Identification, Characterization, and Site-Specific Mutagenesis of a Thermostable ω-Transaminase from Chloroflexi bacterium.

In the present study, we have identified an ω-transaminase (ω-TA) from Chloroflexi bacterium from the genome database by using two ω-TA sequences (ATA117 Arrmut11 from Arthrobacter sp. KNK168 and amine transaminase from Aspergillus terreus NIH2624) as templates in a BLASTP search and motif sequence alignment. The protein sequence of the ω-TA from C. bacterium (CbTA) shows 38% sequence identity to that of ATA117 Arrmut11. The gene sequence of CbTA was inserted into pRSF-Duet1 and functionally expressed in Escherichia coli BL21(DE3). The results showed that the recombinant CbTA has a specific activity of 1.19 U/mg for (R)-α-methylbenzylamine [(R)-MBA] at pH 8.5 and 45 °C. The substrate acceptability test showed that CbTA has significant reactivity to aromatic amino donors and amino receptors. More importantly, CbTA also exhibited good affinity toward some cyclic substrates. The homology model of CbTA was built by Discovery Studio, and docking was performed to describe the relative activity toward some substrates. CbTA evolved by site-specific mutagenesis and found that the Q192G mutant increased the activity to (R)-MBA by around 9.8-fold. The Q192G mutant was then used to convert two cyclic ketones, N-Boc-3-pyrrolidinone and N-Boc-3-piperidone, and both the conversions were obviously improved compared to that of the parental CbTA.

Introduction

Chiral amines are very important intermediates, which have a wide range of applications in medicine and fine chemical industries.[1] The production of chiral amines includes chemical and biochemical methods. As far as chemical methods are concerned, chiral amines are usually produced by the asymmetric catalysis of prochiral molecules, such as hydrogenation of imine or enamine, alkylation of imine, amino hydroxylation, and reductive amination.[2] In order to overcome the drawbacks of low enantioselectivity, need for noble-metal catalysts, harsh reaction conditions, and environmental concerns in asymmetric synthesis, several biochemical methods using efficient biocatalysts have been developed and managed to replace chemical methods. These methods mainly involve enzymes such as amine oxidases, ammonia lyases, amine dehydrogenases, and transaminases.[3−5] Among them, ω-transaminases (ω-TAs), which catalyze the transfer of ketone and amino groups, have received extensive attention.[6−9] The well-known example of the synthesis of chiral amines by ω-TAs is the manufacturing of sitagliptin, an oral antihyperglycemic drug.[10]

Interestingly, ω-TAs originating from various sources have shown distinct enantioselectivity, that is, (R)-selective or (S)-selective. Hence, both enantiomers of specific amines can be produced by ω-TAs with different enantioselectivities. Although the ω-TA Ata-117 Arrmut11 from Arthrobacter sp. KNK168 is commercially available now, only a few (R)-selective ω-TAs have been identified.[11,12] Therefore, the discovery of novel R-selective ω-TAs has still been the focus issue in the past few years. Bornscheuer et al.[13,14] developed a method for predicting and screening enzyme functions on the basis of a key motif in sequence, which has been proved to be effective and successfully used to search and identify 17 (R)-selective ω-TAs by database mining. The method has also been applied to discover five other different sources of (R)-selective ω-TAs.[15] In recent years, more and more (R)-selective ω-TAs have been discerned and investigated, but their wide applications are still limited by their poor properties.[16−18] In order to improve the performance of enzymes, protein engineering has usually been adopted to conduct enzyme design and optimization. An (R)-selective ω-TA has been created by bioinformatic analysis combined with the computational redesign of the d-amino acid aminotransferase, exhibiting a specific activity close to those of natural (R)-selective ω-TAs.[19] On the basis of a fluorescence-based screening system, a KnowVolution campaign has been carried out to optimize a (R)-selective ω-TA from Mycobacterium vanbaalenii, and the best resulting mutant showed specific activity to acetonaphthone more than 100 times higher than that of the parental enzyme.[20] In another study, a (R)-selective ω-TA from Arthrobacter cumminsii ZJUT212 has been modified using a semirational protein design, and a mutant has been screened to produce the sitagliptin intermediate on a kilogram scale with >99% e.e. and approximately 80% conversion.[21]

In the current study, BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed against an NCBI nonredundant protein sequence library to identify a putative ω-TA sequence from Chloroflexi bacterium (CbTA) using Ata-117 Arrmut11 from Arthrobacter sp. KNK168 and amine transaminase from Aspergillus terreus NIH2624 as templates. Subsequently, the gene of CbTA was cloned into Escherichia coli for expression, and its kinetic parameters and substrate spectra were characterized. Thereafter, CbTA was improved by site-specific mutagenesis on the basis of the reported results and modeling analysis.

Results and Discussion

Sequence Analysis

Nowadays, more and more protein sequences are stored in the database, which provides a huge resource for the mining of biocatalysts. Ata-117 Arrmut11 from Arthrobacter sp. KNK168 (PDB ID: 5FR9), a variant of the KNK168 with mutations in 27 positions with excellent stereoselectivity, has been successfully applied to industrial synthesis and a series of kinetic resolution.[10,22] The amine transaminase fromA. terreus NIH2624 (PDB ID: 4CE5) is another important template used to explore the functional motif of (R)-selective ω-TAs and has been shown to be highly active.[14] Two independent BLAST searches were performed using two crystallized (R)-selective ω-TAs, Ata-117 Arrmut11 from Arthrobacter sp. KNK168 and amine transaminase from A. terreus NIH2624 as templates. Then, rational analysis of the aligned sequences was performed using a motif sequence alignment, following the criteria previously established by Höhne and Bornscheuer.[14]Four putative sequences derived from thermophilic bacteria were obtained from the NCBI nonredundant protein sequence library: EAY57657.1 (Leptospirillum rubarum), EES52356.1 (Leptospirillum ferrodiazotrophum), RIK47101.1 (Chloroflexi bacterium), and RLT41415.1 (Chloroflexi bacterium). The construction and expression of the four enzymes are the same. According to the preliminary activity test, the crude enzyme activities of these four enzymes were 0.207, 0.124, 0.91, and 0.248 U/mg, respectively. Among them, CbTA, the ω-TA from C. bacterium (RIK47101.1) with the highest activity, was selected as the target for further study.

CbTA showed identity with amine transaminases from the following microorganisms: Arthrobacter sp. KNK168[23] (PDB ID: 3WWH; 38%), A. terreus NIH2624[24] (PDB ID: 4CE5; 36.1%) Aspergillus fumigatus Af293[25] (GenBank accession no. EAL86783; 37.2%), Archaeoglobus fulgidus DSM 4304[26] (PDB ID: 5MQZ; 37.8%), Nectria haematococca MPVI[27] (PDB: 4CMD; 36.8%), M. vanbaalenii PYR-1 (GenBank accession no. ABM15291; 35.9%), and Geoglobus acetivorans(26) (PDB ID: 5E25; 35.8%). Although the identity with the reported sequence is not high, the functional sites of R86, K188, and E221 are conservative after alignment. Phylogenetic tree analysis is performed to verify the taxonomic and evolutionary relationship of CbTA to previously reported transaminases, and the results are presented in Figure . Nine amino acid sequences with different degrees of sequence identity to CbTA were collected and compared. CbTA was found to have a close evolutionary relationship with thermophilic archaea Geoglobus acetivorans.

Figure 1

Phylogenetic analysis of CbTA from C. bacterium and related proteins. The phylogenetic tree was constructed using the neighbor-joining algorithm in the molecular evolutionary genetic analysis package (MEGA 7) and the relative positions of the proteins were highlighted and labeled with species names.

Purification and Identification of the Recombinant CbTA

The recombinant E. coli BL21(DE3)/pRSF-CbTA cells were cultured, collected, and sonicated. The expressed recombinant protein with a theoretical molecular weight of 44.3 kDa and was detectable in the soluble part of E. coli BL21 (DE3)/pRSF-CbTA cells (Figure ). The recombinant protein is purified by immobilized metal ion affinity chromatography with the assistance of His-tag at the N-terminus of the sequence, and the purification results are shown in Figure . The followed activity test indicated that the specific activity of CbTA to (R)-α-methylbenzylamine [(R)-MBA] was 1.19 U/mg at pH 8.5, 45 °C.

Figure 2

SDS-PAGE of CbTA. Lane 1, protein markers; lane 2, crude extract; and lane 3, purified enzyme.

Enzymatic Properties of CbTA

As shown in Figure a, the effect of pH on CbTA activity is assessed over the range of pH 6.0 to 10.5, and CbTA has the highest activity at pH 8.5, which is similar to the known ω-TAs.[28,29] For example, a typical (R)-selective ω-TA from Arthrobacter sp. KNK168 has shown an optimal pH of 8.0 to 9.0.[30] Besides, a branched-chain amino acid aminotransferase TUZN1299 identified from the genome of the hyperthermophilic archaeon Thermoproteus uzoniensis has exhibited an optimal pH of 8.0.[31] The influence of temperature on CbTA activity was evaluated over a range of 25–65 °C, and the highest activity was detected at 45 °C (Figure b). Figure c presents the temperature profile of CbTA, and results show that it has good thermal stability. Interestingly, with the increase of incubation time at 45 °C, the highest activity of CbTA can reach twice of the original activity. The possible mechanisms for stability are high bulk density, optimal charge pattern or ion pair, and minimized hydrophobic surface area and helical stability.[32] This increase in activity is probably caused by temperature-related folding that increases stabilizing interactions, thus improving its reactivity; we also noticed a similar increase in an thermophilic transaminase from Geobacillus thermodelinificans.[33] The temperature dependence also showed that CbTA retains 50% activity after 12 h of incubation at 50–55 °C, but it was significantly inactivated at above 60 °C. Similarly, most of the thermophilic TAs have shown significant thermal stability at about 55 °C.[34] For instance, it has been found that a transaminase identified from Thermomicrobium roseum retained 50% activity after 5 h of incubation at 70 °C.[35] A β-amino acid transaminase Ms-TA2, which has been discovered in the genome of the Meiothermus strain isolated in an Icelandic hot spring, kept around 60% activity after incubation at 50 °C for 3 h.[36] However, few thermophilic TAs remain active after incubation at temperatures above 65 °C. A novel amine transaminase has demonstrated spectacular thermostability, and its activity can be maintained at 85% or around 40% after being incubated at 80 °C for 5 or 14 days.[37]

Figure 3

Characterization of enzymatic properties of CbTA. (a) Optimum pH; (b) optimum temperature; (c) temperature stability; and (d) effect of organic cosolvent concentration on CbTA activity.

The organic cosolvent is very necessary to increase the solubility of substrates. The influence of the organic cosolvent on CbTA activity was determined by adding methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile in reaction solutions (Figure d). Results showed that the activity of CbTA increased significantly in all four 20–40% organic cosolvents, while it decreased in 60% organic cosolvents. This indicates that CbTA is well tolerable to the organic solvents and the results are superior to that of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic Period salt mine.[38]

Kinetic Parameters of CbTA

The kinetic parameters are helpful to evaluate the catalytic ability of enzymes. Given that transaminases operate via a typical dual substrate recognition, the Km and kcat to either substrate of CbTA were determined by changing the concentration of (R)-MBA or pyruvate from 2.5 to 40 mM, respectively. With 20 mM pyruvate as a cosubstrate, the Km and kcat of CbTA to (R)-MBA are 14.68 mM and 3397.63 s–1, while the Km and kcat of CbTA for pyruvate are 2.834 mM and 1617.5 s–1, respectively. Compared with the ATA117 from Arthrobacter sp. KNK168, CbTA showed similar affinity for pyruvate, but its Km value for (R)-MBA was significantly higher than that of ATA117, which indicated that CbTA had a slightly lower affinity for amine. However, CbTA showed lower Km to pyruvate and higher kcat than a transaminase fromFusarium oxysporum,[39] which means higher reactivity on pyruvate (Figure ).

Figure 4

Nonlinear regression fitting of the Michaelis–Menten equation.

Substrate Specificity of CbTA

Previous studies on the substrate range and stereoselectivity have shown that the active center of transaminase is composed of two active pockets. The large pocket can embrace the large group and the small pocket is usually limited to methyl-sized substituents.[40] Considering the binding of the substrate to the ω-TA active center, we resolved the structure of the substrate on the basis of the ω-TA active site model, and the ω-TA’s large pocket embraces R1, while the ω-TA’s small pocket contains R2. All substrates were divided into four groups as follows: aromatic and aliphatic amines, amino acids, ketones, and aldehydes to allow us to compare reactivity chemically.

As shown in Tables –4, we can find that CbTA can utilize a wide range of amino donors and acceptors for the substrate range, which is consistent with the literature41, indicating their potential for synthetic applications. It can be seen from Table that CbTA has good activity for most aliphatic and aromatic amines, but there is a marked difference among the various aliphatic or aromatic amines, which is similar to the results reported by Jiang et al.[15] A comparison of the relative activity of CbTA to aliphatic amines revealed that it showed a higher activity for the long-chain amines (A5–A8), while it exhibited a very low reactivity against the short-chain amines, isopropylamine (A3) and 2-butylamine (A4), except for propylamine (A2). Interestingly, CbTA displayed higher reactivity to propylamine but extremely low to isopropylamine unlike other amine transaminases. Furthermore, CbTA is quite reactive to cyclic amines, and its relative activity is normally better with the increase of the ring (A9–A13). Unexpectedly, when amino acids are used as the amino donors (B1–B4), the activity given by CbTA is not very high, or even very low, for example, 2-aminobutyric acid (B5).

Table 1

Amino Donor Specificity of CbTA for Aliphatic and Aromatic Amines

Reaction conditions in Tables and 2: 0.1 mg mL–1 purified enzyme, 25 mM pyruvate and 25 mM amino donor, 1 mL of glycine–NaOH buffer (50 mM, pH 8.5), and 40 °C. One unit of enzyme activity was defined as the conversion of 1 μmol pyruvate per minute. The relative activity for (R)-MBA was designated as 100%.

Table 4

Amino Acceptor Specificity of CbTA for Aldehydes

amino acceptor	R1	R2	relative activity (%)a
D1	–CH2CH3	–H	1.2
D2	–(CH2)2CH3	–H	6
D3	–CH(CH3)2	–H	5.7
D4	–CH(CH2)4(CH3)2	–H	5.8
D5	–C6H5	–H	272
D6	–C6H3(2-OH) (5-NO2)	–H	250
D7	–CH2C6H5	–H	26

Reaction conditions in Tables and 4: 0.1 mg mL–1 purified enzyme, 25 mM (R)-MBA and 25 mM amino acceptor, 1 mL of glycine–NaOH buffer (50 mM, pH 8.5) and 40 °C. One unit of enzyme activity was defined as the conversion of 1 μmol acetophenone per minute. The relative activity for pyruvate was designated as 100%.

Table 2

Amino Donor Specificity of CbTA for Amino Acids

amino donor	R1	R2	relative activity (%)a
B1	–(CH2)2COOH	–COOH	36
B2	–(CH2)3NHNH2NH	–COOH	38
B3	–CH2SH	–COOH	48
B4	–CH2COOH	–CH3	4

Reaction conditions in Tables and 2: 0.1 mg mL–1 purified enzyme, 25 mM pyruvate and 25 mM amino donor, 1 mL of glycine–NaOH buffer (50 mM, pH 8.5), and 40 °C. One unit of enzyme activity was defined as the conversion of 1 μmol pyruvate per minute. The relative activity for (R)-MBA was designated as 100%.

Table 3

Amino Acceptor Specificity of CbTA for Keto Acids and Ketones

Reaction conditions in Tables and 4: 0.1 mg mL–1 purified enzyme, 25 mM (R)-MBA and 25 mM amino acceptor, 1 mL of glycine–NaOH buffer (50 mM, pH 8.5), and 40 °C. One unit of enzyme activity was defined as the conversion of 1 μmol acetophenone per minute. The relative activity for pyruvate was designated as 100%.

The amino receptor specificity of CbTA is studied and the results are presented in Tables and 4. CbTA appears to have similar amino receptor specificity for Capronia semiimmersa.[42] The amino receptor spectrum shows that CbTA has low reactivity to nearly all aliphatic, cyclic, and aromatic ketones except for 2′-nitroacetophenone (C7), 4′-methoxyacetophenone (C8), benzaldehyde (D5), and 5-nitrosalicylaldehyde (D6). According to the results given in tables, amino acceptors with an aryl group have comparatively good reactivity for CbTA, and aryl ketones and aromatic aldehydes present similar results. However, low activity for CbTA is observed in the case where the selected aliphatic ketones or aldehydes were used as amino acceptors. In comparison, the reactivity of CbTA toward aliphatic ketones and aldehydes was different from previously reported results.[43] It should be noted that in most cases benzene ring with methyl substituent group in aryl ketones would dramatically inhibit the reactivity of amino acceptors, and the existence of methylene between aryl group and aldehyde group also significantly reduced the activity of CbTA to aryl aldehydes. Additionally, CbTA appears to be reactive to two selected cyclic substrates with ketone groups.

Discovery studio proposed an active site model of CbTA binding to pyridoxal 5′-phosphate (PLP). The most favorable binding model contains several reported key residues. Previous study has reported that residue K252 shows significant catalytic reactivity with coenzymes and substrates.[44] As is shown in Figure , K252, which binds to the center of the pocket, has a strong hydrogen bond force and attractive charge with PLP. At the same time, a strong hydrogen bond force was also found between PLP and residues G288 and S346. Residue E285 is located in the substrate-cofactor binding pocket and has a conventional hydrogen bonding with the amino group of (R)-MBA. In addition, it is observed that the force contribution of the oxygen atom of the phosphate group in PLP was provided by K252, G288, and E285, which is similar to other (R)-selective transaminase.[45]

Figure 5

Model of the active site of CbTA binding with PLP and (R)-MBA. The key residues in the active site are shown in green.

To manage to explain the possible cause of substrate specificity, some selected amino donors and acceptors were docked with CbTA and PLP, and the results were visualized by Discovery Studio (Figure and Figure ). The most favorable docking configuration was selected to analyze the forces. As is shown in Figure , from the docking results of aliphatic amines (A2–A8), residues in binding sphere of CbTA might have Pi–alkyl and attractive charge on the carbon chain, except isopropylamine (A3), which explains the low reactivity for A3. According to the docking results of cyclic amines, PLP has strong acting forces, such as Pi–cation interaction and conventional hydrogen bond, on the amino groups of cyclic substrates cyclobutanamine (A10) and cyclopentylamine (A11), while the acting force on amino groups of cyclopropylamine (A9) is only the C–H bond. It seems that with the increase of the size of the cyclic substrate, the L-pocket becomes more suitable for binding ligands. When aromatic substrates were involved, there were many intermolecular forces between residues in binding sphere and benzene ring, including T-shaped Pi–Pi stack and Pi–alkyl interactions. Taking (R)-1-(3-chlorophenyl) ethylamine (A17) as an example, we could see that residues R287 and Q259 have a strong conventional hydrogen bond with the amino group of A17 and that the chloro group in benzene ring is an electron-withdrawing group forming a Pi–alkyl force with residue H260, which may lead to high reactivity of A17. In addition, the low activity of benzylamine (A14) is probably because of the fact that coenzyme PLP has no interaction with it, and residue E259 also has an unfavorable donor–donor to its amino group.

Figure 6

Docking results of CbTA with selected amino donors listed in Tables and 2.

Figure 7

Docking results of CbTA with selected amino acceptors listed in Tables and 4.

The docking results of selected amino acceptors with the active site model of CbTA binding to PLP are shown in Figure . We could observe that pyridoxamine 5′-phosphate (PMP) has a strong hydrogen bond with the aldehyde group in the typical acceptor pyruvate (C1), and residues T242, N253, and H258 also bear the corresponding hydrogen bond with the aldehyde group and carboxyl group; we infer that this leads to strong affinity for pyruvic acid. As for α-ketoglutarate (C6), the interaction between PMP and the substrate was not found, which may be the reason why CbTA has no activity on C6. From the possible docking diagrams of ketones, it could be perceived that the active site of PMP and the enzyme tended to bind to stable structures containing benzene or rings and only exhibited weak van der Waals for straight chain substrates, which might be the reason why most of the aliphatic ketones were low or even inactive, for example, pentanone (C3). For the docking results of aromatic ketones, the electron-withdrawing groups of 2′-nitroacetophenone (C7) with good reactivity show the attractive charge and salt bridge with PMP. In the case of 4′-methoxyacetophenone (C8), PMP has a Pi–alkyl interaction with its benzene ring and other residues have a Pi–Pi T-shaped interaction and hydrogen bond with the benzene ring and ketone groups, with its methoxy group having an additional C–H interaction binding with CbTA. In contrast to other acceptors, the substituted R2 groups show little interaction with CbTA and PMP, suggesting that the small pocket of CbTA is not suitable for substrates with R2 more than one methyl group, which may be responsible for the lack of activity of 2-hydroxyacetophenone (C13) and valerophenone (C14). For aldehydes, residues in both PMP and binding sphere have a conventional hydrogen bond with the aldehyde group except for propionaldehyde (D1) and 2-ethylhexanal (D4), which might explain the low reactivities of D1 and D4. With regard to the most reactive amino acceptors benzaldehyde (D5) and 5-nitrosalicylaldehyde (D6), it is found during the docking that residue G259 appears to exert a strong force on the D5 and D6 aldehyde groups, which is not available for other aldehydes during the docking. Besides, the nitro group of D6 appears to have an additional force on the active site as an electron-withdrawing group.

Site-Specific Mutagenesis of CbTA

The enzymatic transamination of ketones to amines is an important method to obtain various chiral building blocks or pharmaceuticals. In this study, a new ω-TA, CbTA, was obtained and its enzymatic properties and substrate spectrum were determined, showing the potential of CbTA for the production of chiral amines and unnatural amino acids. However, the unsatisfactory activity of CbTA on many ketones is a vital obstacle for its application in chiral amine production. In order to improve its activity, site-saturation mutagenesis was conducted on the basis of the previous results and homology modeling.

As confirmed by Voss et al., residues F130, Y190, Q190, G288, and A348 showed significant influence on the activity of amine transaminase. The docking result of CbTA with PLP and (R)-MBA indicated that residues K252, E285, G288, Q192, and Y190 are all located in the small pocket of the active site. Hereby, residues Y190, Q192, and G288 are selected for site-specific mutagenesis, and the results are presented in Figure . The results showed that the changes in residues Y190 and Q192 did have a significant effect on the activity of CbTA to (R)-MBA. Among them, mutant Q192G was the most beneficial mutation. The activity of the mutant Q192G is 9.8 times than that of the parental enzyme. However, there was no significant change in the activity of mutants G288 for CbTA.

Figure 8

Results of site-specific mutagenesis of CbTA.

Chiral N-heterocyclic molecules, especially compounds with amino groups, such as 3-aminopiperidines, are valuable intermediates in the production of biologically active compounds with pharmacological properties.[46] Subsequently, CbTA and mutant Q192G are further applied to evaluate the conversion of cyclic substrates with a ketone group, and the results are shown in Figure . After 36 h of reaction, the conversion of N-Boc-pyrrolidinone and N-Boc-piperidone by CbTA was about 15% and 12%, and the conversion of N-Boc-pyrrolidinone and N-Boc-piperidone by mutant Q192G was increased to 23% and 30%, respectively. Compared with the parental CbTA, the reactivity of the mutant Q192G to cyclic substrates was obviously improved.

Figure 9

Conversion of N-Boc-3-pyrrolidinone and N-Boc-3-piperidone by the ω-TA and its Q192G mutant: (a) N-Boc-3-pyrrolidinone and (b) N-Boc-3-piperidone. Reaction conditions: 0.1 mg mL–1 purified enzyme, 25 mM D-Ala, 25 mM of either N-Boc-pyrrolidinone or N-Boc-piperidone, 0.02 mM PLP, 1 mL of glycine–NaOH buffer (50 mM, pH 8.5), and 45 °C.

Materials and Methods

Reagents

All the chemicals used were of analytical grade and purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. or Sigma-Aldrich, Inc. unless otherwise noted.

The Ata-117 Arrmut11 sequences from Arthrobacter sp. KNK168 and amine transaminase from A. terreus NIH2624 were used as templates for BLAST search in NCBI to find new ω-TA sequences. After removing incomplete and redundant sequences, MEGA 7.0 was used to align all the unknown sequences in the identity range of 20−60% toward the templates, and the target sequences that conform to the functional characterstics of (R)-selective ω-TA with conserved sites R86, K188, and E221 were screened out according to the motif sequence [31 H/R****Y*V/*S(T/A/H/P), 95 F(Y)VE(ANQ)] as confirmed in ref (14). Among them, a putative (R)-selective ω-TA from C. bacterium (CbTA) was chosen for further study, which shows 38% identity with Ata-117 Arrmut11. A phylogenetic tree was also generated with MEGA 7.0 by using the neighbor-joining algorithm.[47,48]

Plasmid and Strain Construction

The gene sequence of the putative CbTA was further codon-optimized and synthesized by GenScript (Nanjing, China). The encoding region was inserted between the BamH I and Xho I, with a 6-histidine tag at the N-terminal, resulting in plasmid pRSF-CbTA. The recombinant plasmid was transformed into E. coli BL21(DE3) to generate the recombinant strain of E. coli BL21(DE3)/pRSF-CbTA.

Expression and Purification of Recombinant CbTA

Recombinant E. coli BL21(DE3)/pRSF-CbTA was cultured in 100 mL of LB medium (1.0% peptone, 0.5% yeast extract, and 1.0% NaCl) with kanamycin (50 μg/mL) at 37 °C and 200 rpm. When the OD600 of the culture reaches about 0.6, 0.02 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the culture was cultivated at 20 °C for another 24 h. Thereafter, the cells were harvested, re-suspended in 20 mL of glycine–NaOH buffer (50 mM, pH 8.5), and sonicated in an ice bath for 30 min. The cell debris were removed by centrifugation at 8000 × g at 4 °C for 40 min. The supernatant was passed through a 5 mL Ni-NTA column to purify recombinant CbTA, and then the purified CbTA solution was stored at 4 °C for further experiments. The molecular weight of the purified CbTA was determined by 12% (w/v) SDS-PAGE.[49] A control experiment was performed under the same conditions using an E. coli transformant carrying empty pRSFDuet-1.

Characterization of Biochemical Properties

By using the following buffers with different pH values of 6.0–10.5, 50 mM phosphate buffer, pH 6.0–8.0, and 50 mM glycine–NaOH buffer, pH 8.5–10.5, the optimal pH of CbTA was determined at 40 °C. The optimal temperature for CbTA was investigated from 25 to 65 °C at pH 8.5. Relative activity (%) was calculated using the maximal activity as a control (100%). The temperature stability was measured by incubating CbTA for a period in the temperature range of 40–65 °C.

The tolerance of CbTA to organic solvents is determined by incubating 100 μL of CbTA in glycine–NaOH buffer (50 mM, pH 8.5) for 5 h at room temperature. The buffer contains 0, 5, 10, 20, 40, 60, and 80% (v/v) organic cosolvents (acetonitrile or DMSO or methanol or ethanol). After that, residual activities were evaluated using the assay method of enzyme activity described below.

Assay of Enzyme Activity

Enzyme activity analysis was performed in 500 μL of glycine–NaOH buffer (50 mM, pH 8.5) containing PLP (20 μM), (R)-MBA (10 mM), and sodium pyruvate (10 mM). The reaction started at 45 °C by adding 0.05 mg of the purified CbTA to the mixture. After 30 min, the mixture was heated and boiled for 20 min to stop the reaction. All the experiments were performed in triplicate. The resulting acetophenone was analyzed by high-performance liquid chromatography (HPLC). One unit of enzyme activity was defined as the production of 1 μmol acetophenone per minute.

Determination of Kinetic Parameters

The kinetic parameters of CbTA to (R)-MBA or pyruvate were measured at 45 °C and pH 8.5. According to the method described above, the initial reaction rate was determined under the conditions of different concentrations of (R)-MBA or pyruvate. The production of acetophenone and the consumption of pyruvate were analyzed by HPLC. In order to determine the kinetic parameters of CbTA to (R)-MBA, the reaction solution was 500 μL of glycine–NaOH buffer (50 mM, pH 8.5) containing 20 mM pyruvate, 0.05 mg of CbTA, 0.02 mM PLP, and different concentrations of (R)-MBA. The kinetic parameters of CbTA to pyruvate was measured in 500 μL of glycine–NaOH buffer (50 mM, pH 8.5) containing 20 mM (R)-MBA, 0.05 mg of CbTA, 0.02 mM PLP, and different concentrations of sodium pyruvate. The Km and kcat values of (R)-MBA and pyruvate were calculated according to the nonlinear regression fitting of the Michaelis–Menten equation.

Characterization of the Substrate Specificity of CbTA

Substrate specificity of CbTA was tested by reactions between different groups of amino donors and amino receptors. All the experiments were performed in triplicate. When the specificity of amino donors is studied, amino donors used are listed in Tables , 2. The activity assay for each reaction was carried out in a 50 mM glycine–NaOH buffer (pH 8.5) containing 25 mM amino donor, 25 mM sodium pyruvate, 0.02 mM PLP, and a suitable amount of purified CbTA, with a final volume of 500 μL at 40 °C for 30 min. The activity of CbTA against different amino receptors was compared by measuring the amount of acetophenone produced. When the specificity of amino receptors is studied, amino receptors used are listed in Tables , 4. The activity assay for each reaction was carried out in a 50 mM glycine–NaOH buffer (pH 8.5) containing 25 mM (R)-MBA, 25 mM amino receptor, 0.02 mM PLP, and a suitable amount of purified CbTA, with a final volume of 500 μL at 40 °C for 30 min. The activity of CbTA against different amino donors was compared by measuring the reduction in pyruvate.

Molecular Modeling and Substrate Docking

Molecular modeling was performed using Modeller 9.24 and I-Tasser (https://zhanglab.ccmb.med.umich.edu/I-Tasser/). The homology model was based on the crystal structure (PDB: 3WWH,[23] 4CE5,[24] 4CMD,[27] 4UUG,[25] and 5E25[26]), and the best model was selected by Ramachandran analysis.

Site-Specific Mutagenesis

The amino acid residues Y190, Q192, and G288 were selected for site-specific mutagenesis on the basis of the previous results and modeling analysis.[19] The site-specific mutagenesis was designed by a NDT codon design. All primers are listed in Table . Site-specific mutagenesis was performed according to the instructions of a site-directed mutagenesis kit (MutExpress II Fast Mutagenesis Kit V2, Vazyme, Nanjing, China). Mutations were introduced by the mutagenesis polymerase chain reaction. The amplified fragments were digested with DpnI at 37 °C for 1 h, and the recombinant plasmid was transformed into E. coli BL21(DE3) for screening. Individual colonies of the transformants were transferred to 96-well plates containing 300 μL of LB medium with kanamycin (50 μg/mL), 96-well plates were incubated overnight (37 °C, 200 rpm). Subsequently, 30 μL of that culture suspension was transferred to a medium containing 300 μL of fresh LB in each well for culture for 2 h (37 °C, 200 rpm), and then 300 μL of LB containing 0.04 mM IPTG was added and induced at 20 °C for 24 h and the sludge was collected by centrifugation at 4200 rpm for 20 min. The cells were fragmented according to a fragmentation kit (xTractor Buffer Kit, TAKARA, Japan). After the cells were allowed to stand at room temperature for 30 min, the crude mutant CbTA solution was collected by centrifugation. Thereafter, the activity of the mutants was tested as described above.

Table 5

List of Primer Sequences for the Creation of CbTA Variants

primer name	5′ → 3′ nucleotide sequence
Y190R F	GAT GCG CGT GTT CAG GTG ATC GTT ACC CGT
Y190R R	CTG AAC ACG CGC ATC ACG GTG ACC CGC ACG
Y190F F	GAT GCG TTT GTT CAG GTG ATC GTT ACC CGT
Y190F R	CTG AAC AAA CGC ATC ACG GTG ACC CGC ACG
Y190L F	GAT GCG CTT GTT CAG GTG ATC GTT ACC CGT
Y190L R	CTG AAC AAG CGC ATC ACG GTG ACC CGC ACG
Y190I F	GAT GCG ATT GTT CAG GTG ATC GTT ACC CGT
Y190I R	CTG AAC AAT CGC ATC ACG GTG ACC CGC ACG
Y190V F	GAT GCG GTT GTT CAG GTG ATC GTT ACC CGT
Y190V R	CTG AAC AAC CGC ATC ACG GTG ACC CGC ACG
Y190S F	GAT GCG AGT GTT CAG GTG ATC GTT ACC CGT
Y190S R	CTG AAC ACT CGC ATC ACG GTG ACC CGC ACG
Y190H F	GAT GCG CAT GTT CAG GTG ATC GTT ACC CGT
Y190H R	CTG AAC ATG CGC ATC ACG GTG ACC CGC ACG
Y190N F	GAT GCG AAT GTT CAG GTG ATC GTT ACC CGT
Y190N R	CTG AAC ATT CGC ATC ACG GTG ACC CGC ACG
Y190D F	GAT GCG GAT GTT CAG GTG ATC GTT ACC CGT
Y190D R	CTG AAC ATC CGC ATC ACG GTG ACC CGC ACG
Y190C F	GAT GCG TGT GTT CAG GTG ATC GTT ACC CGT
Y190C R	CTG AAC ACA CGC ATC ACG GTG ACC CGC ACG
Y190G F	GAT GCG GGT GTT CAG GTG ATC GTT ACC CGT
Y190G R	CTG AAC ACC CGC ATC ACG GTG ACC CGC ACG
Q192R F	TAC GTT CGT GTG ATC GTT ACC CGT GGT CTG
Q192R R	GAT CAC ACG AAC GTA CGC ATC ACG GTG ACC
Q192F F	TAC GTT TTT GTG ATC GTT ACC CGT GGT CTG
Q192F R	GAT CAC AAA AAC GTA CGC ATC ACG GTG ACC
Q192L F	TAC GTT CTT GTG ATC GTT ACC CGT GGT CTG
Q192L R	GAT CAC AAG AAC GTA CGC ATC ACG GTG ACC
Q192I F	TAC GTT ATT GTG ATC GTT ACC CGT GGT CTG
Q192I R	GAT CAC AAT AAC GTA CGC ATC ACG GTG ACC
Q192V F	TAC GTT GTT GTG ATC GTT ACC CGT GGT CTG
Q192V R	GAT CAC AAC AAC GTA CGC ATC ACG GTG ACC
Q192S F	TAC GTT AGT GTG ATC GTT ACC CGT GGT CTG
Q192S R	GAT CAC ACT AAC GTA CGC ATC ACG GTG ACC
Q192Y F	TAC GTT TAT GTG ATC GTT ACC CGT GGT CTG
Q192Y R	GAT CAC ATA AAC GTA CGC ATC ACG GTG ACC
Q192H F	TAC GTT CAT GTG ATC GTT ACC CGT GGT CTG
Q192H R	GAT CAC ATG AAC GTA CGC ATC ACG GTG ACC
Q192N F	TAC GTT AAT GTG ATC GTT ACC CGT GGT CTG
Q192N R	GAT CAC ATT AAC GTA CGC ATC ACG GTG ACC
Q192D F	TAC GTT GAT GTG ATC GTT ACC CGT GGT CTG
Q192D R	GAT CAC ATC AAC GTA CGC ATC ACG GTG ACC
Q192C F	TAC GTT TGT GTG ATC GTT ACC CGT GGT CTG
Q192C R	GAT CAC ACA AAC GTA CGC ATC ACG GTG ACC
Q192G F	TAC GTT GGT GTG ATC GTT ACC CGT GGT CTG
Q192G R	GAT CAC ACC AAC GTA CGC ATC ACG GTG ACC
G288R F	AGC CGT CGT GCG AAC GTG TTT CTG ATT CAA
G288R R	GTT CGC ACG ACG GCT TTC GGT CAG GTA ACC
G288F F	AGC CGT TTT GCG AAC GTG TTT CTG ATT CAA
G288F R	GTT CGC AAA ACG GCT TTC GGT CAG GTA ACC
G288L F	AGC CGT CTT GCG AAC GTG TTT CTG ATT CAA
G288L R	GTT CGC AAG ACG GCT TTC GGT CAG GTA ACC
G288I F	AGC CGT ATT GCG AAC GTG TTT CTG ATT CAA
G288I R	GTT CGC AAT ACG GCT TTC GGT CAG GTA ACC
G288V F	AGC CGT GTT GCG AAC GTG TTT CTG ATT CAA
G288V R	GTT CGC AAC ACG GCT TTC GGT CAG GTA ACC
G288S F	AGC CGT AGT GCG AAC GTG TTT CTG ATT CAA
G288S R	GTT CGC ACT ACG GCT TTC GGT CAG GTA ACC
G288Y F	AGC CGT TAT GCG AAC GTG TTT CTG ATT CAA
G288Y R	GTT CGC ATA ACG GCT TTC GGT CAG GTA ACC
G288H F	AGC CGT CAT GCG AAC GTG TTT CTG ATT CAA
G288H R	GTT CGC ATG ACG GCT TTC GGT CAG GTA ACC
G288N F	AGC CGT AAT GCG AAC GTG TTT CTG ATT CAA
G288N R	GTT CGC ATT ACG GCT TTC GGT CAG GTA ACC
G288D F	AGC CGT GAT GCG AAC GTG TTT CTG ATT CAA
G288D R	GTT CGC ATC ACG GCT TTC GGT CAG GTA ACC
G288C F	AGC CGT TGT GCG AAC GTG TTT CTG ATT CAA
G288C R	GTT CGC ACA ACG GCT TTC GGT CAG GTA ACC

HPLC Analysis

The acetophenone was measured at 254 nm by HPLC (Agilent 1260) using an Agilent C18 column (250 mm × 4.6 mm) at 30 °C.[41] The mobile phase is water/acetonitrile (50:50, v/v) and the flow rate is 1 mL/min.

The pyruvate was measured at 210 nm by HPLC (Agilent 1260) using an Agilent C18 column (250 mm × 4.6 mm) at 30 °C. The mobile phase is methyl alcohol/water/phosphoric acid (20:80:0.03, v/v) and the flow rate is 0.8 mL/min.

The chiral MBA were detected by HPLC (Agilent 1260) with a CR-I(+) chiral column (150 mm × 3.0 mm) (Daicel Corp.) at 254 nm and 30 °C. The eluent is water/acetonitrile (70:30, v/v) containing 0.36% trifluoroacetic acid at a flow rate of 0.4 mL/min.

The analyses of N-Boc-3-pyrrolidinone, N-Boc-3-piperidone, N-Boc-3-aminopyrrolidine, and N-Boc-3-aminopiperidine were performed on a HPLC system (Agilent 1260) with an Agilent C18 column (250 mm × 4.6 mm) at 220 nm and 25 °C. Elution was carried out with a mobile phase of water/acetonitrile/diethanolamine (70:30:0.1, v/v/v) at a rate of 1 mL/min.[46]

Conclusions

(R)-selective ω-TAs has great potential for industrial applications. In this work, CbTA, a hypothetical (R)-selective ω-TA from C. bacterium was found by the motif sequence BLAST from genome mining. On the basis of the amino acid sequences, a phylogenetic tree was constructed to verify the taxonomic and evolutionary relationships of CbTA with other amine transaminase family homologues. Nine amino acid sequences with different degrees of sequence identity to CbTA were selected and analyzed, and CbTA showed a close evolutionary relationship to pyruvate transferase from M. vanbaalenii PYR-1 and Ata-117 Arrmut11. By characterizing the enzymatic properties of CbTA, results showed that CbTA has good thermal stability, organic solvent tolerance, and broad substrate specificity. Thereafter, the site-specific mutagenesis of CbTA was conducted and the mutant Q192G with higher activity was screened, which was applied in conversions of N-Boc-pyrrolidinone and N-Boc-piperidone. The results showed that CbTA can be used as a valuable catalyst for the asymmetric synthesis of chiral amines from the corresponding aldehydes or ketones.