pubs.acs.org/journal/ascecg Research Article

Rapidly and Precisely Cross-Linked Enzymes Using Bio-Orthogonal Chemistry from Cell Lysate for the Synthesis of (S)‑1-(2,6-Dichloro-3- fluorophenyl) Ethanol Huimin Li, Ru Wang, Anming Wang,* Jing Zhang, Youcheng Yin, Xiaolin Pei, and Pengfei Zhang*

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ABSTRACT: To develop a method for preparing rapidly, precisely, and bio-orthogonally cross-linked enzymes (RP-CLEs), nonstandard amino acids (NSAAs) were inserted into the enzyme protein, and microwave irradiation was used to accelerate its site- specific linkage through Cu-free strain-promoted alkyne−azide cycloaddition (SPAAC). To this end, we selected aldehyde ketone reductase (AKR) as model enzyme, and AKR mutants were obtained by five-point insertion of p-azido-L-phenylalanine (pAzF) which were subsequently cross-linked to form RP-CLEs from cell lysate supernatant under microwave irradiation. The AKR five-point mutant and corresponding RP-CLEs were characterized using MALDI-TOF MS, SEM, and FT-IR, respectively. The specific activities of RP-CLEs of three-point and five-point AKR were 1.27 and 2.06 U·mg−1, 1.21- and 2.16-fold those of the corresponding free enzymes, respectively. In the asymmetric synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol, the yield was up to 90.8%, and the ee was 99.98%. Moreover, after 6 consecutive 12 h reaction cycles, AKR five-point RP-CLEs still retained 80% of their initial activity. Thus, depending on the enzyme structure analysis, different numbers of NSAAs could be reasonably incorporated into the protein to accurately guide and control the covalent linkage to form RP-CLEs. This green method could be further developed both to generate bio-orthogonally cross-linked enzyme and separate them from nontarget proteins for industrial biocatalysis. KEYWORDS: Cross-linked enzyme aggregates, Nonstandard amino acids, Crizotinib, Aldehyde ketone reductase, p-Azido-L-phenylalanine, Bio-orthogonal chemistry, Microwave irradiation

■ INTRODUCTION specificity, and reduce inhibition.10 Even purification of the fi Enzyme biocatalysts are made from available renewable enzyme may be achieved in the one-step puri cation and Downloaded via HANGZHOU NORMAL UNIV on May 19, 2020 at 07:25:59 (UTC). ’ resources, are biodegradable, and are essentially harmless and immobilization reported in Barbosa s work and our previous nontoxic. Unlike chemical synthesis, it avoids the use of rare work, which facilitates the enzyme immobilization after See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. precious metals and the costs associated with removing trace chemical or genetically modification using functionalized precious metals from end products.1 With high catalytic supports.11,12 However, preparation and functionalization of efficiency, as well as chemo-, regio-, and stereoselectivities, supports are often lengthy and costly, which will increase the enzyme-mediated biocatalytic reactions have become more and cost of the immobilized enzyme. more widely applied in the fermentation, chemical, food, and Support-free immobilizations of enzymes have attracted 2−6 environmental industries. However, their practical applica- great interest since no supports were used for immobiliza- − tion is hampered by the fragility of enzymes, such as low tion.13 16 The most common and facile of these is cross-linked thermal stability, a narrow optimal pH range, and low tolerance enzyme aggregates (CLEAs).14 CLEAs were first developed in to most organic solvents and many metal ions. In addition, the early 2000s, formed by physical precipitation of the enzyme enzymes themselves are a source of contamination in the target product necessitating purification and separation steps.7 Enzyme immobilization frequently offers considerable advan- Received: February 6, 2020 tages in terms of reusability and enzyme stability by improving Revised: April 3, 2020 their organic solvent resistance, pH tolerance, and thermal Published: April 7, 2020 stability and facilitating their separation from reaction mixture.8,9 In addition, a proper immobilization may also permit the enzyme improvement of activity, selectivity,

© 2020 American Chemical Society https://dx.doi.org/10.1021/acssuschemeng.0c00987 6466 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article

Scheme 1. Scheme for the Formation of RP-CLEs of AKR

in water-miscible solvents. Compared with the parent enzyme, very stable immobilized GAC, and almost 60% of the initial a CLEA is generally stable in organic solvents, remains activity was recovered by this methodology.26 Chemical unchanged, and can even enhance activity in some cases.17 amination using ethylenediamine and carbodiimide by a CLEAs are extremely resistant to organic solvents, extremely solid-phase strategy has also been used to enrich the amino high pH, and high temperatures, because immobilization groups in the enzyme surface for facile cross-linking.27,29 reduces the flexibility of the enzyme and inhibits the However, chemical amination and coaggregation with feeder development of the tertiary structure required for catalytic often result in the decrease of the catalytic activity, which may − activity.18 20 be attributed to this random chemical modification reaction, However, several problems remain in the traditional method amination, and cross-linking reaction occurred to the for preparing CLEAs. One is the use of organic solvents or undesired amino acid residues using glutaraldehyde.23,30 fi saturated (NH4)2SO4 aqueous solution, and the other is the In contrast, the site-speci c incorporation of nonstandard random and unwanted cross-links derived from cross-linkers amino acids (NSAAs) allows protein immobilization in a such as glutaraldehyde. Organic solvents, especially toxic controlled manner.31,32 Genetic code expansion uses orthog- solvents, are increasingly being recognized as unsuitable for onal aminoacyl-tRNA synthetase (aaRS)−tRNA pairs to guide green and sustainable chemical manufacturing.21 Unwanted the insertion of NSAAs into proteins in response to unassigned cross-linking due to glutaraldehyde also usually decreases the codons (commonly the amber stop codon, UAG) introduced activity of the obtained CLEAs.22,23 Additionally, under neutral in the targeted gene.33,34 For the immobilization of these conditions, Schiff base may be formed by a nucleophilic proteins bearing NSAAs, Cu(I)-catalyzed azide−alkyne cyclo- reaction between the ε-amino group of lysine and addition (CuAAC) and its strain-promoted variant (SPAAC), glutaraldehyde to achieve the covalent cross-links.23 The Schiff both effective click chemistry reactions, are generally used as − base is often unstable and apt to hydrolysis, and some the cross-linking reactions.35 38 However, cytotoxicity from procedures suggest the use of NaBH4 or NaBCNH3 reduction, copper(I) salts has prevented their use in cellular biochemical which is the last step to stabilize the formed Schiff base to a reactions and imaging studies in living organisms.39 As an secondary amine.12,23 Furthermore, glutaraldehyde at high alternative, SPAAC is an effective strategy and can promote concentrations perhaps inactivates the enzyme because of formation of reactive ring strain, a physical force that is put on excessive cross-linking. Additionally, the excessive and the ring when a ring stretches. In 1961, Wittig and Krebs uncontrollable cross-linking that has been used for decades pointed out that the reaction between pure cyclooctyne (the probably increases the diffusional limitations of substrates and smallest stable cycloalkyne) and benzene azide “produced a products.24,25 Additionally, it is also a problem that the cross- single product like an explosion, Triazole”.40 The alkyne can be linking efficiency always depends on the amount of Lys activated by this ring strain rather than metal catalysis, and the residues in the used enzyme.26 In preparing classical CLEAs, biocompatibility of the azide−alkyne cycloaddition is also some enzymes such as glutaryl acylase (GAC) and Lipase B substantially higher. The wide bond angle deformation of from Candida antarctica (CALB) are not adequate to form the acetylene to 163° results in a ring strain of almost 18 kcal/mol. cross-linked enzyme aggregates (CLEAs) due to the low This destabilization of strained alkynes of the ground state number of Lys residues in them.26,27 To carry out efficient versus the transition state just provides a dramatic rate cross-linking of the enzyme molecules, polyethylenimine (PEI) acceleration in linking reaction compared to the general and BSA full of amino groups were often used as feeder to unstrained alkynes.41,42 coaggregate with enzyme, and the coaggregation system Apart from unwanted covalent cross-links in enzyme promotes the high reactivity of PEI and the cross-linking immobilization, multipoint covalent linkage may make the between the abundant primary amino groups on the enzyme enzyme more rigid and less sensitive to conformational surface.26,28 Lopez-Gallegó and co-workers have prepared a changes. Moreover, cross-linking step would cause the

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50 μg·mL−1 ampicillin (MG1655), 34 μg·mL−1 chloramphenicol intermolecular multipoint attachment, which would result in − the efficient suppression of enzyme denaturation, therefore, (pEVOL-pAzF), and 100 μg·mL 1 kanamycin (pZE21-GFPaav). enzyme activity may be higher than free enzymes under harsh Plasmid Construction, Protein Expression, and Character- ization. conditions.43,44 In fact, the cross-linked enzymes are also In order to design and provide a bacterial expression vector 45 2+ derived from aldehyde ketone reductase (AKR, PDB ID: 5dan.1; similar to a copolymer of protein. [Ru(bpy)3] and resolution, 2.0 Å) of from Thermotoga maritima MSB8 and to encode (NH4)2S2O8 are often necessary to initiate and catalyze the a C-terminal His×6 tag, the plasmid pZE21-GFPaav from Addgene polymerization and produce the polymerized protein using 48 46 was selected as the target vector. The target gene was PCR- photo-cross-linking. For enzyme immobilization, this Ru(II)- amplified to obtain akr with restriction sites. We double-digested mediated photo-cross-linking between the soluble monomer pZE21-GFP by using restriction enzymes KpnI and Hind III and enzyme protein to a high-molecular-weight species may be also ligated the akr gene instead of the gdh gene into pZE21 to acquire one of the efficient strategies to inhibit enzyme proteolysis. pZE21-akr. The primers designed for construction of akr-pZE21 are However, the metal−organic catalyst used may be not listed in Table S1. biocompatible with the enzyme protein, which perhaps Site-directed mutagenesis PCR was performed to introduce an denatures the enzyme structure resulting in the decrease of amber codon (UAG) in place of a tyrosine codon at position 114 (Y). activity. In addition, they must purify the enzyme protein The gene of a multipoint mutant was subjected to site-directed before the cross-linking because tyrosine used this cross-linking mutagenesis multiple times based on one-point mutation. We selected multiple mutation sites away from the active site (NADP+) and the method is general and ubiquitous as one of the natural amino active site of AKR, including 110 (Y), 114, 143 (Y), 162 (Q), and 189 acids in protein. 49 fi fi (Q) (N-terminal) of pZE21-akr. We selected three mutation sites In this work, we rst produced AKR ve-point mutant (114Y−143Y−189Q) and five mutation sites (110Y−114Y−143Y− incorporated with p-azido-L-phenylalanine (pAzF) using 162Q−189Q) as the number of different mutations of the akr gene genetic code expansion. Then, a diyne (sym-dibenzo-1,5- and named them akr three-point mutant gene and akr five-point cyclooctadiene-3,7-diyne) was used as the bifunctional azide mutant gene. Site-directed mutagenesis primers are listed in Table S2. cross-linking agent to form RP-CLEs in the supernatant of the The plasmid pEVOL-pAzF (tRNA synthetase/tRNA pair) used for cell lysate, allowing additional purification of the protein the insertion of pAzF in vivo into proteins which responsded to the amber codon, TAG, was from Peter G. Schultz’s group at the Scripps (Scheme 1). Microwave irradiation was also used to further 50 accelerate the cross-linking reaction, and if the reaction is Institute. The multipoint mutant gene and pEVOL-pAzF were sufficiently fast, then hydrolysis of the enzyme protein cotransformed into MG1655 for pAzF incorporation expression, while pZE21-akr was transformed into BL21 for wild-type AKR (WT-AKR) promoted by some protease contaminants in the cell lysate expression.51 can be avoided, resulting in better retention of the structure The specific protein expression and purification experimental and enzymatic activity. In fact, as reported by Barbosa et al., if method is derived from our previous work.52 Plasmid preculture rapid cross-linking is used for forming CLEAs, then proteolysis containing the akr multipoint mutant gene was obtained by overnight (due to autolysis if the enzyme is a protease or due to the growth in LB, shaking at 34 °C with appropriate antibiotics action of some contaminant protease) of enzyme protein may (ampicillin, chloramphenicol, and kanamycin) added to LB to be suppressed even if it cannot be avoided.10 The AKR RP- maintain the plasmid. The culture was diluted 100-fold and re- CLEs were characterized using SEM and FT-IR, and the expanded in fresh LB containing antibiotics to expand the plasmid influences of microwave irradiation time and power on the culture. These cultures were grown to an OD600 of 0.5, and 0.2% L- specific activity of the RP-CLEs were also carefully arabinose was added for induction of pEVOL-pAzF for 1 h. To grow · −1 to an OD600 of 0.6, we added 30 ng mL aTc and/or 1 mM pAzF. investigated. In this method, NSAAs are expected to direct ° the cross-linking reaction to avoid random and unwanted Protein expression was performed for 16 h at 23 C with continuous shaking. The induction of pZE21-akr-BL21 only requires addition of covalent linkage, and the location and number of these μ · −1 100 g mL kanamycin to grow to an OD600 of 0.6, followed by substitutions are also expected to allow precise and control- addition of 30 ng·mL−1 aTc and shaking for 16 h. The induced lable multipoint immobilization and additional one-step enzymes were treated with ultrasonic disruption and purified by Ni purification. column. We use SDS-PAGE to verify the protein expression and matrix-assisted laser desorption/ionization time-of-flight mass spec- ■ EXPERIMENTAL SECTION trometry (MALDI-TOF MS; Bruker Ultraflextreme) to examine the Materials and General Methods. The bacteria (E. coli DH5α actual relative molecular mass of the obtained protein. and E. coli BL21(DE3)) used in studies were ordered from Stratagene Covalent Cross-Linking of Multipoint AKR Mutants under and Novagen. The used endonucleases and primers in the study were Consecutive Microwave Irradiation. In a preliminary study, we · −1 synthesized or purchased from Shanghai Generay Biotech Co. Ltd. dissolved the diyne in isopropanol (8 mmol L ) and then added this · −1 (Shanghai). Antibiotics (chloramphenicol, kanamycin, and ampicillin) solution to 1 mL of 0.1 mol L KPB, pH 7.0, containing the were purchased from Sangon Biotech. (Shanghai). Nonstandard multipoint mutants. The molar ratio of the azide to the alkynyl groups amino acid p-azide-L-phenylalanine (pAzF) was obtained from Artis in the reaction was 1:1. The vessel containing this mixture was placed Biotech Co. Ltd. The inducer anhydrotetracycline hydrochloride in a microwave reactor equipped with a cooling module and irradiated (aTc) and dihydro-4,4-dimethyl-2,3-furandione (Ketopantolactone) for a certain period (Discover CoolMate, CEM, USA). The system is were obtained from Sigma-Aldrich. NADPH was purchased from J&K continuously illuminated to control the temperature gap between the Scientific Ltd. sym-Dibenzo-1,5-cyclooctadiene-3,7-diyne (diyne) and microwave reaction reactor and the cooling module. Then, the cross- −1 all other chemical reagents were bought from Sinopharm Chemical linked enzymes were separated and washed with 2 mol·L NaCl Reagent Ltd. (Shanghai) unless otherwise stated, while all other solution and deionized water until no protein was detected in the biological reagents were purchased from Sangon Biotech. The whole- supernatant. Bradford analysis was used to determine the amount of plasmid site-directed mutagenesis PCR was performed to mutate the enzyme remaining in the supernatant. For a control, the same amount target gene.47 E. coli BL21 (DE3) is used for general cloning and of WT-AKR as AKR mutant was used to react with dyene under the DNA propagation. C321.ΔA (MG1655) is used for the expression of same conditions. NSAAs-modified enzymes to increase protein expression. In our To indicate the activity difference between the confined enzyme in experiments, deionized water was used as medium to prepare all RP-CLEs and free enzyme, relative activity was used to characterize solutions. Bacterial resistance(s) of plasmid and concentrations were the activity alteration as in the previous reports.53,54 Coupled yield in

6468 https://dx.doi.org/10.1021/acssuschemeng.0c00987 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article terms of enzyme recovery and relative activity for RP-CLEs were autosampler was kept at 20 °C, and the volume of each injection was calculated using the following equations: 10 μL. Data were collected at 226 nm, and the corresponding AB− standards were used to verify retention times to the substrate and Coupled yield (%)= × 100% target product standards. For the reuse stability measurement of RP- A (1) CLEs, RP-CLEs were separated from the reaction mixture by C centrifugation after the enzymatic synthesis reaction of 12 h every Relative activity (%)= × 100% time, and the product in the supernatant of reaction mixture was then AB− (2) extracted with 10 mL of anhydrous heptane. The product content was In the above equations, A represents the total activity of AKR mutant examined by HPLC, and the product yield (%) was determined and from cell lysate, B is the activity of the same amount of enzyme in the calculated according to the standard curve. In the case of the kinetic supernatant after cross-linking, and C is the activity of the CLEs. After parameter analysis, catalysis using RP-CLEs was performed at 30 °C immobilization, the amount of enzyme protein in the supernatant was with ketopantolactone as the substrate. The concentration of the determined by Bradford analysis. substrate used ranged from 0.4 to 31.2 mM. On the basis of the To verify the covalent cross-linking of the AKR multipoint mutants relationship between the concentration and the reaction rate, a under continuous microwave irradiation, the cross-linked enzymes substrate saturation graph was used to calculate the K and V − m max were hydrolyzed using 6 mol·L 1 HCl at 110 °C for 24 h. The values of the AKR five-point RP-CLEs. hydrolyzed sample was then washed 4 times with deionized water and analyzed by mass spectrometry using acetonitrile as the solvent. ■ RESULTS AND DISCUSSION Enzymatic Activity Assay and Thermal Stability of the AKR Preparations. The AKR mutants bearing pAzF were purified by Ni- Expression and Characterization of AKR Containing NTA affinity chromatography at 4 °C. Enzyme activity is examined pAzF. To prevent the active sites of the enzymes from being based on the previous literature as follows: 100 μL of NADPH buried or blocked, the sites of amino acid mutation are solution (2.5 mg·mL−1) and 100 μL of ketopantolactone (5 mg· preferred for covalent immobilization of the protein.56 We can − mL 1) were transferred into 0.7 mL of potassium phosphate buffer mutate the corresponding bases for encoding the chosen initial (KPB; 0.1 mol·L−1, pH 7.0).55 The mixture was first preheated at 30 ° native amino acid residues to the TAG codon by site-directed C for 3 min using a water incubator at 150 rpm. Then, AKR was mutagenesis. Thus, we generated a primitive plasmid, akr- added to the mixture and incubated for 1 min. The decrease in 114Y-pZE21, and on this basis, we obtained AKR three-point absorbance at 340 nm was monitored through the entire reaction, fi making it a measure of the conversion of cofactor (NADPH) to mutant and AKR ve-point mutant. However, when the NADP+. One unit of activity for AKR is defined as the amount of generated plasmid is transformed into BL21 cells to express the AKR required for catalyzing 1 μmol of dihydro-4,4-dimethyl-1,2,3- target enzyme protein, the upregulation of the natural furandione per minute. suppression mechanisms (e.g., ssrA) is problematic because To evaluate the thermal stability of AKR, the free and rapidly and these mechanisms promote the formation of truncation precisely cross-linked enzymes were transferred to reaction media and products.57,58 The genomically re-coded organism (GRO) ° ff incubated at 30, 40, 50, and 60 C using water bath for di erent fully recoded C321. The ΔA strain (MG1655) can avoid these periods of time. Then, samples from the suspension were periodically problems, as it allows the release of release factor 1 (RF1) and − withdrawn, and the activity of the aliquot was determined as described the redistribution of the UAG translation function.59 61 This above. The initial activity was set as 100%, and thermostability is strain can eliminate the TAG termination function in E. coli interpreted as residual activity (% basis), which is the ratio of the 51 remained activity of the RP-CLEs or the soluble enzyme after heating and generate only the UAA stop codon linkage. Therefore, to their initial activity, respectively. we cotransformed the plasmid akr-114Y-pZE21 with pEVOL- General Procedure for the Enzymatic Synthesis of (S)-1- pAzF to MG1655 and inserted pAzF to generate full-length (2,6-Dichloro-3-fluorophenyl) Ethanol Using AKR RP-CLEs. aldehyde ketone reductases including AKR three-point mutant The enzymatic synthesis of (S)-1-(2,6-dichloro-3-fluorophenyl) and AKR five-point mutant. ethanol was performed by the obtained AKR RP-CLEs in the We grew MG1655 cells cotransformed with akr three-point presence of NADPH (Scheme 2). To examine the optimal mutant gene and pEVOL-pAzF in LB medium containing ° S fl azido-phenylalanine at 37 C. Consistent with the above in vivo Scheme 2. Synthesis of ( )-1-(2,6-Dichloro-3- uorophenyl) enzyme assay data, the full-length AKR mutant was produced Ethanol Using RP-CLEs in an overall purified yield of 100 mg·L−1, whereas the previous BL21 system yielded only 2 mg·L−1 of one mutant protein for GFP with NSAA incorporated at one UAG site.62 The free enzyme activity of the AKR mutants was also determined depending on the above procedure. The activities of the AKR three-point mutant and AKR five-point mutant are very close to that of WT-AKR (1.07 U·mg−1)(Table 1). According to fi this study, the substrate speci city of the orthogonal MjTyrRS is concentration of the RP-CLEs for this reaction, AKR RP-CLEs directed by tyrosine in the protein containing natural amino were suspended in 5.0 mL of KPB (pH 7.0) containing 2 mg·mL−1 μ substrate, 10 mg of NADPH, and 500 L of isopropanol. The Table 1. Expression and Activity of WT-AKR, Free AKR, reactions were carried out at 37 °C at 250 rpm for 12 h, and then we centrifuged the reaction mixture to separate the RP-CLEs. The and the Cross-Linked Enzyme product in the supernatant of reaction mixtures were extracted with purified enzyme free enzyme RP-CLEs activity 10 mL of anhydrous heptane and analyzed by chiral HPLC. During · −1 · −1 · −1 content analysis, the proportions of isomers can be determined by enzyme (mg L ) activity (U mg ) (U mg ) high-performance liquid chromatography (HPLC, Agilent 1260). The WT AKR 180 ± 3.0 1.07 ± 0.02 final products were separated by liquid chromatography on a Daicel AKR three-point 100 ± 2.0 1.05 ± 0.01 1.27 ± 0.01 IC column (250 mm × 4.6 mm, 5 μm particle size). The pump was mutant run at a flow rate of 1 mL·min−1. Solvent A was hexane; solvent B was AKR five-point 82 ± 2.5 0.95 ± 0.04 2.06 ± 0.02 isopropanol. The volume ratio of n-hexane to isopropanol is 98:2. The mutant

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Figure 1. SDS-PAGE analysis (A) and MALDI-TOF of AKR five-point mutant (B). Lane M: protein marker; lane 1: culture supernatant of AKR five-point mutant + pAzF; lane 2: cellular soluble fraction of AKR five-point mutant + pAzF; lane 3: cellular insoluble fraction of AKR five-point mutant + pAzF; lane 4: culture supernatant of AKR five-point mutant − pAzF; lane 5: cellular soluble fraction of AKR five-point mutant − pAzF; lane 6: cellular insoluble fraction of AKR five-point mutant − pAzF; lane 7: AKR purified five-point mutant + pAzF.

Figure 2. SEM of AKR cross-linked enzyme derived from different enzyme concentrations (A, cell lysate supernatant, 18.75 mg·mL−1; B, purified enzyme, 18.75 mg·mL−1; C, cell lysate supernatant, 37.5 mg·mL−1; and D, purified enzyme, 37.5 mg·mL−1). acid.63 We found three tyrosine sites (110, 114, and 143) away In addition, as shown in Table 1, the AKR was mutated at from the active center through 3D structure simulation; thus, three and five points, respectively, depending on only a these residues were replaced by the desired tyrosine analogue preliminary analysis of the enzyme structure, and both the pAzF. However, the mutant sites cannot be limited to tyrosine obtained cross-linked enzymes presented enhanced catalytic residues due to the sequence of the enzyme protein, and activities. The activities of the three- and five-point mutants sometimes other amino acids, such as glutamine, were also were slightly lower than that of WT-AKR (Table 1). The selected for mutation based on the immobilization require- insertion of multiple NSAA may disrupt the natural proteins ments. structure to some extent, leading to a possible decrease in

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Figure 3. (A) FT-IR spectra of AKR RP-CLEs and AKR mutants (a, AKR three-point mutant without cross-linking; b, microwave-assisted AKR three-point RP-CLEs; and c, microwave-assisted AKR five-point RP-CLEs) and (B) mass spectrum of the acid hydrolysis products of the AKR RP- CLEs. enzyme activity. Thus, in future studies further optimization of and five UAG sites can undergo strain-promoted “double-click” the choice of the mutation sites for the enzyme could (SPDC) reactions with the diyne. By this novel convergent ameliorate these negative effects. However, after the novel method, we are also able to conjugate three molecules using supramolecular structures of cross-linked enzymes (CLEs) the Sondheimer diyne and cross-link spontaneously the azido- were formed, intermolecular cross-linking at multiple sites biomolecule using a small bifunctional azido molecule.42 would mimic macromolecular crowding effects of the enzymes To directly obtain the RP-CLEs from the cell lysate in the CLEs, which perhaps increases the rates of refolding and supernatant in an ecofriendly manner and avoid the use of − the yields of native state proteins.64 66 When enzyme proteins precipitation agents such as ammonium sulfate and organic are purified and removed from cells, their catalytic activities solvents, the cell lysate supernatant containing the AKR may decrease dramatically due to the absence of crowding mutants was concentrated, and the diyne was added to achieve environments.67,68 the intermolecular cross-linking of the AKR mutants. As a All the obtained purified protein was analyzed using SDS- control, AKR mutants purified by Ni-NTA chromatography PAGE (Figure S1A), and the apparent molecular weight of the were also cross-linked using a diyne cross-linker in buffer AKR-pZE21-BL21 protein was approximately 32 kDa (Figure solution. The morphologies of the obtained rapidly and S1B, Table S3). The arrow above the SDS-PAGE represents precisely cross-linked enzyme (RP-CLEs) were characterized the high expression of the multipoint mutants, and the by scanning electron microscopy (SEM), and Figure 2 shows induction temperature was selected according to our previous the micrographs of the RP-CLEs prepared under different work.69 As shown in the photographs of the SDS-PAGE results protein concentrations. The visible dimensions of the RP- (Figure 1A), the protein bands for the AKR five-point mutant CLEs are shown below the picture. We prepared RP-CLEs were all at approximately 32 kDa (Figure 1B, Table S3). with crude enzyme at different concentrations and with pure Similarly, bands for the AKR three-point mutant were also enzyme at different concentrations. observed at this location (Figure S2A). The actual molecular Figure 2A,BshowstheRP-CLEspreparedfromthe weight in the purified AKR mutants determined by MALDI- concentrated cell lysate supernatant and purified enzyme TOF analysis closely matched its theoretical molecular weight with protein concentrations of 18.75 mg·mL−1. Figure 2C,D (Figure S2B, Table S3). In addition, a lot of additional peaks shows the RP-CLEs prepared from the crude enzyme can be observed, and these peaks may be sodium adducts as supernatant and purified enzyme, respectively, at concen- mentioned earlier. To examine the insertion of NSAAs, control trations of 37.5 mg·mL−1. The RP-CLEs prepared from the expression experiments were carried out by simultaneous crude enzyme supernatant shown in Figure 2 are spherical contrast induction without NSAA pAzF. No band was found at particles, which is consistent with the shapes of CLEAs − approximately 32 kDa, and AKR mutants were not produced described in previous works.70 72 Most importantly, as shown when no pAzF was supplemented into the culture medium. in Figure 2A,C, the target enzyme protein AKR mutants can Therefore, we can reconfirm the successful expression of our also be cross-linked by Cu-free bio-orthogonal click reactions AKR mutants by MALDI-TOF and the insertion of pAzF into to form an almost uniformly structured RP-CLEs from the cell the AKR mutants. lysate. No metal salts, ammonium sulfate, or organic solvents Rapid and Precise Cross-Linking of AKR Mutants were used to prepare the RP-CLEs in this method. Using sym-Dibenzo-1,5-cyclooctadiene-3,7-diyne Moreover, as the protein concentration was increased, the under Consecutive Microwave Radiation. Under con- probability that the CLEAs form larger aggregates correspond- tinuous microwave irradiation at 0−25 °C, the diyne was used ingly increases.73 While CLEAs prepared with pure enzyme as a linker to form intermolecular cross-links between the AKR present larger particles and appear as small clusters, the size of multipoint mutants and the diyne. In this work, AKR the larger clusters may be more than 10−20 μm, which will derivatives incorporated with azido-phenylalanine at three cause significant mass transfer restrictions.74 By comparing

6471 https://dx.doi.org/10.1021/acssuschemeng.0c00987 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article crude and pure enzymes, we determined that higher enzyme the enzyme and decreases the effective Michaelis constant.80 concentrations can promote cross-linking and the formation of Macromolecular crowding can also increase the intrinsic RP-CLEs. The spherical structure of RP-CLEs provides a new activity of an enzyme by inducing conformational changes perspective into the CLEAs, because they have a higher and possibly other structural changes in the enzyme.81 specific surface area and therefore more catalytic sites per unit Effect of Temperature on the Coupled Yield and area, which is beneficial as a biocatalyst and reduces the Relative Activity of AKR Cross-Linked Enzymes. Figure diffusion limit in enzymatic reactions.75 4A presents the effect of temperature on the immobilization To verify the covalent linkages among the intermolecular yield and activity of the RP-CLEs obtained under microwave AKR multipoint mutants using the diyne under continuous irradiation. The optimal temperature for maintaining the microwave irradiation, we characterized the corresponding activity of AKR RP-CLEs was 10 °C. At this temperature, the samples using IR spectroscopy (Figure 3A). The existence of a specific activity of AKR in the RP-CLEs was 2.06 U·mg−1, N-containing heterocycle, derived from the click reaction of which means that the enzyme activity of the AKR enzyme the azide and alkyne in the AKR multipoint cross-linked aggregate can reach approximately 220% of that of the free enzymes, was identified based on the signals in the regions of enzyme. Moreover, microwave radiation effectively heats the − ∼2900 cm 1, which is characteristic of the stretching vibrations mixture without degrading the enzyme, as it does not adversely − of CC olefins, and ∼2800 cm 1, characteristic of CH affect the properties of the enzyme, such as stability or stretching vibrations of olefins, as shown in Figure 3A. These activity.82 At higher temperatures, microwave irradiation leads bands were not observed in the IR spectrum of the AKR to higher frequency factors, which increases the number of multipoint mutants before cross-linking. This further verified collisions between molecules and increases the energies of the that AKR RP-CLEs were successfully formed under microwave collisions. When the molecules have more energy, the electrons irradiation. Furthermore, the proteins generated from acid in the reacting molecules are more likely to jump to higher hydrolysis of the obtained RP-CLEs were characterized using energy levels. This movement of electrons increases the MS. In the intermolecular cross-links of the AKR mutants, randomness of the system, inevitably increasing the entropy of triazole structures will form due to the reaction of the diyne the system and reducing the activation energy, resulting in and two azides by click chemistry (Scheme 1). The mass faster reactions83 spectrum showed a protonated ion at 675, which is not However, at higher temperatures, although the change in the consistent with the target peak (Figure 3B). The obtained MS immobilization yield was not large, substantial inactivation of + peaks could be consistent with [M + Na ]and [M + CH3CN] the AKR RP-CLEs was also observed. The higher temperature because in our work, sodium dihydrogen phosphate and causes a significant decrease in the activity of AKR RP-CLEs. sodium hydrogen phosphate were used as components in the High-temperature microwave treatment slows down the 76−79 buffer and acetonitrile was used as the solvent. reaction by denaturing the protein structure of the enzyme The Km value of the free enzyme AKR used in a previous through thermal destruction and breaking down weak ionic 69 report is higher than that of AKR RP-CLEs. If the substrate and hydrogen bonds. enzyme. These bonds typically stabilize concentration necessary to achieve the enzyme saturation state the three-dimensional structure of the enzyme.84 is lower, then the apparent Km will decrease. However, if the Effect of Power for Microwave Irradiation on the substrate concentration necessary to achieve that state is Coupled Yield and Relative Activity of AKR Cross- higher, then K m will increase. However, in both situations, Kcat Linked Enzymes. The effect of microwave irradiation power ’ will remain unchanged. The RP-CLEs Km being lower than on the immobilization yield and activity of the AKR RP-CLEs that of the soluble enzyme indicates a higher apparent affinity was investigated in the power range from 5 to 40 W. The of the enzyme by the substrate used, probably by a crowded results in Figure 4B show that 10 W is the optimal power for environment, which mimics the natural environment of the AKR RP-CLEs. At this microwave power level, the activity of 10 enzyme in the cell. This indicates that the AKR five-point the obtained RP-CLEs was 2.06 U·mg−1 after covalent cross- RP-CLEs lead to increased substrate affinity in its catalytic linking for 4 min, which was 220% that of the free enzyme. reaction (Table 2, Figure S3). Moreover, the Kcat/Km value of However, at higher power, the activities of the RP-CLEs decreased dramatically even though the immobilization yield Table 2. Michaelis−Menten Kinetic Parameters of Free continued to increase slightly. AKR and AKR RP-CLEs If the microwave power is high and exceeds 50% (exceeding by more than 48 W) of the microwave capacity, then it Kcat/Km −1 −1· −1 ffi enzyme Km (mM) Kcat (s ) (mM s ) ref becomes di cult to control the temperature. Therefore, in fi order to consume less energy and maintain a more uniform AKR ve-point ± ± ± this RP-CLEs 1.99 0.02 10.22 0.25 5.26 0.13 work temperature distribution, a proper input power must be 85 free AKR 5.36 ± 0.12 7.71 ± 0.18 1.44 ± 0.03 69 reasonably selected. In addition, the three-dimensional structure of the enzyme undergoes major structural changes AKR RP-CLEs is 3.6-fold greater than that of free enzyme, at high microwave power.86,87 Experiments conducted under which means that the catalytic efficiency of the RP-CLEs is low microwave power, below 50 W, have shown ideal results at substantially higher and that using the RP-CLEs in catalysis optimal parameters. Trypsin activity toward the digestion of can significantly improve the catalytic efficiency and reduce casein has been reported to increase very significantly following costs. This enhancement in catalytic efficiency of the obtained exposure to low-power microwave irradiation (10 W) when the RP-CLEs may be attributed to their precise and bio-orthogonal reaction temperature is kept constant.88 When conducted at chemical cross-linking at preselected sites. Additionally, the the optimum temperature, suitable microwave power can give crowded environment resulting from the intermolecular cross- acceptable enzyme immobilization results. links would increase the volume fraction between enzyme Effect of Time for Microwave Irradiation on the proteins, which substantially influences substrate binding to Coupled Yield and Relative Activity of AKR Cross-

6472 https://dx.doi.org/10.1021/acssuschemeng.0c00987 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article

Figure 4. Effects of temperature (A, 4 min and 10 W) and microwave power (B, 4 min and 10 °C) on the coupled yield and relative activity of AKR RP-CLEs.

Linked Enzymes. To avoid the adverse effects of strong not occur with wild-type AKR (with nonreactive residues of microwave radiation, when studying the effect of microwave azide groups for the click reaction). Regarding the AKR three- radiation time on the AKR multipoint mutants, we chose 10 W point RP-CLEs, the enzyme confined in CLEs presents 1.27 U and 10 °C as the main power and temperature, respectively.89 mg−1 catalytic activity under optimal conditions, and the As shown in Figure 5, 4 min is long enough to achieve relative activity is 121.0%, which indicates that the structure of AKR mutants is not destroyed in the cross-linking. Thermal Sability of the AKR Mutants’ Preparations. Figure 6 demonstrates the thermal stability of the AKR RP- CLEs and free enzyme incubated at 30, 40, 50, and 60 °Cin 0.1 M KPB at pH 7.0. Compared to the free enzymes, AKR RP-CLEs present higher thermal stability. After 16 h of heating at 60 °C, the residual activity of the AKR five-point RP-CLEs under continuous microwave irradiation was still 70.3%. However, free AKR preparations lost more than 90% of their initial activities. Both Cesar Mateo et al. and Lopez-Gallego et al. have demonstrated that multipoint covalent immobilization can provide better thermal stability for immobilized enzymes.92,93 We prepared a semilogarithmic plot of the percentage of residual activity versus time, calculating the denaturation rate Figure 5. Effects of time on the coupled yield and relative activity of constant (KD) as the slope and the time (t1/2) required to AKR RP-CLEs (10 °C and 10 W). reduce the activity to half its original activity. In addition, the reaction activation energies (Ea values) using the free enzyme and the enzyme aggregate were calculated from their Arrhenius complete cross-linking of AKR to form RP-CLEs using the diagrams. For AKR five-point RP-CLEs cross-linked for 4 min diyne linker. Excessive microwave irradiation does not increase under 10 W of microwave radiation power, the half-life time the enzymatic activity because microwave radiation can ° enhance the kinetics of protein unfolding.90 The molecular (t1/2)at60 C is 26.76 h, which is approximately 4.5 times (t1/2 = 5.97 h) that of the corresponding free enzyme rearrangement during microwave processing changes the fi intermolecular spacing between proteins, thereby changing preparation (Table S4). The half-lives (t1/2) of our ve-point 91 RP-CLEs at 30, 40, 50, and 60 °C were 5.45, 4.60, 4.71, and their quaternary and tertiary structure. To investigate the fi effect of irradiation time on the size of the RP-CLEs, 1, 2, 4, 4.48 times those of the corresponding AKR ve-point mutant, and 6 min of irradiation were tested, and the obtained RP- respectively. The results shown in Table S4 indicate that the Ea CLEs were characterized using SEM (Figure S4). The results of the RP-CLEAs gradually increases, which may be attributed show that the size of the RP-CLEs gradually increased until 4 to the covalent binding of the azide and alkynyl groups min. The diameter of the RP-CLEs was approximately 10 μm, increasing the rigidity of the enzyme protein. Enhanced ff which suggests that the most complete AKR RP-CLEs were structural sti ness and entropy stability correspond to 94,95 formed in 4 min. enhanced thermal stability. Additionally, both crowding Before we carried out the rapid and precise cross-linking of and confinement can significantly influence the ubiquitous enzymes aggregates using bio-orthogonal chemical reaction, we cellular functions such as association, activation, stabilization, 96 finished a control experiment. The wild-type AKR was mixed and conformation. This increase in stability was due to the and allowed to react with cross-linker diyne (sym-dibenzo-1,5- decrease in the configurational entropy of the unfolded state, cyclooctadiene-3,7-diyne) under consecutive microwave irra- which is substantially lower than that of the native state.97 By diation at 4 °C for 2 min. However, the mixture remained favoring lower entropy and lower energy complexes, macro- relatively as clear as the original, and no aggregates were molecular crowding can also increase the association obtained because the bio-orthogonal cross-linking reaction did constant.96,98

6473 https://dx.doi.org/10.1021/acssuschemeng.0c00987 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article

Figure 6. Thermal stability of AKR preparations. A, 30 °C; B, 40 °C; C, 50 °C; D, 60 °C. FP-CLEs, five-point AKR RP-CLEs; TP-CLEs, three- point RP-CLEs; Free AKR, free wild-type AKR; Free TP AKR mutant, free three-point AKR mutant; Free FP AKR mutant, free five-point AKR mutant.

Figure 7. HPLC analysis of the synthesized (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol using AKR mutant preparations biocatalysts (A, free AKR; B, RP-CLEs, 12 h per cycle at 37 °C).

Enzymatic Synthesis of (S)-1-(2,6-Dichloro-3-fluoro- fluorophenyl) ethanol, we used standard samples, 2,6-dichloro- phenyl) Ethanol Using AKR Cross-Linked Enzyme. For 3-fluoroacetophenone, (S)-1-(2,6-dichloro-3-fluorophenyl) industrial applications, the reusability of immobilized enzyme ethanol and (±)-1-(2,6-dichloro-3-fluorophenyl) ethanol, to preparations is the key to cost-effective use.99 The catalytic perform high-performance liquid phase examination. Retention performance of AKR five-point RP-CLEs for the synthesis of times of substrate, S-enantiomer product, and R-enantiomer (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at 37 °C using product were about 4.8, 6.2, and 6.6 min, respectively (Figures NADPH as a cofactor was assessed in this work. In order to S5−S7). determine the retention time of the substrate 2,6-dichloro-3- When equimolar amounts of the substrate and NADPH are fluoroacetophenone and the product (S)-1-(2,6-dichloro-3- used, the yield of the target product after 12 h reaction reaches

6474 https://dx.doi.org/10.1021/acssuschemeng.0c00987 ACS Sustainable Chem. Eng. 2020, 8, 6466−6478 ACS Sustainable Chemistry & Engineering pubs.acs.org/journal/ascecg Research Article 90.8% using RP-CLEs, which was 8.4-fold higher than that ■ AUTHOR INFORMATION using free AKR (HPLC chromatograms of reaction medium Corresponding Authors after 12 h of reaction are shown in Figure 7B,A, respectively), − and the ee value is as high as 99.98% in the first 12 h batch Anming Wang College of Materials, Chemistry and Chemical (close to 100%, Figure 8), higher than what can be achieved Engineering, Hangzhou Normal University, Hangzhou 311121, Zhejiang, PR China; orcid.org/0000-0002-8480-5783; Phone: +86-571-28865978; Email: [email protected]; Fax: +86-571-28865630 Pengfei Zhang − College of Materials, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, Zhejiang, PR China; orcid.org/0000-0001-9859-0237; Phone: +86-571-28865978; Email: [email protected]; Fax: +86-571-28865630 Authors Huimin Li − College of Materials, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, Zhejiang, PR China Ru Wang − Holistic Integrative Pharmacy Institutes, College of Medicine, Hangzhou Normal University, Hangzhou 311121, Figure 8. Reusability of the AKR RP-CLE for the enzymatic synthesis Zhejiang, PR China of (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol (12 h per cycle at 37 Jing Zhang − College of Materials, Chemistry and Chemical ° C). Engineering, Hangzhou Normal University, Hangzhou 311121, Zhejiang, PR China Youcheng Yin − Holistic Integrative Pharmacy Institutes, College 100 of Medicine, Hangzhou Normal University, Hangzhou 311121, with chemical methods. Moreover, after 6 consecutive 12 h Zhejiang, PR China reaction cycles, the yield of (S)-1-(2,6-dichloro-3-fluorophen- − fi Xiaolin Pei College of Materials, Chemistry and Chemical yl) ethanol using AKR ve-point RP-CLEAs still retained 80% Engineering, Hangzhou Normal University, Hangzhou 311121, of its initial activity, and all the ee values are over 99.5% in the Zhejiang, PR China; orcid.org/0000-0002-1953-4323 6 consecutive 12 -h reaction cycles (Figure 8). In the future, with the regeneration of NADPH, the cost of producing the Complete contact information is available at: corresponding drug intermediate and other fine chemicals https://pubs.acs.org/10.1021/acssuschemeng.0c00987 would be substantially decreased using this efficient and low- cost RP-CLEs prepared directly from a cell lysate. Author Contributions The manuscript was written through contributions of all ■ CONCLUSIONS authors. All authors have given approval to the final version of the manuscript. In this study, NSAAs with reactive groups were incorporated into the enzyme protein and directed the precise intermo- Notes lecular cross-linking of the AKR mutants to avoid random and The authors declare no competing financial interest. unwanted covalent linkages. Using this Cu-free biorthogonal click reaction, AKR RP-CLEs were prepared from the cell ■ ACKNOWLEDGMENTS lysate, which allows a convenient one-step purification and immobilization process. No inorganic salts, including metal This study was supported by the National Natural Science ions, and no organic solvents, were used; additionally, RP- Foundation of China (21576062), the Natural Science CLEs can be formed rapidly under consecutive microwave Foundation of Zhejiang Province (LY18B060009, irradiation at low temperature. The obtained RP-CLEs present LY15B060011), the Technology Research and Development enhanced catalytic efficiencies and thermal stability, which may Program of Hangzhou (20191203B09), the Key Projects of be a result of the macromolecular crowding from the proximity National Natural Science Foundation of China (81730108), of the AKR mutants. These RP-CLEs also demonstrate fair the Research Plan for Sprout Talents in University in Zhejiang stability for reuse for many cycles in the biocatalytic synthesis Province (2019R426081), the National Innovation and of high value-added drug intermediates. This green method for Entrepreneurship Training Program for Undergraduate “ ” preparing RP-CLEs from cell lysates could be further improved (201810346008), and the Star and light Project for Talent and expanded to other enzymes for efficient biocatalysis. Students in Hangzhou Normal University (2019).

■ ASSOCIATED CONTENT ■ LIST OF ABBREVIATIONS *sı Supporting Information AKR, aldehyde ketone reductase; CLEAs, cross-linked enzyme The Supporting Information is available free of charge at aggregates; RP-CLEs, rapidly, precisely, and bio-orthogonally https://pubs.acs.org/doi/10.1021/acssuschemeng.0c00987. cross-linked enzymes; pAzF, p-azido-L-phenylalanine; diyne, sym-dibenzo-1,5-cyclooctadiene-3,7-diyne; SPAAC, strain-pro- fi Primers used for preparing AKR mutants and gures of moted alkyne−azide cycloaddition; NSAAs, nonstandard some characterization information and catalysis of AKR amino acids; aTc, anhydrotetracycline hydrochloride; KPB, CLEs (PDF) potassium phosphate buffer

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