b)). changes FTIR spectrum in the characteristic of QNR and peaks GDH vibration of free enzymes indicated that the NH2− group of QNR and GDH reacted with the NH2− beforeof functional (Figure2 A(c))groups. and Espe aftercially immobilization decreasing of (Figure intensity2A(d)) at 1500 presented and 1550 changes cm−1 correspond in the characteristic to N-H

vibration of free enzymes indicated that the NH2− group of QNR and GDH reacted with the NH2−

Catalysts 2018, 8, 334 5 of 16 peaksCatalysts of 2018 functional, 8, x FOR PEER groups. REVIEW Especially decreasing of intensity at 1500 and 1550 cm−1 correspond5 of to16 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 group− attached on Fe3O4@SiO2 by GA cross-linking. XRD was used to identify the crystalline structure NHgroup2 group attached attached on Fe3 onO4@SiO Fe3O24 by@SiO GA2 cross-linking.by GA cross-linking. XRD was XRDused to was identify used tothe identify crystalline the structure crystalline of all samples. As can be seen in Figure 2B, the bare Fe3O4 exhibited characteristic crystalline peaks at structureof all samples. of all samples. As can be As seen can in be Figure seen in 2B, Figure the bare2B, theFe3O bare4 exhibited Fe 3O4 characteristicexhibited characteristic crystalline peaks crystalline at around 30.40°, 35.80°,◦ 43.60°,◦ 53.70°,◦ 57.20°◦ and 62.70°,◦ which◦ are consistent with the standard Fe3O4 peaksaround at around 30.40°, 30.4035.80°,, 35.8043.60°,, 43.6053.70°,, 57.20° 53.70 ,and 57.20 62.70°,and which 62.70 are, which consistent are consistent with the withstandard the standard Fe3O4 FeXRD3XRDO 4dataXRD data (JCPDS, data(JCPDS, (JCPDS, card card card89-0950) 89-0950) 89-0950) [46].[46]. [ 46InIn]. addition,addition, In addition, itit waswas it was foundfound found (Figure(Figure (Figure 2B(c)) 2B(c))2B(c)) that that that the thethe coating coatingcoating 3 4 2 procedureprocedure did did notnot not affectaffect affect thethe the phase phasephase change changechange ofof FeFe33OO44 nanoparticlesnanoparticles because becausebecause the thethe SiO SiOSiO2 and2 and APTES APTES coatingscoatings are are amorphous. amorphous. Therefore, Therefore, newnew peakspeaks attrib attributableutable toto these these groups groups could could not not be be detected detected by by XRDXRD analysis. analysis. For For free free enzyme enzyme (Figure (Figure 2 2B(a)),2B(a)),B(a)), theirtheir trendstrendstrends werewere similar similar to toto the thethe XRD XRDXRD of ofof the thethe magnetic magneticmagnetic combi-CLEAscombi-CLEAs (Figure(Figure (Figure2 B(d)).2B(d)). 2B(d)). Nevertheless, Nevertheless, Nevertheless, the the crystallinity crystallinity of theofof the samplesthe samples samples was was clearlywas clearly clearly decreased decreased decreased after theafterafter coating the the coating coating or/and or/and or/and immobilization immobilization immobilization process process process (Figure (Figure2B(c,d)), 2B(c,d)),2B(c,d)), mainly mainly mainly due to due due the to partialto the the partial partial destruction destruction destruction of the of ofthe the regularity regularity of of the the Fe Fe3O3O4crystal4crystal structurestructure after coatingcoating withwith aa silica silica shell shell and and APTES APTES and and regularity of the Fe3O4crystal structure after coating with a silica shell and APTES and subsequently subjectedsubsequentlysubsequently to the subjected subjected GA cross-linking to to the the GA GA procedure.cross-linking cross-linking procedure.

Figure 2. (A) FTIR spectra of (a) Fe3O4; (b) Fe3O4@SiO2-NH2; (c) free enzyme; (d) magnetic combi- Figure 2. (A) FTIR spectra of (a)3 Fe43O4; (b)3 Fe4 3O4@SiO2 2-NH2; (c) free enzyme; (d) magnetic FigureCLEAs. 2. (AB)) XRDFTIR of spectra (a) free ofenzyme; (a) Fe (b)O ;Fe (b)3O 4Fe; (c)O Fe@SiO3O4@SiO-NH2-NH; (c)2; free(d) magnetic enzyme; combi-CLEAs. (d) magnetic combi- combi-CLEAs.CLEAs. (B) XRD (B )of XRD (a) free of (a) enzyme; free enzyme; (b) Fe (b)3O4 Fe; (c)3O Fe4;3 (c)O4@SiO Fe3O42-NH@SiO2;2 (d)-NH magnetic2; (d) magnetic combi-CLEAs. combi-CLEAs. The magnetic behaviors were confirmed by the magnetization curves (Figure 3a), which were typicalThe magneticmagneticplot of magnetization behaviors behaviors were were against confirmed confirmed applied by themagneticby the magnetization magnetization ﬁeld (M− curvesH loop) curves (Figure at room (Figure3a), temperature, which 3a), werewhich typicaland were plottypicalcrossed of magnetizationplot the of zero magnetization point, against inductive appliedagainst of the applied magnetic superpar magnetic fieldamagnetic (M −ﬁeldH properties. loop) (M−H at loop)room The saturationat temperature, room temperature, magnetization and crossed and thecrossedvalue zero the point,of Fezero3O inductive4 @SiOpoint,2-NH inductive of2 theand superparamagnetic magneticof the superpar combi-CLEAsamagnetic properties. was properties. about Thesaturation 66.79 The emu/g saturation magnetization and 40.88 magnetization emu/g, value of respectively.3 4 The decreased2 2 superparamagnetic behaviors of the magnetic combi-CLEAs could be Fevalue3O4 @SiOof Fe2-NHO @SiO2 and-NH magnetic and magnetic combi-CLEAs combi-CLEAs was about was 66.79 about emu/g 66.79 and 40.88emu/g emu/g, and 40.88 respectively. emu/g, Therespectively.explained decreased due The superparamagnetic to decreased cross-linking superparamagnetic of proteins behaviors on the of the surfacebehaviors magnetic of Fe of3O combi-CLEAsthe4@SiO magnetic2-NH2 nanoparticles couldcombi-CLEAs be explained quenching could due be the magnetic moment. Although the superparamagnetic behaviors of the magnetic combi-CLEAs toexplained cross-linking due to of cross-linking proteins on theof proteins surface ofon Fethe3O surface4@SiO2 of-NH Fe23Onanoparticles4@SiO2-NH2 nanoparticles quenching the quenching magnetic moment.thedisplayed magnetic Although decreasing moment. the superparamagnetic manner,Although they the were superparamag behaviors well dispersed ofnetic the in magnetic behaviors phosphate combi-CLEAs of buffered the magnetic saline displayed (PBS) combi-CLEAs decreasing(Figure 3b) and the superparamagnetic properties were high enough to make easy separation from liquid manner,displayed they decreasing were well manner, dispersed they in phosphate were well buffered dispersed saline in (PBS) phosphate (Figure 3bufferedb) and the saline superparamagnetic (PBS) (Figure media (about 10 s) in the present of an external magnetic field (Figure 3c), suggesting the as- properties3b) and the were superparamagnetic high enough to makeproperties easy separation were high from enough liquid to mediamake (abouteasy separation 10 s) in the from present liquid of synthesized magnetic combi-CLEAs could be efficiently applied in asymmetric synthesis of (R)-3- anmedia external (about magnetic 10 s) in field the (Figure present3c), of suggesting an external the as-synthesizedmagnetic fieldmagnetic (Figure 3c), combi-CLEAs suggesting could the beas- quinuclidinol. efficientlysynthesized applied magnetic in asymmetric combi-CLEAs synthesis could of (beR)-3-quinuclidinol. efficiently applied in asymmetric synthesis of (R)-3- quinuclidinol.

Figure 3. (a) Magnetic hysteresis curves of Fe3O4@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 by an external magnetic ﬁeld. Figureexternal 3. ( magnetica) Magnetic field. hysteresis curves of Fe3O4@SiO2-NH2 and magnetic combi-CLEAs, (b) PBS- dispersivity 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 TEMTEM images (Figure4 4a)a) suggested suggested that that nearly nearly monodisperse monodisperse Fe FeO3O@SiO4@SiO2-NH-NH2 nanoparticles were successfully prepared and the average size was about 50 nm. A relatively3 4 2smooth2 surface was were successfully prepared and the average size was about 50 nm. A relatively smooth surface was obtained because of silica coating. Compared with these bare particles, it was obviously observed obtainedobtained because because of of silica coatin coating.g. Compared Compared with with these these bare bare particles, it it was obviously observed that combi-CLEAs were inlayed within magnetic nanoparticles (The combi-CLEAs embedded in the thatthat combi-CLEAs combi-CLEAs were were inlayed inlayed within within magnetic magnetic nanoparticles nanoparticles (The (The combi-CLEAs combi-CLEAs embedded embedded in in the the nanoparticles was indicated by the red arrow), clearly showing a well-defined spherical appearance nanoparticlesnanoparticles was was indicated indicated by by the the red red arrow), clea clearlyrly showing a well-defined well-defined spherical appearance (Figure 4b). Based on morphology, the magnetic combi-CLEAs are classified as type 1 which permit (Figure(Figure 44b).b). BasedBased onon morphology,morphology, thethe magneticmagnetic combi-CLEAscombi-CLEAs areare classifiedclassified asas typetype 11 whichwhich permitpermit better mass transfer. Furthermore, the surface of the magnetic combi-CLEAs showed relatively high betterbetter mass mass transfer. transfer. Furthermore, the surface of the magnetic combi-CLEAs showed showed relatively high numbers of perforations that might facilitate the substrates/cofactors transfer, leading to superior numbersnumbers of perforations that might facilitate the substrates/cofactorssubstrates/cofactors transfer, transfer, leading leading to to superior catalytic efficiency. catalyticcatalytic efficiency. efficiency.

Figure 4. (a) TEM images of the Fe3O4@SiO2-NH2 and (b) FE-SEM images of the magnetic combi- Figure 4. (a) TEM images of the Fe3O4@SiO2-NH2 and (b) FE-SEM images of the magnetic combi- CLEAs.Figure 4. (a) TEM images of the Fe3O4@SiO2-NH2 and (b) FE-SEM images of the magnetic combi- CLEAs. CLEAs. 2.2.2.2. Screening Screening of of Precipitant Precipitant Agents Agents for for Magnetic Magnetic Combi-CLEAs Preparation 2.2. Screening of Precipitant Agents for Magnetic Combi-CLEAs Preparation ActivityActivity recovery recovery of of the the immobilized immobilized enzyme enzyme is isa key a key cost cost determining determining parameter parameter of CLEAs. of CLEAs. To Activity recovery of the immobilized enzyme is a key cost determining parameter of CLEAs. To preserveTo preserve the maximum the maximum enzyme enzyme activity activity in magnetic in magnetic combi-CLEAs, combi-CLEAs, it is essential it isessential to optimize to optimizedifferent preserve the maximum enzyme activity in magnetic combi-CLEAs, it is essential to optimize different parametersdifferent parameters for the preparation for the preparation of magnetic of magnetic combi-CLEAs. combi-CLEAs. Firstly, Firstly, an efficient an efficient precipitation precipitation of all of parameters for the preparation of magnetic combi-CLEAs. Firstly, an efficient precipitation of all enzymesall enzymes in its in active its active form form is of is ofsignificant significant import importanceance in inthe the preparation preparation of of magnetic magnetic combi-CLEAs. combi-CLEAs. enzymes in its active form is of significant importance in the preparation of magnetic combi-CLEAs. Therefore,Therefore, five five precipitants including ammonium su sulfate,lfate, acetone, acetone, ethanol, ethanol, isopropanol, isopropanol, DMSO DMSO were were Therefore, five precipitants including ammonium sulfate, acetone, ethanol, isopropanol, DMSO were testedtested 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 to evaluate their abilities of precipitating QNR and GDH. After the formation of physical aggregates,aggregates, the the precipitate precipitate was was redissolved, redissolved, then then th thee remaining activities were measured. Figure Figure 55 aggregates, the precipitate was redissolved, then the remaining activities were measured. Figure 5 showedshowed ammonium ammonium sulfate sulfate acting acting as as the the best best precip precipitatingitating agent agent compared compared to to four four organic organic solvents, solvents, showed ammonium sulfate acting as the best precipitating agent compared to four organic solvents, andand 97.2% andand 98.5%98.5% of of recovered recovered activity activity was was respectively respectively achieved achieved for QNR for QNR and GDH,and GDH, which which agrees and 97.2% and 98.5% of recovered activity was respectively achieved for QNR and GDH, which agreeswith previous with previous studies studies [47,48]. [47,48]. Ammonium Ammonium sulfate wassulfate the was best the option best because option because it can usually it can preserveusually agrees with previous studies [47,48]. Ammonium sulfate was the best option because it can usually preservethe enzyme the activity enzyme of theactivity biocatalyst of the [ 47biocatalyst,48]. While [47,48]. ethanol, While isopropanol, ethanol, DMSO isopropanol, and acetone DMSO exhibited and preserve the enzyme activity of the biocatalyst [47,48]. While ethanol, isopropanol, DMSO and acetonerelatively exhibited lower the relatively recovered lower activity: the 35.3%recovered and 27.6%,activity: 58.7% 35.3% and and 36.1%, 27.6%, 54.8% 58.7% and and 57.9%, 36.1%, and 54.8% 85.6% acetone exhibited relatively lower the recovered activity: 35.3% and 27.6%, 58.7% and 36.1%, 54.8% andand 57.9%, 76.2% forand QNR 85.6% and and GDH, 76.2% respectively. for QNR and Therefore, GDH, respectively. ammonium Therefore, sulfate was ammonium chosen as sulfate precipitant was and 57.9%, and 85.6% and 76.2% for QNR and GDH, respectively. Therefore, ammonium sulfate was chosenagent for as furtherprecipitant magnetic agent combi-CLEAsfor further magnetic preparation. combi-CLEAs preparation. chosen as precipitant agent for further magnetic combi-CLEAs preparation.

FigureFigure 5. 5. EffectEffect of of precipitants precipitants on on activity activity recovery recovery of of the the magnetic magnetic combi-CLEAs. combi-CLEAs. The The measurements measurements Figure 5. Effect of precipitants on activity recovery of the magnetic combi-CLEAs. The measurements werewere 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 Combi- CLEAs2.3. 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 CLEAs.GA However, is a bialdehyde it is important functional to moleculenote that commonlyif too little cross-linker used as a cross-linking and cross-linking reagent time to prepareare not enoughCLEAs. to However, form CLEAs it is importantthat leads to leaching note that of if enzyme too little molecules, cross-linker while and too cross-linking much cross-linker time are and not cross-linkingenough to form time CLEAs can cause that the leads destruction to leaching of enzyme’s of enzyme flexibility molecules, which while is essential too much for cross-linker its activity [39,49,50].and cross-linking To preserve time maximum can cause en thezyme destruction activity ofin enzyme’sthe biocatalysts, ﬂexibility therefore, which iswe essential optimized for the its concentrationactivity [39,49 ,of50 ].cross-linker To preserve and maximum cross-linking enzyme time. activity As shown in the biocatalysts,in Figure 6, therefore,the activity we recovery optimized of QNRthe concentration and GDH in of the cross-linker magnetic combi-CLEAs and cross-linking was time. increased As shown with inincrease Figure 6in, the GA activity concentration recovery and of cross-linkingQNR and GDH time in and the magneticover 93% combi-CLEAs activity recovery was for increased the two with enzymes increase was in obtained GA concentration at 20 mM andGA andcross-linking 3 h cross-linking time and over time. 93% Therefore, activity recovery in the forfurther the two optimization enzymes was of obtained magnetic at 20combi-CLEAs mM GA and preparation3 h cross-linking process, time. 20 Therefore, mM GA in and the further3 h cross-linking optimization time of magnetic were determined combi-CLEAs as the preparation optimal conditions.process, 20 mM GA and 3 h cross-linking time were determined as the optimal conditions.

Figure 6. Effect ofof GAGA concentrationconcentration ( a()a and) and cross-linking cross-linking time time (b )(b on) on the the activity activity recovery recovery of QNR of QNR and andGDH GDH in the in magneticthe magnetic combi-CLEAs. combi-CLEAs. The The measurements measurements were were performed performed in triplicate. in triplicate.

2.4. Optimal Optimal pH and Temperature for Magnetic Combi-CLEAs ActivityActivity RecoveryRecovery The influence influence of pH on enzyme activity was was invest investigatedigated in in the the range range from from 5.0 5.0 to to 10.0 10.0 at at 25 25 °C.◦C. As can bebe seenseen fromfrom FigureFigure7 a,7a, for for the the free free enzyme enzyme the the optimized optimized pH pH was was at 7.0,at 7.0, which which shifted shifted to pH to pH 8.0 8.0for thefor the magnetic magnetic combi-CLEAs. combi-CLEAs. The The shift shift in pH in could pH could be due be to due the to change the change in acidic in acidic and basic and amino basic aminoacid side acid chain side ionization chain ionization in the microenvironment in the microenviron aroundment thearound active the site, active and site, the interactionand the interaction between betweenthe enzyme the and enzyme cross-linking and cross-linking agent [51]. agent In addition, [51]. In theaddition, relative the activity relative of magneticactivity of combi-CLEAs magnetic combi- was CLEAsalways keptwas always higher thankept thosehigher of than free those enzyme of atfree various enzyme pH at value. various Nevertheless, pH value. Nevertheless, at pH 9.0–10.0 at both pH 9.0–10.0the free enzymeboth the and free theenzyme magnetic and combi-CLEAsthe magnetic co hadmbi-CLEAs significant had loss significant of activity, loss but of the activity, activity but of the activitylatter decreases of the latter slower decreases than the slower former. than The influencethe former. of The temperature influence on of the temperature relative activity on the of relative the free activityenzyme of and the the free magnetic enzyme combi-CLEAs and the magnetic was monitored combi-CLEAs by measuring was monitored enzyme by activity measuring as a functionenzyme activityof temperature as a function between of 15 temperature and 50 ◦C after between 4 h incubation. 15 and 50°C The after optimum 4 h incubation. temperature The of theoptimum former temperaturewas 25 ◦C, whereas of the former the latter was showed 25 °C, whereas highest activitythe latter at showed 30 ◦C (Figure highest7b). activity Nevertheless, at 30 °C the(Figure relative 7b). Nevertheless,activity of both the the relative free enzyme activity and of the both magnetic the free combi-CLEAs enzyme and was the gradually magnetic decreased combi-CLEAs with further was graduallyincrease in decreased temperature, with but further the activity increase of in the temperat latter stillure, decreases but the activity slower of than the thelatter former. still decreases The data slowersuggested than that the the former. magnetic The combi-CLEAs data suggested had that contributed the magnetic to the combi-CLEAs resilience of the had enzyme contributed against to high the resiliencetemperature, of the which enzyme agreed against with thehigh results temperature, obtained which by XRD agreed analysis. with Thisthe results was probably obtained due by toXRD the analysis. This was probably due to the fact that the Fe3O4@SiO2-NH2 nanoparticles could retarded fact that the Fe3O4@SiO2-NH2 nanoparticles could retarded heat transfer and collapse of the CLEAs heatstructure transfer at test and temperatures, collapse of the and CLEAs the rigidity structure of the at active test temperatures, conformation was and enhanced the rigidity by cross-linkingof the active conformationduring the formation was enhanced of enzyme by aggregates cross-linking [52]. during In addition, the formation good storage of enzyme stability aggregates is another essential[52]. In addition,requirement good for storage a biocatalyst stability to is be another employed essential in industrial requirement applications. for a biocatalyst The free to enzymebe employed and the in industrialmagnetic combi-CLEAsapplications. The were free stored enzyme in PBS and at the 4 ◦ magneticC for a 70 combi-CLEAs days period. Storagewere stored stability in PBS of theat 4 free °C forenzyme a 70 days and magneticperiod. Storage combi-CLEAs stability of was the presented free enzyme in Figureand magnetic7c. The magneticcombi-CLEAs combi-CLEAs was presented still inretained Figure about 7c. The 80% magnetic of its initial combi-CLEAs activity after still 70 retained days. However, about 80% the of free its initial enzyme activity retained after only 70 28.6%days. However,and 30.5% the of itsfree initial enzyme activity retained for QNR only and28.6% GDH, and respectively.30.5% of its initial The improved activity for storage QNR stabilityand GDH, of respectively. The improved storage stability of magnetic combi-CLEAs might be due to the covalent

Catalysts 2018, 8, 334 8 of 16

Catalysts 2018, 8, x FOR PEER REVIEW 8 of 16 magnetic combi-CLEAs might be due to the covalent cross-linking between the enzyme aggregates by cross-linkingGA, which avoided between enzyme the enzyme leaching, aggregates denaturation by GA, and which autodigestion, avoided enzyme and limited leaching, the conformational denaturation andchanges autodigestion, of the enzyme. and limited the conformational changes of the enzyme.

Figure 7. Effect ofof pHpH andand temperature temperature on on the the free free enzyme enzyme and and magnetic magnetic combi-CLEAs combi-CLEAs activity. activity. (a) pH,(a) (pH,b) temperature (b) temperature and (andc) storage (c) storage stability stability of the of free the enzymefree enzyme and magneticand magnetic combi-CLEAs. combi-CLEAs. Activity Activity was wasassayed assayed at 25 ◦atC 25 in the°C standardin the standard reaction reaction medium medium at pH 7.5–8.0. at pH The 7.5–8.0. measurements The measurements were performed were performedin 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 substrate- The 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 (Scheme2). 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 (Figure8a) 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(Figure8b 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 Catalystscombi-CLEAs 2018, 8, x isFOR an PEER effective REVIEW strategy for asymmetric synthesis (R)-3-quinuclidinol. 9 of 16 Catalysts 2018, 8, x FOR PEER REVIEW 9 of 16

Scheme 2. The magnetic combi-CLEAs biocatalysts for the production of (R)-3-quinuclidinol with Scheme 2. The magnetic combi-CLEAs biocatalysts for the production of (R)-3-quinuclidinol with cofactorScheme regeneration 2. The magnetic in situ. combi-CLEAs biocatalysts for the production of (R)-3-quinuclidinol with cofactorcofactor regenerationregeneration inin situ.situ.

Figure 8. Reaction time-course for the bioreduction of 3-quinuclidinone into (R)-3-quinuclidinol catalyzedFigureFigure 8.8. byReactionReaction the free time-coursetime-course enzyme or forforthe thethemagnetic bioreductionbiored combi-CLEAs,uction ofof 3-quinuclidinone3-quinuclidinone respectively. intoSamplesinto (R(R)-3-quinuclidinol)-3-quinuclidinol (0.5 mL) were withdrawncatalyzedcatalyzed byby at thethe freeappropriatefree enzymeenzyme intervals oror thethe magneticmagnetic and analyzed combi-CLEAs,combi-CLEAs, by GC (a) respectively.orrespectively. by TLC (b, SamplescSamples). (0.5(0.5 mL)mL) werewere withdrawnwithdrawn atat thethe appropriateappropriate intervalsintervals andand analyzed analyzed by by GC GC ( a(a)) or or by by TLC TLC ( b(b,c,c).). For applications of ketoreductase, recycling is of significant importance. The main advantage of For applications of ketoreductase, recycling is of significant importance. The main advantage of the magneticFor applications combi-CLEAs of ketoreductase, is its easy recycling separation is offrom signiﬁcant the reaction importance. mixtures. The This main resulted advantage in ofa the magnetic combi-CLEAs is its easy separation from the reaction mixtures. This resulted in a completethe magnetic recovery combi-CLEAs of the immobilized is its easy separation enzymes fromand cost-effective the reaction mixtures. industrial This applications resulted in [51]. a complete In our complete recovery of the immobilized enzymes and cost-effective industrial applications [51]. In our experience,recovery of thethe immobilizedmagnetic combi-CLEAs enzymes and are cost-effective eminently industrial recyclable. applications After each [51 cycle,]. In our the experience, magnetic experience, the magnetic combi-CLEAs are eminently recyclable. After each cycle, the magnetic combi-CLEAsthe magnetic combi-CLEAscould be successfully are eminently separated recyclable. from Afterthe reaction each cycle, medium the magnetic using a combi-CLEAsmagnet, and combi-CLEAs could be successfully separated from the reaction medium using a magnet, and employedcould be successfully again in the separatednext cycle. from As shown the reaction in Figure medium 9a, the usingmagnetic a magnet, combi-CLEAs and employed biocatalyst again was in employed again in the next cycle. As shown in Figure 9a, the magnetic combi-CLEAs biocatalyst was ablethe nextto cycle cycle. up Asto 10 shown runs, in and Figure the complete9a, the magnetic conversion combi-CLEAs time for each biocatalyst run was 3, was 3, 3.5, able 3.5, to cycle4, 4, 4, up 5, 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, 5, 5.5to 10and runs, 7 h, and respectively. the complete After conversion the successive time for each7th cycle, run was reduction 3, 3, 3.5, of 3.5, 0.33 4, 4,g 4, (2.0 5, 5.5 mmol) and 73- h, 5.5 and 7 h, respectively. After the successive 7th cycle, reduction of 0.33 g (2.0 mmol) 3- quinuclidinonerespectively. After with the 50 successive mg of the 7th magnetic cycle, reduction combi-CLEAs of 0.33 in g (2.0the mmol)presence 3-quinuclidinone of 3 mmol of glucose with 50 and mg quinuclidinone with 50 mg of the magnetic combi-CLEAs in the presence of 3 mmol of glucose and 6.6of themg magneticreduced nicotinamide combi-CLEAs adenine in the presence dinucleotide of 3 mmol (NAD of+) glucoseafforded and over 6.6 100% mg reducedconversion nicotinamide efficiency 6.6 mg reduced nicotinamide adenine dinucleotide (NAD+) afforded over 100% conversion efficiency andadenine (R)-3-quinuclidinol dinucleotide (NAD in 100%+) afforded ee at 300 over min. 100% However, conversion at 10th efﬁciency cycle, it took and 420 (R)-3-quinuclidinol min to reach 100% in and (R)-3-quinuclidinol in 100% ee at 300 min. However, at 10th cycle, it took 420 min to reach 100% yield,100% ee180 at min 300 longer min. However, than the at7th 10th cycle. cycle, The it resi tookdual 420 activity min to reachof the 100%magn yield,etic combi-CLEAs 180 min longer during than yield, 180 min longer than the 7th cycle. The residual activity of the magnetic combi-CLEAs during thethe reaction 7th cycle. cycles The residualwas also activity presented of the in Figure magnetic 9b. combi-CLEAsIt was found that during the activity the reaction of magnetic cycles was combi- also the reaction cycles was also presented in Figure 9b. It was found that the activity of magnetic combi- CLEAspresented was in well-maintained Figure9b. It was up found to the that 7th the batch activity and of decreased magnetic combi-CLEAsto approximately was 59.1%, well-maintained 54.3% and CLEAs was well-maintained up to the 7th batch and decreased to approximately 59.1%, 54.3% and 43.5%up to theof its 7th original batch and activity decreased for QNR, to approximately and 60.9%, 58.3% 59.1%, and 54.3% 45.8% and for 43.5% GDH of itsat original8, 9 and activity 10 cycles, 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.QNR, and 60.9%, The obvious 58.3% and decrease 45.8% forof the GDH catalytic at 8, 9 acti andvity 10 cycles, after the respectively. 7th cycle, especially The obvious in the decrease 9th and of respectively. The obvious decrease of the catalytic activity after the 7th cycle, especially in the 9th and 10ththe catalytic runs, could activity be due after to the the 7th enzyme cycle, especiallydenaturati inon, the partial 9th and collapse 10th runs, of the could magnetic be due combi-CLEAs to the enzyme 10th runs, could be due to the enzyme denaturation, partial collapse of the magnetic combi-CLEAs anddenaturation, substantial partial leakage collapse from the of the support magnetic intocombi-CLEAs the reaction batch and substantialduring repeated leakage use. 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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FigureFigure 9. 9. ReusabilityReusability of ofmagnetic magnetic combi-CLEAs combi-CLEAs for the for bioreduction the bioreduction of 3-quinuclidinone of 3-quinuclidinone into (R into)-3- quinuclidinol,(R)-3-quinuclidinol, (a) conversion (a) conversion yield yield of 3-quinuclidinone of 3-quinuclidinone and and ee eeof of(R ()-3-quinuclidinol;R)-3-quinuclidinol; (b (b) )relative relative activityactivity of of QNR QNR and and GDH GDH in magnetic magnetic combi-CLEAs.

In addition, we also utilized the FE-SEM to further investigate the change of the magnetic combi- InIn addition, addition, we we also also utilized utilized the the FE-SEM FE-SEM to furt toher further investigate investigate the change the change of the magnetic of the magnetic combi- CLEAs surface structure during the cycles of repeated use. The results revealed that almost no CLEAscombi-CLEAs surface surfacestructure structure during during the cycles the cyclesof repe ofated repeated use. The use. results The results revealed revealed that thatalmost almost no obvious morphologic change was observed, and the magnetic biocatalyst presented a spherical or obviousno obvious morphologic morphologic change change was was observed, observed, and and the the magnetic magnetic biocatalyst biocatalyst presented presented a a spherical spherical or or nearly spherical structure even after the seventh cycle (Figure 10a–g), indicating that the morphologic nearlynearly spherical spherical structure structure even even after after the the seventh seventh cy cyclecle (Figure (Figure 10a–g),10a–g), indicating indicating that that the the morphologic morphologic integrity of the magnetic combi-CLEAs was obviously in close agreement with its enzyme activity integrityintegrity of of the the magnetic magnetic combi-CLEAs combi-CLEAs was was obviously obviously in in close close agreement agreement with with its its enzyme enzyme activity activity (Figure 9b). As can be seen from Figure 10h–j, the eighth, ninth and tenth cycles experienced partial (Figure(Figure 99b).b). AsAs cancan bebe seenseen fromfrom FigureFigure 1010h–j,h–j, thethe eighth,eighth, ninthninth andand tenthtenth cyclescycles experiencedexperienced partialpartial collapse of spherical structure, the disappearance of small hole present on the surface of the magnetic collapsecollapse of of spherical spherical structure, structure, the the disappearance disappearance of small small hole hole present present on on the the surface surface of of the the magnetic magnetic combi-CLEAs and then aggregation into a bigger size of the combi-CLEAs, respectively. Because combi-CLEAscombi-CLEAs and and then then aggregation aggregation into into a bigger si sizeze of the combi-CLEAs, respectively. Because particle size of the combi-CLEAs can affect mass transfer of the substrates and prevent substrates particle size size of of the combi-CLEAs can can affect mass transfer of the substrates and prevent substrates contacting with the inner enzymes of the combi-CLEAs, the inner enzymes present in the bigger size contactingcontacting with the inner enzymesenzymes ofof thethe combi-CLEAs,combi-CLEAs, the the inner inner enzymes enzymes present present in in the the bigger bigger size size of of the magnetic combi-CLEAs were inaccessible and could not form complexes with the substrates ofthe the magnetic magnetic combi-CLEAs combi-CLEAs were were inaccessible inaccessible and couldand could not form not complexesform complexes with the with substrates the substrates [48,54], [48,54], hence, leading to the loss of enzyme activity of the magnetic combi-CLEAs. From another [48,54],hence,leading hence, toleading the loss to of the enzyme loss of activity enzyme of theactivity magnetic of the combi-CLEAs. magnetic combi-CLEAs. From another From point another of view, point of view, the change in surface structure of magnetic combi-CLEAs supports the value of using pointthe change of view, in surfacethe change structure in surface of magnetic structure combi-CLEAs of magnetic supports combi-CLEAs the value supports of using the combi-CLEAs value of using in combi-CLEAs in combination with the Fe3O4@SiO2-NH2 to yield magnetic combi-CLEAs with combi-CLEAscombination with in combination the Fe O @SiO with-NH theto Fe yield3O4@SiO magnetic2-NH2 combi-CLEAs to yield magnetic with excellent combi-CLEAs resistance with to excellent resistance to mechanical3 4 2 stress,2 such as shake or agitate and wash, allowing high recovery excellentmechanical resistance stress, such to mechanical as shake or stress, agitate such and as wash, shake allowing or agitate high and recovery wash, allowing of the biocatalyst high recovery after of the biocatalyst after many cycles and providing (R)-3-quinuclidinol in high ee and yield. ofmany the biocatalyst cycles and providingafter many ( Rcycles)-3-quinuclidinol and providing in high(R)-3-quinuclidinol ee and yield. in high ee and yield.

Figure 10. Cont.

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

3. Materials and Methods 3. Materials and Methods

3.1. Materials Materials Recombinant plasmid plasmid pET28a-QNR pET28a-QNR and and pET28a-GDH pET28a-GDH was was provided provided by by Taihe Taihe (Beijing, (Beijing, China). China). Competent cells cells E.E. coli coli BL(21)BL(21) (DE3) (DE3) was was from from Tiangen Tiangen (Beijing, (Beijing, China). China). Page Ruler Page Prestained Ruler Prestained Protein LadderProtein Ladderwas form was Thermo form Thermo Fisher Fisher Scientific Scientiﬁc (Waltham, (Waltham, MA, MA, USA). USA). Isopropy-l- Isopropy-l-ββ--DD-thiogalacto--thiogalacto- pyranoside (IPTG),(IPTG), 3-aminopropyltriethoxysilane 3-aminopropyltriethoxysilane (APTES), (APTES), Tetraethylorthosilicate Tetraethylorthosilicate (TEOS), (TEOS), all reagents all reagentsfor sodium for sodium dodecyl dodecyl sulfate sulfate polyacrylamide polyacrylamide gel electrophoresis gel electrophoresis (SDS-PAGE), (SDS-PAGE), 3-quinuclidone 3-quinuclidone and andstandard standard (R)-3-quinuclidinol (R)-3-quinuclidinol were obtainedwere obtained from Sigma-Aldrich from Sigma-Aldrich (Shanghai, (Shanghai, China). Other China). chemicals Other chemicalsand reagents and were reagents the highest were the purity highest available. purity The available. deionized The water deionized was ultrapure water was produced ultrapure by producedMillipore systemby Millipore (Bedford, system MA, (Bedford, USA). 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 cell cellss were respectively denoted as E. coli (QNR)(QNR) and and E. coli (GDH)(GDH) and and cultivatedcultivated inin Luria Luria Broth Broth medium medium supplemented supplemented with with 100 µ g/mL100 μg/mL of kanamycin of kanamycin at 37 ◦C. at The 37 overnight °C. The overnightculture was culture inoculated was inoculated and grown and at grown 37 ◦C. at Expression 37 °C. Expression of the targetof the target enzyme enzyme was induced was induced until untilOD600 OD600 reached reached about about 0.6 by 0.6 adding by adding IPTG IPTG up to up a final to a concentrationfinal concentration of 0.2 of mM. 0.2 CellsmM. growthCells growth along alongwith enzyme with enzyme expression expression continued continued for 48 h atfor 25 48◦C. h Theat 25 cells °C. were The then cells pelleted were then by centrifugation pelleted by centrifugation(12,000× g for (12,000× 5 min) at g for 4 ◦C, 5 min) washed at 4 °C, using washed ice PBS, using 7.4) ice and PBS, resuspended 7.4) and resuspended in 50 mL PBS in 50 per mL gram PBS perwet gram cells. wet Then cells. the Then cells the were cells lysed were using lysed XC-CD2500 using XC-CD2500 Ultrasonic Ultrasonic Oscillator Oscillator (Ningbo, (Ningbo, China) China) on ice. onThe ice. supernatant The supernatant was collected was collected by centrifugation by centrifugation (15,000 (15,000×× g for g20 for min) 20 min) at 4 at◦C. 4 °C. After After filtrated filtrated by by0.22 0.22µm μ filterm filter membrane, membrane, the the supernatant supernatant was was analyzed analyzed by SDS-PAGEby SDS-PAGE and and the the concentration concentration of the of theproteins proteins was was determined determined by Bradfordby Bradford protein protein assay assay [55]. [55].

3.3. Enzyme Enzyme Activity Activity Assay Assay Enzyme activity activity was was assayed assayed spectrophotometrically spectrophotometrically at at 25 25 °C◦ Cby by monitoring monitoring the the absorbance absorbance at 340at 340 nm nm corresponding corresponding to tothe the change change in in oxidiz oxidizeded nicotinamide nicotinamide adenine adenine dinucleotide dinucleotide (NADH) (NADH) concentrationconcentration [[17].17]. 11 mLmL of of assay assay mixture mixture was was consisted consisted of PBS,of PBS, 3 µ mol3 μmol 3-quinuclidinone 3-quinuclidinone and 0.3andµ mol0.3 μNADHmol NADH for QNR for (orQNR 10 µ(ormol 10 glucoseμmol glucose and 1 µ andmol 1 NAD μmol+ forNAD GDH),+ for GDH), and an and appropriate an appropriate amount amount of QNR 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

Catalysts 2018, 8, 334 12 of 16 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 Fe3O4 Nanoparticles (Fe3O4@SiO2-NH2)

Magnetic Fe3O4 nanoparticles were prepared according to the pervious reported method [56]. In brief, 0.27 g FeCl3·6H2O 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 Fe3O4 nanoparticles with -NH2 groups, 100 mg of freshly prepared Fe3O4 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·H2O. 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 Fe3O4@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·H2O, followed by the drop-wise addition of 300 µL APTES with vigorous stirring. After 6 h, the precipitate was washed and denoted as Fe3O4@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 Fe3O4@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)2SO4, 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].

( ) = Total activity of each enzyme in magnetic combi−CLEAs(U) × Activity recovery % Total starting activity of each enzyme used for 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 CH2Cl2 and CH3OH (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. Catalysts 2018, 8, 334 13 of 16

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 Fe3O4@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 ofFe3O4, Fe3O4@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 Fe3O4@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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