A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity

A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity ruixue yang, jianing wu, yunhe zhang, zhaohui zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5761267/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Methyl ( R, S )-2, 6-dimethylphenylaminopropionate (( R, S )-1), is an intermediate in the production of the agricultural fungicide ( R, S )-metalaxyl. ( R, S )-1 can be hydrolyzed enantioselectively by some hydrolases to produce ( R )-1, which was used for production of ( R )-metalaxyl. In this work, a strain Burkholderia sp. YD106 that could hydrolyze ( R, S )-1 was screened from the activated sludge, but it had almost no enantioselectivity. The intracellular active esterase WZest was successfully heterologous expressed in the recombinant E. coli BL21 (DE3)-pET-28a (+)-GE04845. Using the recombinant strain as the parent strain, the mutants were constructed by site-directed mutation. Among all 33 mutants, 7 had altered enantioselectivity, of which 4 mutants were ( R )-enantioselective, and 3 were ( S )-enantioselective. The mutant WZest-W23T had the highest ( R )-enantioselectivity. When it catalyzed hydrolysis of ( R, S )-1 at 44.6% substrate conversion, e.e. p reached 94.70% with enantiomeric ratio (E) of 85.0. WZest showed significant amino acid sequence differences from the two reported esterases capable of hydrolyzing ( R, S )-1. It was both active in two kinds of solutions. One was emulsion with the substrate ( R, S )-1 emulsified with Tween80, the other was homogeneous solution with acetone as co-solvent. The activity of WZest in the former was higher than that in the latter. Methyl (R S)-2 6-dimethylphenylaminopropionate (R)-metalaxyl Esterase Strain screening Site-directed mutant Enantioselectivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Esterase (EC 3.1.1.1)-catalyzed reactions have the advantages of high stereoselectivity and no need for coenzymes, and play an important role in the production of chiral intermediates or products in pharmaceutical [1 ~ 3], chemical [ 4 , 5 ] and other industries. The esterase from Pseudomonas CGMCC No.4184 [ 3 ] enantioeselectively hydrolyzed rac -2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester ( rac -CNDE) to produce ( S )-CNDE, which is an important intermediate for the synthesis of pregabalin. Ma et al. [ 4 ] heterologous expressed Pseudomonas putida ECU1011 esterase rPPE01 in E.coli which catalyzed the selective deacetylation of different acetyl carboxylic esters to produce α-hydroxyl acids, with e.e. p >99% and E > 200 at conversion rates close to 50%. (±)- trans -chrysanthemic acid is an important intermediate for the preparation of pyrethroids. Mitsukura et al. [ 5 ] used Alcaligenes sp. NBRC 14130 esterase to hydrolyze the mixture of (±)- trans -chrysanthemic acid ethyl ester and (±)- cis -chrysanthemic acid ethyl ester, finally obtained ( 1R, 3R )-(+)- trans -chrysanthemic acid. ( R, S )-2, 6-dimethylphenyl aminopropionic acid methyl ester (( R, S )-1) is an intermediate for production of agricultural fungicide ( R, S )-metalaxyl. ( R )-1 can be used to produce R -metalaxyl which is several times more effective than ( S )-metalaxyl. The enzymes reported that were able to eantioselectively hydrolyze ( R, S )-1 included lipases (such as Lipase PS, Lipase OF, Lipase QLM [ 6 ], Lipozyme RMIM [ 7 ] and lipases from Burkhloderia sp. MC16-3 and 99-2-1) [ 8 ]), the alkaline protease Alcalase ( Bacillus licheniformis ) and Acylase Amano ( Aspergillus melleus ) [ 6 ], esterases (such as EHest ( Achromobacter denitrificans 1104) [ 9 ] and PAE07 ( Pseudochrobactrum asaccharolyticum WZZ003) [ 7 ]). Some of them (Lipase PS, EHest and PAE07) had ( R )-enantioselectivity which catalyze the reaction shown in Scheme 1 . Some have ( S )-enantioselectivity. Park et al. [ 10 ] studied the process of ( R )-metalaxyl production on a small pilot scale through resolution of ( R, S )-1 by Lipase PS, but the process had the disadvantages of low reaction rate, large enzyme dosage and high production cost. To make the process more valuable, new biocatalysts need to be found and developed. The rational design for changing the chiral selectivity of enzymes usually modified the substrate binding pocket, such as controlling the size and shape of the substrate binding pocket or regulating the volume of amino acid residues or improving the activity of the preferred substrate or reducing that of the non-preferred substrate [11 ~ 13]. Park et al. [ 13 ] proved that it was more effective to improve enzyme’s enatioselectivity (E) by making mutation at the substrate binding sites than doing that at random sites. In addition to rational design, semi-rational design and directed evolution were widely used in the modification of enzyme chiral selectivity. Mutant libraries can be constructed by random or semi-rational methods [14 ~ 19]. Kim et al. [ 15 ] changed the enantioselectivity of the thermophilic esterase Est-AF toward ( R, S )-ketoprofen ethyl ester by error-prone PCR, and the enantioselectivity of the mutants V138G and V138G/L200R toward S-ketoprofen ethyl ester were significantly improved. Studies [ 11 , 16 ] showed that several mutants of Bacillus subtilis esterase BS2 had high activity and enantioselectivity for tert-butanol esters. The enantiomeric ratio of mutants G105A, E188D and E188D/M193C were E > 100. By directed evolution, enantioselectivity can even be reversed. Ivancic et al. [ 17 ] designed the evolution of esterase EstB from Burkholderia gladioli , the enantiomeric ratio of the enzyme toward the substrate ( R, S )-methyl-β-hydroxyisobutyrate changed from E ( S ) = 6.1 to E ( R ) = 28.9. In this work, a novel esterase from Burkholderia sp. YD106 was found, which hydrolyzed ( R, S )-1 with almost no enantioselectivity. It was heterologous expressed in the recombinant Escherichia. coli . Its enantioselectivity can be changed to either ( R ) - or ( S )-type by Site-directed mutation. A mutant with high ( R )-enantioselectivity was obtained. Materials and Methods Materials ( R, S )-1 was given by a metalaxyl-manufacturing plant. All other chemicals were reagent grade and were obtained from commercial sources. Biochemicals, QuikChange® Site-Directed Mutagenesis Kit, DEAE SefinoseTM 6FF (anion exchange column material) and Butyl SefinoseTM 4 Fast Flow (hydrophobic column material) were purchased from Sangon Biotech Corporation (China). Strain screening and assay of activity of strain YD106 cells In the enrichment medium (inoculated with the activated sludge from a wastewater treatment plant), ( R, S )-1 was the sole carbon source. After the enrichment culture was carried for several weeks, the culture solution was appropriately diluted and spread on LB medium agar plates containing 20 g/L ( R, S )-1 and 10 mg/L rhodamine B. The plates were incubated at 30℃ for 4 ~ 7 days and then observed under ultraviolet light at 365 nm. The strain YD106 with the largest fluorescent circle were inoculated into 50 mL of LB medium, incubated at 30℃ and 200 rpm for 24 h. After collected by centrifugation, the cells were added to a 10 mL reaction solution, with ( R, S )-1 of 5 g/L and Tween 80 of 5 g/L in 100 mM sodium phosphate buffer (pH 7.0), for determining its catalytic activity. The reaction was carried out at 30℃ and 200 rpm for 12h, stopped by 0.1 mL of 2 M hydrochloric acid. The catalytic activity of the cells was obtained by detecting the change of the concentration of ( R, S )-2 by HPLC. Enrichment medium (g/L): NH 4 Cl 0.8 g/L, K 2 HPO 4 1.5 g/L, KH 2 PO 4 1.0 g/L, NaCl 1.0 g/L, MgSO 4 0.2 g/L, ( R, S )-1 4.0 g/L (concentration gradually increased to 10.0 g/L during enrichment process), pH 7.0. Purification of intracellular active enzyme from strain YD106 The strain was inoculated into LB medium and cultured overnight at 30°C, 180 rpm. The culture solution was ultrasonicated to break the cells, and then centrifuged. The supernatant was added with (NH 4 ) 2 SO 4 powder to 70% saturation under stirring in an ice bath. The precipitate was dissolved in an equal amount of Tris-HCl buffer (pH 8.0), then dialyzed and concentrated with a 10 kDa ultrafiltration centrifuge tube. The concentrate was applied to DEAE ion exchange column, the proteins were eluted with 0, 0.2, 0.4, 0.5 M NaCl sequentially. The active fraction was applied to the DEAE anion ion exchange column again, the proteins were eluted with 0, 0.1, 0.13, 0.15, 0.2 M NaCl sequentially. The new active fraction was applied to hydrophobic column, the protein was eluted with 0.8, 0.6, 0.4, 0.2, 0 M (NH 4 ) 2 SO 4 (pH7.0) sequentially. The active fraction from the hydrophobic column was subjected to Native-PAGE with esterase activity staining [ 20 ]. Assay of activity of esterase WZest It was the same as that of the activity of stain YD106 cells described above, except that the cells were replaced with 100 µL of enzyme solution and the reaction time reduced to be 10 min. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol of ( R, S )-2 per min under the above reaction condition. Construction of the Recombinant E . coli BL21 (DE3)-pET-28a (+)-GE04845 The gene GE04845 of Burkholderia sp. YD106 was amplified by PCR using EcoR I-forward primer (5’-CGCGGATCCGAATTCGAGATGGAGACGAACGTAACCGC-3’) and Hind III-reverse primer (5’-CGAGTGCGGCCGCAAGCTTGTCAGCTTTTCGCGATATCCG-3’). The amplified fragment with a size of 900 bp was ligated into pET-28a (+) vector and transformed into E . coli BL21 (DE3). Construction of WZest mutants Mutants were constructed by QuikChange® Site-Directed Mutagenesis Kit using the recombinant E. coli BL21 (DE3)-pET-28a (+)-GE04845 as the parent strain. For mutation W23→T (as an example), the forward primer was (5’ - GGCGCG ACT CACGGTGCGTGGTG - 3’), the reverse primer was (5’ - CACCGTG AGT CGCGCCGTGCACG - 3’). The underlined and bolded letters indicated the mutation site. Assay of activity of recombinant E . coli and its mutants It was the same as that of the activity of strain YD106 cells described above, except that the strain cells were replaced with cells of recombinant E. coli and its mutants, respectively, and the reaction time reduced to be 10 min. Determination of kinetic parameters of esterase WZest The recombinant esterase WZest was purified by Ni-NTA affinity column chromatography. 52.7 mg/L (1.5×10 − 3 mM) of the purified enzyme and the range of concentration of ( R, S )-1 was 0 ~ 24 mM were used for determining kinetic parameters. The rate of the enzyme reaction was expressed as the rate of ( R, S )-2 production in 5 min. The kinetic parameters was obtained by Lineweaver–Burk plot. HPLC analysis ( R, S )-1 and ( R, S )-2 were determined by Reversed phase HPLC. ( R )-1, ( S )-1, ( R )-2 and ( S )-2 were determined by Normal phase HPLC. Reversed phase HPLC conditions: acetonitrile/water/trifluoroacetic acid = 60/40/0.1 (v/v/v), flow rate 1.0 mL/min, UV detection wavelength 220 nm, column temperature 25°C, InterSustain ® C18 column (250 mm × 4 mm). Normal phase HPLC conditions: n-hexane/isopropanol/trifluoroacetic acid = 98/2/0.1 (v/v/v), flow rate 0.5 mL/min, UV detection wavelength 220 nm, column temperature 30°C, DAICEL chiral OD-H column (250 mm × 4 mm). The substrate conversion rate ( C ), enantiomeric excess of the product ( e.e . p ) and enantiomeric ratio (E) were calculated as formula (1), (2) and (3), respectively. [ S ] 0 and [ S ] represent the substrate concentration of ( R, S )-1 at 0 and t time, respectively. [ P ] S and [ P ] R represent the product concentration of ( S )-2 and ( R )-2, respectively. Results and Discussion Strain screening ( R, S )-1 was the sole carbon source for strain screening in the enrichment medium, in which rhodamine B played a chromogenic role. Rhodamine B combined with the acidic product produced by hydrolysis of ( R, S )-1 emitted fluorescence under UV light at 365 nm, which efficiently screened out the strains capable of degrading ( R, S )-1. The most active strain YD106 was inoculated to the LB medium and cultivated for 24 h at 30℃, and the cells were collected by centrifugation. The activity and enatioselectivity of hydrolysis of ( R, S )-1 catalyzed by the strain cells was shown in Table 1 . The strain YD106 had almost no enantioselectivity. Table 1 The activity and enatioselectivity of hydrolysis of ( R, S )-1 catalyzing by strain YD106 strains reaction time substrate conversion rate e.e. p E enantioselectivity YD106 12h 49.52% 8.37% 1.27 Almost no Morphological and molecular biological identification of strain YD106 The colonies of strain YD106 on the LB medium agar plates were round, smooth, milky white, opaque, as shown in Fig. 1 . Gram staining showed that the strain was a Gram-negative, and its shape was short rod, as shown in Fig. 2 . The 16S rDNA of strain was amplified by PCR and sequenced. Its nucleotide length was 1526 bp (See Appendix Ⅰ). BLAST at NCBI website found that its sequence similarity was 100% with 16S rDNA of Burkholderia sp. AFS072602, and was more than 99.9% with those of Burkholderia cepacia FDAARGOS_345, Burkholderia ambifaria Q53 and Burkholderia pyrrocinia LWK2. According to the morphological and molecular biological characteristics of the strain YD106, it was identified as Burkholderia , and was named Burkholderia sp. YD106. Purification of intracellular active enzyme of Burkholderia sp. YD106 and acquisition of its gene The culture solution of Burkholderia sp.YD106 was sonicated to break the cells, and then centrifuged. The supernatant was salted out by (NH 4 ) 2 SO 4 . The precipitate was dissolved in pH7.0 buffer and dialyzed and concentrated. The concentrate was applied to DEAE anion exchange column, the fraction from 0.2 M NaCl elution had enzymatic activity. The active fraction was applied to DEAE anion exchange column again, the proteins were eluted with the narrower concentration range of NaCl solution. The fraction from 0.13 M NaCl elution had catalytic activity. The new active fraction was concentrated and applied to hydrophobic column, and the enzymatic activity was found in the fraction from 0.2 M (NH 4 ) 2 SO 4 elution. At the beginning, the active enzyme in intracellular crude enzymes from Burkholderia sp. YD106 had the specific activity of 0.210 U/mg protein. After it was purified sequentially by (NH 4 ) 2 SO 4 precipitation, DEAE anion-exchange, and hydrophobic column chromatography, the final purified enzyme reached the specific enzyme activity of 1.88 U/mg protein, which was 8.94-fold purified. The purified enzyme were subjected to SDS-PAGE and Native-PAGE, and the results were shown in Fig. 3 . Most of the impurity proteins were removed after DEAE anion-exchange (compare lane 3 and lane2 in Fig. 3 a), and the remaining impurities were further removed by hydrophobic column chromatography (compare lane 4 and lane 3 in Fig. 3 a), but a single protein band was still not obtained (see lane 4 in Fig. 3 a). In order to determine which band in lane 4 was of the target enzyme, Native-PAGE analysis with esterase activity staining was performed. The result was shown in Fig. 3 b. The stained band was sliced, and the amino acid sequence of the active protein in it was analyzed by protein mass spectrometer. The obtained amino acid sequence had the greatest homology with gene GE04845 (its nucleotide and amino acid sequences were shown in Appendices Ⅱ and Ⅲ) of Burkholderia sp. YD106. The gene had a base size of 885 bp, and encoded a protein with 294 aa and molecular weight of 35,275 Da which was consistent with the results of SDS-PAGE (see the band in the red box in lane 4 of Fig. 3 a). The esterase protein encoded by gene GE04845 was named WZest. Effect of temperature on the activity and thermal stability of esterase WZest The relative activities of the enzyme (at pH7.0) at different temperatures (with the highest activity as 100%) were measured. Each experiment was repeated twice. The results (see in Fig. 4 a) showed the optimal catalytic temperature of WZest was 30℃. The experimental result of the thermal stability of the enzyme was shown in Fig. 4 b, the enzyme still remained 86% and 80% of the initial activity after incubation at 20℃ and 30℃ for 2 h, respectively. After incubation at 40℃ for 2 h, the activity of the enzyme decreased significantly, while at 50℃ for 2 h, the residual activity was only about 9% of the initial activity. Effect of pH on the activity and stability of esterase WZest The relative activities of the enzyme at different pH were measured at 30℃. The results (see in Fig. 5 A) showed the optimal catalytic pH of WZest was pH 8.0. The residual activity of the enzyme samples were measured after incubation for 1 h at different pH. The results (see in Fig. 5 B) showed that the enzyme still maintained high activity at between pH 7.0 and 9.0, but and the stability of the enzyme decreased rapidly at pH beyond this range. The optimal stable pH of the enzyme was 8.0. Cloning and expression of the gene of WZest The gene GE04845 in Burkholderia sp. YD106 was amplified by PCR using specific primers, and a band of about 900 bp was detected by agarose electrophoresis which was consistent with the expected. Using pET-28a(+) as the vector and E .coli BL21 (DE3) as the host, the recombinant strain was constructed. The obtained E. coli BL21 (DE3)-pET-28a(+)-GE04845 were cultured with and without induction of IPTG respectively, and the cells were ultrasonically broken and centrifuged. The supernatants were analyzed by SDS- PAGE, the result was shown in Fig. 6 . Lane 2 (induced with IPTG) had one over-expressed band with a size of about 38 kDa, which was consistent with the expected. Catalytic activity and enantiomeric ratio (E) of the recombinant cells with IPTG induction was measured. After 15 min of catalytic reaction, the conversion rate of the substrate was 49.56% with e.e . p of 8.21% and E of 1.15, which was consistent with that from strain YD106 cells (see Table 1 ). 3D structure modeling for WZest and its molecular docking with the substrate Modeling for the esterase WZest was performed on SWISS-MODEL website. The template with the highest homology was an alpha/beta fold hydrolase (PDB ID: 8HFW) from Burkholderia pyrrocinia . It had 94% of identity in amino acid sequence with WZest. Both belonged to Burkholderia α/β hydrolase with the consensus motif of GHSMG. Although the 3D structure of 8HFW had been known, there was no research to elucidate its catalytic mechanism and active site. Xu et al. [ 21 ] studied the structure and properties of the esterase 7WWF (alpha/beta fold hydrolase, the second highest homology with WZest, 40% of identity in amino acid sequence). By alignment WZest with 7WWF, the catalytic triad of WZest was determined to be Ser111, Asp241, and His274. Using 8HFW as a template, the 3D structure model of WZest was constructed. Using the region containing the catalytic triad as the docking pocket, WZest was docked with the substrate by AutodockTools 1.5.6. The docking image was shown in Fig. 7 . Mutation of WZest for improving enantioselectivity The following six amino acid residues of WZest were selected as mutation sites: (1) the residues Ala22 and Met112 that made up the oxygen anion hole; (2) the residues Gly21 and Trp23 that were on the sides of Ala22; and (3) the residues His110 and Leu192. The former is on the front side of the active residue S111. The latter is located on the loop close to the hydrophobic moiety of the substrate, 2, 6-dimethylphenyl. The single-point mutation library was shown in Table 2 . The criteria for selecting the amino acids for mutation are their polarity or size are different from the original residue’s. The mutations with changed activity and enantioselectivity were shown in Fig. 8 . Among them, the mutant WZest-W23T had the highest ( R )-enatioselectivity. After 10 min of hydrolysis of ( R, S )-1 catalyzed by it, the product and substrate enantiomers were detected by normal-phase HPLC (see in Fig. 9 ). The substrate conversion reached 44.64% with e.e. p of 94.70% and E ( R ) of 85. The activity of the mutant increased to 1.35 times that of WZest. The mutant WZest-H110I had the highest ( S )-enatioselectivity (see Fig. 8 ). After 15 min of the reaction catalyzed by it, the substrate conversion was 35.86% with e.e. p of 63.07% and E ( S ) of 6.21. The activity of the mutant decreased to 90.5% of WZest. Table 2 Single-point mutation library for WZest Sites for mutation substitute A22 F, I, L, M, A, Q, V, W, D, G, P, S M112 A, I, L, V, W G21 A, L, S, V, W W23 F, G, L, T, Y H110 I, T, Y L192 A, F, S Comparison of WZest with other esterases hydrolyzing ( R, S )-1 Two other esterases, EHest [ 9 ] and PAE07 [ 7 ] can also hydrolyze ( R, S )-1. Multiple sequence alignment of the two and WZest was shown in Fig. 10 . There were large amino acid sequence differences among them. Pairwise sequence alignment showed that WZest had higher homology with EHest (25.18%), while it had no homology with PAE07 (< 20% homology). In Fig. 10 , the red box showed the consensus motif (GXSXG) of the α/β hydrolase family, which was shared by all three esterases. The symbol ▼ indicated the catalytic triad S111, D241, and H274 of WZest. The catalytic residues S and H of EHest and PAE07 can be found at the aligned positions, but no catalytic residue D (or E) of the two esterases was found. The kinetic parameters of WZest and PAE07 catalyzing hydrolysis of ( R, S )-1 was shown in Table 3 . The characteristic catalytic rate (K cat /K m ) of WZest was one order of magnitude higher than that of PAE07. That was mainly due to the much smaller K m of the former. Because the substrate ( R, S )-1 was a water-insoluble oil, it was emulsified by Tween80 (0.05%) when determining the kinetics parameters of WZest. That meant the reaction mainly occurred at the oil-water interface. The activity of WZest was also measured in a homogeneous solution with 10% acetone as co-solvent. The relationship between the WZest-catalyzed reaction rate and substrate concentration in both kinds of solutions was shown in Fig. 11 . The enzyme was active in both of them, the maximum activity in the emulsion was about 2.8 times higher than that in the homogeneous solution. In general, lipase is active in the emulsion but not in the homogeneous solution. Because there is a hydrophobic “lid” over its active center, only when the “lid” opens at the oil-water interface, the substrate can enter. The enzyme WZest had not such a “lid” (see in Fig. 7 ), but it was active in the both solutions. Contrary to lipase, most of the esterases are active only in homogeneous solutions and inactive in the emulsion solution. However, the solubility of the oil substrate is low in homogeneous solutions even if the cosolvent is added, and the cosolvent may inhibit the enzyme activity. In this work, the esterase WZest was active in the emulsion solution, in which the oil substrate was dispersed in the solution by the surfactant. That can avoid the above problems. Table 3 The kinetic parameters of hydrolysis of ( R, S )-1 catalyzed by WZest and PAE07 esterase K m (mM) K cat (s -1 ) K cat /K m ((mM) -1 s -1 ) enantioselctivity E WZest* 1.10 2.68 2.44 / 1.15 PAE07 [ 7 ] 35.7 ± 2.3 7.2 ± 1.2 0.202 R 1393 *recombinant, purified by Ni-NTA affinity column chromatography Conclusions A strain Burkholderia sp. YD106 that could hydrolyze ( R, S )-1 was screened from the activated sludge. The intracellular active esterase WZest from the strain had almost no enantioselectivity towards the substrate ( R, S )-1. Its optimal catalytic temperature and pH was 30℃ and pH 8.0, respectively. It was both active in the emulsion with the substrate ( R, S )-1 emulsified with Tween80, and the homogeneous solution with acetone as co-solvent. WZest was successfully heterologous expressed in the recombinant E. coli BL21 (DE3)-pET-28a (+)-GE04845. By site-directed mutation, WZest can be changed to be either ( R )-selective or ( S )-selective. The Mutant WZest-W23T had a great improvement in ( R )-enantioselectivity, with the enantiomeric ratio (E) of 85. WZest showed significant sequence differences from the two reported esterases capable of hydrolyzing ( R, S )-1. In the future, the catalytic properties of WZest on oil substrates in emulsions will be further investigated and compared with lipases’. In addition, the relationship between mutation and enzyme activity/enantioselectivity will be further investigated, which will help to rapidly improve the enantioselectivity of the enzymes. Declarations Ethics Approval Not applicable. Consent to Participate Not applicable. Consent to Publish Not applicable. Competing Interests The authors declare no competing interests. Fundings This work was supported by no funds, grants, or other support. Data Availability All data generated during this study are included in this published article and its supplementary information files. 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Journal of the American Chemical Society , 132(20), 7038-7042. Kim, J., Kim, S., Yoon, S., Hong, E., & Ryu, Y. (2015). Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl easer by directed evolution and characterization of mutant esterases. Applied Microbiology and Biotechnology , 99, 1-9. Kourist, R., Bartsch, S., & Bornscheuer, U. T. (2007). Highly enantioselective synthesis of arylaliphatic tertiary alcohols using mutants of an esterase from Bacillus subtilis . Advanced Synthesis and Catalysis , 349(8-9), 1393–1398. Ivancic, M., Valinger, G., Gruber, K., & Schw, H. (2007). Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution. Journal of Biotechnology , 129, 109–122. Yu, S. S., Li, J. L., Yao, P. Y., Feng, J. H., & Zhu, D. M. (2021). Inverting the Enantiopreference of Nitrilase-Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror-Image Strategy. Angewandte Chemie International Edition , 60, 3679– 3684. Zhang, H. J., Cheng, Z. G., Wei, L. T., Yu, X. J., Wang, Z., Zhang Y. J. (2022). Semi-rational protein engineering of a novel esterase from Bacillus aryabhattai (BaCE) for resolution of (R,S)-ethyl indoline-2-carboxylate to prepare (S)-indoline-2-carboxylic acid. Bioorganic Chemistry , 120:105602. Choi, G. S., Kim, J. Y., Kim, J. H., Ryu, Y. W., & Kim, G. J. (2003). Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester. Protein Expression and Purification , 29, 85–93. Xu, Y., Yang, J., Li, W., Song, S., Shi, Y., & Feng, Y. (2022). Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target. PLoS Pathogens , 18(7), e1010615. Scheme 1 Scheme 1 is available in the Supplementary Files section. Supplementary Files Scheme1.png Scheme 1 ( R )-enantioselective hydrolysis of ( R, S )-1 catalyzed by some lipases or esterases. Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Applied Biochemistry and Biotechnology → Version 1 posted Reviewers agreed at journal 27 Mar, 2025 Reviewers invited by journal 24 Mar, 2025 Editor invited by journal 24 Mar, 2025 First submitted to journal 23 Mar, 2025 Editorial decision: Accept with revisions 05 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5761267","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433045548,"identity":"4d3a1872-b634-4ea0-9003-56beec89352e","order_by":0,"name":"ruixue yang","email":"","orcid":"","institution":"Zhejiang University of Technology Chaohui Campus: Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"ruixue","middleName":"","lastName":"yang","suffix":""},{"id":433045549,"identity":"8db1d9e9-9e8f-4715-abf5-b8bf5c27c6db","order_by":1,"name":"jianing wu","email":"","orcid":"","institution":"Zhejiang University of Technology Chaohui Campus: Zhejiang University of Technology","correspondingAuthor":false,"prefix":"","firstName":"jianing","middleName":"","lastName":"wu","suffix":""},{"id":433045550,"identity":"f2af1b03-f5d2-41d2-b930-62bbda1b83f4","order_by":2,"name":"yunhe zhang","email":"","orcid":"","institution":"Zhejiang University - University of Edinburgh Institute","correspondingAuthor":false,"prefix":"","firstName":"yunhe","middleName":"","lastName":"zhang","suffix":""},{"id":433045551,"identity":"65391013-cfd3-42dc-8bd2-4b1ffd6fbb57","order_by":3,"name":"zhaohui zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFACHsYHDDxglgHRWpgNgFokSNLCBlJOghb5/rPHKn/IHK5jYG/eJsFQc4ewFsaGc2k3JHgOSzDwHCuTYDj2jLAWZsYesxsGIC0SOWYSjA2HCWthY+YxK0gAaZF/Q6QWHjYeM4YDYFt4iNQiwcNjLNnAky7ZxpNWbJFwjAgt8v1nDD/+7LHm52c/vPHGhxoitIABYw/QUyBGApEagOAH8UpHwSgYBaNgBAIAhfMtnNjfwV4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0002-4766-1140","institution":"Zhejiang University of Technology","correspondingAuthor":true,"prefix":"","firstName":"zhaohui","middleName":"","lastName":"zhang","suffix":""}],"badges":[],"createdAt":"2025-01-04 04:15:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5761267/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5761267/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12010-025-05292-3","type":"published","date":"2025-06-18T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79179709,"identity":"7faa6b5d-a1a3-4903-b224-679a404f9754","added_by":"auto","created_at":"2025-03-25 10:17:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":176369,"visible":true,"origin":"","legend":"\u003cp\u003eColony morphology of strain YD106 on the LB medium agar plate\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/3bc515297dc7355bc1c17d16.png"},{"id":79178530,"identity":"e541aacd-4495-4db9-a615-8ef6f8e2bfb8","added_by":"auto","created_at":"2025-03-25 10:09:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":213272,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of Gram-stained strain YD106 under microscope\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/48285f8d75b975eb9098eea2.png"},{"id":79178568,"identity":"741e1b87-950e-4908-9e00-374402d0285e","added_by":"auto","created_at":"2025-03-25 10:09:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154025,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE (a) and Native-PAGE (b) of active fractions from different purification steps.\u003c/p\u003e\n\u003cp\u003e(a) SDS-PAGE：lane1, intracellular crude enzymes from \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106; lane 2, fraction after (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e precipitation; lane 3, active fraction of 0.13 M NaCl elution from DEAE anion exchange column; lane 4, active\u003csub\u003e \u003c/sub\u003efraction from hydrophobic column.\u003c/p\u003e\n\u003cp\u003e(b) Native-PAGE with esterase activity staining：lane 1, fraction after (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e precipitation; lane 2, active fraction of 0.13 M NaCl elution from DEAE anion exchange column; lane 3, active fraction from hydrophobic column\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/ebd48749702425d759c9723b.png"},{"id":79178558,"identity":"6a3aa426-fdb2-4c86-bc8b-db2badf9a540","added_by":"auto","created_at":"2025-03-25 10:09:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56424,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of temperature on activity (a) and thermal stability (b) of esterase WZest(at pH7.0). Symbols in (b): 20℃ (■); 30℃ (●); 40℃ (▲); 50℃ (▼)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/19c8ae58a99db4bb1000bf13.png"},{"id":79178549,"identity":"fbb3f873-48d3-4487-9dc2-af82276c9439","added_by":"auto","created_at":"2025-03-25 10:09:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47368,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pH on the activity (a) and stability (b) of esterase WZest (at 30℃). Symbols in (b): pH6.0 (■); pH7.0 (●); pH8.0 (▲); pH9.0 (▼); pH10.0 (◢)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/8b21ef37ac65705b8842f18a.png"},{"id":79178574,"identity":"be9f3ec7-83c5-48aa-872f-1c9c24dfb365","added_by":"auto","created_at":"2025-03-25 10:09:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":119491,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of expressed proteins by the recombinant \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)-pET-28a (+)-GE04845 with and without IPTG induction and purified recombinant protein by Ni-NTA affinity column chromatography. M, Marker; lane1, without IPTG induction; lane2, with IPTG induction; lane3, purified recombinant protein by Ni-NTA affinity column chromatography\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/586845dd38ca0a88be6fa9d5.png"},{"id":79178556,"identity":"594efa0b-4dc4-4900-b6ee-ec2640bf6a46","added_by":"auto","created_at":"2025-03-25 10:09:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":241521,"visible":true,"origin":"","legend":"\u003cp\u003eThree-sided views of the esteraseWZest. (a) Front view: Molecular docking diagram of WZest with (\u003cem\u003eR\u003c/em\u003e)-1 (grey). Ser111 (cyan), Asp241 (yellow), and His274 (purple) were catalytic triad. (b) Side view. (c) Top veiw.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/513a43e7acdf7460f38dbb62.png"},{"id":79179710,"identity":"4118cb1b-eb90-46ed-9ba1-0f6bb119abcd","added_by":"auto","created_at":"2025-03-25 10:17:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":63211,"visible":true,"origin":"","legend":"\u003cp\u003eThe mutations of WZest with changed activity and enantioselectivity. Mutants with (\u003cem\u003eR\u003c/em\u003e)-enantioselectivity (●) and (\u003cem\u003eS\u003c/em\u003e)-enantioselectivity (■) were above and below zero respectively. The Relative activity of WZest was 100%\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/a9c1a22593c84298995339e4.png"},{"id":79178569,"identity":"e7c8d765-be02-4d70-9085-e2b2820242f0","added_by":"auto","created_at":"2025-03-25 10:09:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":56275,"visible":true,"origin":"","legend":"\u003cp\u003eThe product and substrate enantiomers detected by normal-phase HPLC after 10 min of hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzed by mutant WZest-W23T\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/62b11fbeabbb1817b78af761.png"},{"id":79178560,"identity":"b3f7cec9-4bd8-46bc-8cbc-1224a4453086","added_by":"auto","created_at":"2025-03-25 10:09:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":475757,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of esterases WZest, EHest and PAE07 which can catalyzing hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1. Symbol \u003cem\u003e▼\u003c/em\u003eindicated the catalytic triad S111, D241, and H274 of WZest\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/fc97050d9d920fa5a9a8d6cc.png"},{"id":79178577,"identity":"109baffd-de36-4c84-9971-040d7b89909d","added_by":"auto","created_at":"2025-03-25 10:09:07","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":53513,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between the WZest-catalyzed reaction rate and substrate (\u003cem\u003eR, S\u003c/em\u003e)-1 concentration in two kinds of solutions. One was oil-in-water emulsified with 0.05% Tween80 (●), the other was homogeneous with 10% acetone as co-solvent (■)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/553c70cb8d9f6c5191e87011.png"},{"id":85231564,"identity":"8d57448e-d495-46ab-b615-2246a7fe3a92","added_by":"auto","created_at":"2025-06-23 16:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2726999,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/78afb567-648e-45b7-a4d7-4e136f8101f4.pdf"},{"id":79178546,"identity":"d43cd040-e3be-49e0-8610-ba5768e10927","added_by":"auto","created_at":"2025-03-25 10:09:01","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":56557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e (\u003cem\u003eR\u003c/em\u003e)-enantioselective hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzed by some lipases or esterases.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-5761267/v1/93ed3eca10d3af6cbc561a62.png"}],"financialInterests":"","formattedTitle":"A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsterase (EC 3.1.1.1)-catalyzed reactions have the advantages of high stereoselectivity and no need for coenzymes, and play an important role in the production of chiral intermediates or products in pharmaceutical [1\u0026thinsp;~\u0026thinsp;3], chemical [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and other industries. The esterase from \u003cem\u003ePseudomonas\u003c/em\u003e CGMCC No.4184 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] enantioeselectively hydrolyzed \u003cem\u003erac\u003c/em\u003e-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (\u003cem\u003erac\u003c/em\u003e-CNDE) to produce (\u003cem\u003eS\u003c/em\u003e)-CNDE, which is an important intermediate for the synthesis of pregabalin. Ma et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] heterologous expressed \u003cem\u003ePseudomonas putida\u003c/em\u003e ECU1011 esterase rPPE01 in \u003cem\u003eE.coli\u003c/em\u003e which catalyzed the selective deacetylation of different acetyl carboxylic esters to produce α-hydroxyl acids, with \u003cem\u003ee.e.\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e \u0026gt;99% and E\u0026thinsp;\u0026gt;\u0026thinsp;200 at conversion rates close to 50%. (\u0026plusmn;)-\u003cem\u003etrans\u003c/em\u003e-chrysanthemic acid is an important intermediate for the preparation of pyrethroids. Mitsukura et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] used \u003cem\u003eAlcaligenes\u003c/em\u003e sp. NBRC 14130 esterase to hydrolyze the mixture of (\u0026plusmn;)-\u003cem\u003etrans\u003c/em\u003e-chrysanthemic acid ethyl ester and (\u0026plusmn;)-\u003cem\u003ecis\u003c/em\u003e-chrysanthemic acid ethyl ester, finally obtained (\u003cem\u003e1R, 3R\u003c/em\u003e)-(+)-\u003cem\u003etrans\u003c/em\u003e-chrysanthemic acid.\u003c/p\u003e \u003cp\u003e(\u003cem\u003eR, S\u003c/em\u003e)-2, 6-dimethylphenyl aminopropionic acid methyl ester ((\u003cem\u003eR, S\u003c/em\u003e)-1) is an intermediate for production of agricultural fungicide (\u003cem\u003eR, S\u003c/em\u003e)-metalaxyl. (\u003cem\u003eR\u003c/em\u003e)-1 can be used to produce \u003cem\u003eR\u003c/em\u003e-metalaxyl which is several times more effective than (\u003cem\u003eS\u003c/em\u003e)-metalaxyl. The enzymes reported that were able to eantioselectively hydrolyze (\u003cem\u003eR, S\u003c/em\u003e)-1 included lipases (such as Lipase PS, Lipase OF, Lipase QLM [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Lipozyme RMIM [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and lipases from \u003cem\u003eBurkhloderia\u003c/em\u003e sp. MC16-3 and 99-2-1) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]), the alkaline protease Alcalase (\u003cem\u003eBacillus licheniformis\u003c/em\u003e) and Acylase Amano (\u003cem\u003eAspergillus melleus\u003c/em\u003e) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], esterases (such as EHest (\u003cem\u003eAchromobacter denitrificans\u003c/em\u003e 1104) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and PAE07 (\u003cem\u003ePseudochrobactrum asaccharolyticum\u003c/em\u003e WZZ003) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]). Some of them (Lipase PS, EHest and PAE07) had (\u003cem\u003eR\u003c/em\u003e)-enantioselectivity which catalyze the reaction shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Some have (\u003cem\u003eS\u003c/em\u003e)-enantioselectivity. Park et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] studied the process of (\u003cem\u003eR\u003c/em\u003e)-metalaxyl production on a small pilot scale through resolution of (\u003cem\u003eR, S\u003c/em\u003e)-1 by Lipase PS, but the process had the disadvantages of low reaction rate, large enzyme dosage and high production cost. To make the process more valuable, new biocatalysts need to be found and developed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe rational design for changing the chiral selectivity of enzymes usually modified the substrate binding pocket, such as controlling the size and shape of the substrate binding pocket or regulating the volume of amino acid residues or improving the activity of the preferred substrate or reducing that of the non-preferred substrate [11\u0026thinsp;~\u0026thinsp;13]. Park et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] proved that it was more effective to improve enzyme\u0026rsquo;s enatioselectivity (E) by making mutation at the substrate binding sites than doing that at random sites.\u003c/p\u003e \u003cp\u003eIn addition to rational design, semi-rational design and directed evolution were widely used in the modification of enzyme chiral selectivity. Mutant libraries can be constructed by random or semi-rational methods [14\u0026thinsp;~\u0026thinsp;19]. Kim et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] changed the enantioselectivity of the thermophilic esterase Est-AF toward (\u003cem\u003eR, S\u003c/em\u003e)-ketoprofen ethyl ester by error-prone PCR, and the enantioselectivity of the mutants V138G and V138G/L200R toward S-ketoprofen ethyl ester were significantly improved. Studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] showed that several mutants of \u003cem\u003eBacillus subtilis\u003c/em\u003e esterase BS2 had high activity and enantioselectivity for tert-butanol esters. The enantiomeric ratio of mutants G105A, E188D and E188D/M193C were E\u0026thinsp;\u0026gt;\u0026thinsp;100. By directed evolution, enantioselectivity can even be reversed. Ivancic et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] designed the evolution of esterase EstB from \u003cem\u003eBurkholderia gladioli\u003c/em\u003e, the enantiomeric ratio of the enzyme toward the substrate (\u003cem\u003eR, S\u003c/em\u003e)-methyl-β-hydroxyisobutyrate changed from E\u003csub\u003e(\u003cem\u003eS\u003c/em\u003e)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.1 to E\u003csub\u003e(\u003cem\u003eR\u003c/em\u003e)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;28.9.\u003c/p\u003e \u003cp\u003eIn this work, a novel esterase from \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 was found, which hydrolyzed (\u003cem\u003eR, S\u003c/em\u003e)-1 with almost no enantioselectivity. It was heterologous expressed in the recombinant \u003cem\u003eEscherichia. coli\u003c/em\u003e. Its enantioselectivity can be changed to either (\u003cem\u003eR\u003c/em\u003e)\u003cem\u003e-\u003c/em\u003e or (\u003cem\u003eS\u003c/em\u003e)-type by Site-directed mutation. A mutant with high (\u003cem\u003eR\u003c/em\u003e)-enantioselectivity was obtained.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003e(\u003cem\u003eR, S\u003c/em\u003e)-1 was given by a metalaxyl-manufacturing plant. All other chemicals were reagent grade and were obtained from commercial sources. Biochemicals, QuikChange\u0026reg; Site-Directed Mutagenesis Kit, DEAE SefinoseTM 6FF (anion exchange column material) and Butyl SefinoseTM 4 Fast Flow (hydrophobic column material) were purchased from Sangon Biotech Corporation (China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStrain screening and assay of activity of strain YD106 cells\u003c/h3\u003e\n\u003cp\u003eIn the enrichment medium (inoculated with the activated sludge from a wastewater treatment plant), (\u003cem\u003eR, S\u003c/em\u003e)-1 was the sole carbon source. After the enrichment culture was carried for several weeks, the culture solution was appropriately diluted and spread on LB medium agar plates containing 20 g/L (\u003cem\u003eR, S\u003c/em\u003e)-1 and 10 mg/L rhodamine B. The plates were incubated at 30℃ for 4\u0026thinsp;~\u0026thinsp;7 days and then observed under ultraviolet light at 365 nm. The strain YD106 with the largest fluorescent circle were inoculated into 50 mL of LB medium, incubated at 30℃ and 200 rpm for 24 h. After collected by centrifugation, the cells were added to a 10 mL reaction solution, with (\u003cem\u003eR, S\u003c/em\u003e)-1 of 5 g/L and Tween 80 of 5 g/L in 100 mM sodium phosphate buffer (pH 7.0), for determining its catalytic activity. The reaction was carried out at 30℃ and 200 rpm for 12h, stopped by 0.1 mL of 2 M hydrochloric acid. The catalytic activity of the cells was obtained by detecting the change of the concentration of (\u003cem\u003eR, S\u003c/em\u003e)-2 by HPLC.\u003c/p\u003e \u003cp\u003eEnrichment medium (g/L): NH\u003csub\u003e4\u003c/sub\u003eCl 0.8 g/L, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1.5 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1.0 g/L, NaCl 1.0 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e 0.2 g/L, (\u003cem\u003eR, S\u003c/em\u003e)-1 4.0 g/L (concentration gradually increased to 10.0 g/L during enrichment process), pH 7.0.\u003c/p\u003e\n\u003ch3\u003ePurification of intracellular active enzyme from strain YD106\u003c/h3\u003e\n\u003cp\u003eThe strain was inoculated into LB medium and cultured overnight at 30\u0026deg;C, 180 rpm. The culture solution was ultrasonicated to break the cells, and then centrifuged. The supernatant was added with (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e powder to 70% saturation under stirring in an ice bath. The precipitate was dissolved in an equal amount of Tris-HCl buffer (pH 8.0), then dialyzed and concentrated with a 10 kDa ultrafiltration centrifuge tube. The concentrate was applied to DEAE ion exchange column, the proteins were eluted with 0, 0.2, 0.4, 0.5 M NaCl sequentially. The active fraction was applied to the DEAE anion ion exchange column again, the proteins were eluted with 0, 0.1, 0.13, 0.15, 0.2 M NaCl sequentially. The new active fraction was applied to hydrophobic column, the protein was eluted with 0.8, 0.6, 0.4, 0.2, 0 M (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (pH7.0) sequentially. The active fraction from the hydrophobic column was subjected to Native-PAGE with esterase activity staining [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAssay of activity of esterase WZest\u003c/h3\u003e\n\u003cp\u003eIt was the same as that of the activity of stain YD106 cells described above, except that the cells were replaced with 100 \u0026micro;L of enzyme solution and the reaction time reduced to be 10 min.\u003c/p\u003e \u003cp\u003eOne unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 \u0026micro;mol of (\u003cem\u003eR, S\u003c/em\u003e)-2 per min under the above reaction condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of the Recombinant\u003c/b\u003e \u003cb\u003eE\u003c/b\u003e. \u003cb\u003ecoli BL21 (DE3)-pET-28a (+)-GE04845\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe gene GE04845 of \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 was amplified by PCR using EcoR I-forward primer (5\u0026rsquo;-CGCGGATCCGAATTCGAGATGGAGACGAACGTAACCGC-3\u0026rsquo;) and Hind III-reverse primer (5\u0026rsquo;-CGAGTGCGGCCGCAAGCTTGTCAGCTTTTCGCGATATCCG-3\u0026rsquo;). The amplified fragment with a size of 900 bp was ligated into pET-28a (+) vector and transformed into \u003cem\u003eE\u003c/em\u003e. coli BL21 (DE3).\u003c/p\u003e\n\u003ch3\u003eConstruction of WZest mutants\u003c/h3\u003e\n\u003cp\u003eMutants were constructed by QuikChange\u0026reg; Site-Directed Mutagenesis Kit using the recombinant \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)-pET-28a (+)-GE04845 as the parent strain. For mutation W23\u0026rarr;T (as an example), the forward primer was (5\u0026rsquo;\u003cb\u003e-\u003c/b\u003eGGCGCG\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eACT\u003c/span\u003eCACGGTGCGTGGTG\u003cb\u003e-\u003c/b\u003e3\u0026rsquo;), the reverse primer was (5\u0026rsquo;\u003cb\u003e-\u003c/b\u003eCACCGTG\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAGT\u003c/span\u003eCGCGCCGTGCACG\u003cb\u003e-\u003c/b\u003e3\u0026rsquo;). The underlined and bolded letters indicated the mutation site.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssay of activity of recombinant\u003c/b\u003e \u003cb\u003eE\u003c/b\u003e. \u003cb\u003ecoli and its mutants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIt was the same as that of the activity of strain YD106 cells described above, except that the strain cells were replaced with cells of recombinant \u003cem\u003eE. coli\u003c/em\u003e and its mutants, respectively, and the reaction time reduced to be 10 min.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of kinetic parameters of esterase WZest\u003c/h2\u003e \u003cp\u003eThe recombinant esterase WZest was purified by Ni-NTA affinity column chromatography. 52.7 mg/L (1.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mM) of the purified enzyme and the range of concentration of (\u003cem\u003eR, S\u003c/em\u003e)-1 was 0\u0026thinsp;~\u0026thinsp;24 mM were used for determining kinetic parameters. The rate of the enzyme reaction was expressed as the rate of (\u003cem\u003eR, S\u003c/em\u003e)-2 production in 5 min. The kinetic parameters was obtained by Lineweaver\u0026ndash;Burk plot.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHPLC analysis\u003c/h3\u003e\n\u003cp\u003e(\u003cem\u003eR, S\u003c/em\u003e)-1 and (\u003cem\u003eR, S\u003c/em\u003e)-2 were determined by Reversed phase HPLC. (\u003cem\u003eR\u003c/em\u003e)-1, (\u003cem\u003eS\u003c/em\u003e)-1, (\u003cem\u003eR\u003c/em\u003e)-2 and (\u003cem\u003eS\u003c/em\u003e)-2 were determined by Normal phase HPLC.\u003c/p\u003e \u003cp\u003eReversed phase HPLC conditions: acetonitrile/water/trifluoroacetic acid\u0026thinsp;=\u0026thinsp;60/40/0.1 (v/v/v), flow rate 1.0 mL/min, UV detection wavelength 220 nm, column temperature 25\u0026deg;C, InterSustain \u0026reg; C18 column (250 mm \u0026times; 4 mm).\u003c/p\u003e \u003cp\u003eNormal phase HPLC conditions: n-hexane/isopropanol/trifluoroacetic acid\u0026thinsp;=\u0026thinsp;98/2/0.1 (v/v/v), flow rate 0.5 mL/min, UV detection wavelength 220 nm, column temperature 30\u0026deg;C, DAICEL chiral OD-H column (250 mm \u0026times; 4 mm).\u003c/p\u003e \u003cp\u003eThe substrate conversion rate (\u003cem\u003eC\u003c/em\u003e), enantiomeric excess of the product (\u003cem\u003ee.e\u003c/em\u003e.\u003csub\u003ep\u003c/sub\u003e) and enantiomeric ratio (E) were calculated as formula (1), (2) and (3), respectively.\u003c/p\u003e\u003cp\u003e\u003cimg 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\" width=\"356\" height=\"246\"\u003e\u003c/p\u003e\u003cp\u003e[\u003cem\u003eS\u003c/em\u003e]\u003csub\u003e0\u003c/sub\u003e and [\u003cem\u003eS\u003c/em\u003e] represent the substrate concentration of (\u003cem\u003eR, S\u003c/em\u003e)-1 at \u003cem\u003e0\u003c/em\u003e and \u003cem\u003et\u003c/em\u003e time, respectively. [\u003cem\u003eP\u003c/em\u003e]\u003csub\u003e\u003cem\u003eS\u003c/em\u003e\u003c/sub\u003e and [\u003cem\u003eP\u003c/em\u003e]\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e represent the product concentration of (\u003cem\u003eS\u003c/em\u003e)-2 and (\u003cem\u003eR\u003c/em\u003e)-2, respectively.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStrain screening\u003c/h2\u003e \u003cp\u003e(\u003cem\u003eR, S\u003c/em\u003e)-1 was the sole carbon source for strain screening in the enrichment medium, in which rhodamine B played a chromogenic role. Rhodamine B combined with the acidic product produced by hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 emitted fluorescence under UV light at 365 nm, which efficiently screened out the strains capable of degrading (\u003cem\u003eR, S\u003c/em\u003e)-1. The most active strain YD106 was inoculated to the LB medium and cultivated for 24 h at 30℃, and the cells were collected by centrifugation. The activity and enatioselectivity of hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzed by the strain cells was shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The strain YD106 had almost no enantioselectivity.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe activity and enatioselectivity of hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzing by strain YD106\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003estrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ereaction time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003esubstrate conversion rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ee.e.\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eenantioselectivity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYD106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.37%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAlmost no\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMorphological and molecular biological identification of strain YD106\u003c/h2\u003e \u003cp\u003eThe colonies of strain YD106 on the LB medium agar plates were round, smooth, milky white, opaque, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Gram staining showed that the strain was a Gram-negative, and its shape was short rod, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The 16S rDNA of strain was amplified by PCR and sequenced. Its nucleotide length was 1526 bp (See Appendix Ⅰ). BLAST at NCBI website found that its sequence similarity was 100% with 16S rDNA of \u003cem\u003eBurkholderia\u003c/em\u003e sp. AFS072602, and was more than 99.9% with those of \u003cem\u003eBurkholderia\u003c/em\u003e cepacia FDAARGOS_345, \u003cem\u003eBurkholderia\u003c/em\u003e ambifaria Q53 and \u003cem\u003eBurkholderia\u003c/em\u003e pyrrocinia LWK2. According to the morphological and molecular biological characteristics of the strain YD106, it was identified as \u003cem\u003eBurkholderia\u003c/em\u003e, and was named \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePurification of intracellular active enzyme of\u003c/b\u003e \u003cb\u003eBurkholderia\u003c/b\u003e \u003cb\u003esp. YD106 and acquisition of its gene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe culture solution of \u003cem\u003eBurkholderia\u003c/em\u003e sp.YD106 was sonicated to break the cells, and then centrifuged. The supernatant was salted out by (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The precipitate was dissolved in pH7.0 buffer and dialyzed and concentrated. The concentrate was applied to DEAE anion exchange column, the fraction from 0.2 M NaCl elution had enzymatic activity. The active fraction was applied to DEAE anion exchange column again, the proteins were eluted with the narrower concentration range of NaCl solution. The fraction from 0.13 M NaCl elution had catalytic activity. The new active fraction was concentrated and applied to hydrophobic column, and the enzymatic activity was found in the fraction from 0.2 M (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e elution.\u003c/p\u003e \u003cp\u003eAt the beginning, the active enzyme in intracellular crude enzymes from \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 had the specific activity of 0.210 U/mg protein. After it was purified sequentially by (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e precipitation, DEAE anion-exchange, and hydrophobic column chromatography, the final purified enzyme reached the specific enzyme activity of 1.88 U/mg protein, which was 8.94-fold purified.\u003c/p\u003e \u003cp\u003eThe purified enzyme were subjected to SDS-PAGE and Native-PAGE, and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Most of the impurity proteins were removed after DEAE anion-exchange (compare lane 3 and lane2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), and the remaining impurities were further removed by hydrophobic column chromatography (compare lane 4 and lane 3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), but a single protein band was still not obtained (see lane 4 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In order to determine which band in lane 4 was of the target enzyme, Native-PAGE analysis with esterase activity staining was performed. The result was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. The stained band was sliced, and the amino acid sequence of the active protein in it was analyzed by protein mass spectrometer. The obtained amino acid sequence had the greatest homology with gene GE04845 (its nucleotide and amino acid sequences were shown in Appendices Ⅱ and Ⅲ) of \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106. The gene had a base size of 885 bp, and encoded a protein with 294 aa and molecular weight of 35,275 Da which was consistent with the results of SDS-PAGE (see the band in the red box in lane 4 of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The esterase protein encoded by gene GE04845 was named WZest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of temperature on the activity and thermal stability of esterase WZest\u003c/h2\u003e \u003cp\u003eThe relative activities of the enzyme (at pH7.0) at different temperatures (with the highest activity as 100%) were measured. Each experiment was repeated twice. The results (see in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) showed the optimal catalytic temperature of WZest was 30℃. The experimental result of the thermal stability of the enzyme was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, the enzyme still remained 86% and 80% of the initial activity after incubation at 20℃ and 30℃ for 2 h, respectively. After incubation at 40℃ for 2 h, the activity of the enzyme decreased significantly, while at 50℃ for 2 h, the residual activity was only about 9% of the initial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH on the activity and stability of esterase WZest\u003c/h2\u003e \u003cp\u003eThe relative activities of the enzyme at different pH were measured at 30℃. The results (see in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) showed the optimal catalytic pH of WZest was pH 8.0. The residual activity of the enzyme samples were measured after incubation for 1 h at different pH. The results (see in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) showed that the enzyme still maintained high activity at between pH 7.0 and 9.0, but and the stability of the enzyme decreased rapidly at pH beyond this range. The optimal stable pH of the enzyme was 8.0.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCloning and expression of the gene of WZest\u003c/h2\u003e \u003cp\u003eThe gene GE04845 in \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 was amplified by PCR using specific primers, and a band of about 900 bp was detected by agarose electrophoresis which was consistent with the expected. Using pET-28a(+) as the vector and \u003cem\u003eE\u003c/em\u003e.coli BL21 (DE3) as the host, the recombinant strain was constructed. The obtained \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)-pET-28a(+)-GE04845 were cultured with and without induction of IPTG respectively, and the cells were ultrasonically broken and centrifuged. The supernatants were analyzed by SDS- PAGE, the result was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Lane 2 (induced with IPTG) had one over-expressed band with a size of about 38 kDa, which was consistent with the expected. Catalytic activity and enantiomeric ratio (E) of the recombinant cells with IPTG induction was measured. After 15 min of catalytic reaction, the conversion rate of the substrate was 49.56% with \u003cem\u003ee.e\u003c/em\u003e.\u003csub\u003ep\u003c/sub\u003e of 8.21% and E of 1.15, which was consistent with that from strain YD106 cells (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3D structure modeling for WZest and its molecular docking with the substrate\u003c/h2\u003e \u003cp\u003eModeling for the esterase WZest was performed on SWISS-MODEL website. The template with the highest homology was an alpha/beta fold hydrolase (PDB ID: 8HFW) from \u003cem\u003eBurkholderia pyrrocinia\u003c/em\u003e. It had 94% of identity in amino acid sequence with WZest. Both belonged to \u003cem\u003eBurkholderia\u003c/em\u003e α/β hydrolase with the consensus motif of GHSMG. Although the 3D structure of 8HFW had been known, there was no research to elucidate its catalytic mechanism and active site. Xu et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] studied the structure and properties of the esterase 7WWF (alpha/beta fold hydrolase, the second highest homology with WZest, 40% of identity in amino acid sequence). By alignment WZest with 7WWF, the catalytic triad of WZest was determined to be Ser111, Asp241, and His274. Using 8HFW as a template, the 3D structure model of WZest was constructed. Using the region containing the catalytic triad as the docking pocket, WZest was docked with the substrate by AutodockTools 1.5.6. The docking image was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMutation of WZest for improving enantioselectivity\u003c/h2\u003e \u003cp\u003eThe following six amino acid residues of WZest were selected as mutation sites: (1) the residues Ala22 and Met112 that made up the oxygen anion hole; (2) the residues Gly21 and Trp23 that were on the sides of Ala22; and (3) the residues His110 and Leu192. The former is on the front side of the active residue S111. The latter is located on the loop close to the hydrophobic moiety of the substrate, 2, 6-dimethylphenyl.\u003c/p\u003e \u003cp\u003eThe single-point mutation library was shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The criteria for selecting the amino acids for mutation are their polarity or size are different from the original residue\u0026rsquo;s. The mutations with changed activity and enantioselectivity were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Among them, the mutant WZest-W23T had the highest (\u003cem\u003eR\u003c/em\u003e)-enatioselectivity. After 10 min of hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzed by it, the product and substrate enantiomers were detected by normal-phase HPLC (see in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The substrate conversion reached 44.64% with \u003cem\u003ee.e.\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e of 94.70% and E\u003csub\u003e(\u003cem\u003eR\u003c/em\u003e)\u003c/sub\u003e of 85. The activity of the mutant increased to 1.35 times that of WZest. The mutant WZest-H110I had the highest (\u003cem\u003eS\u003c/em\u003e)-enatioselectivity (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). After 15 min of the reaction catalyzed by it, the substrate conversion was 35.86% with \u003cem\u003ee.e.\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e of 63.07% and E\u003csub\u003e(\u003cem\u003eS\u003c/em\u003e)\u003c/sub\u003e of 6.21. The activity of the mutant decreased to 90.5% of WZest.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSingle-point mutation library for WZest\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSites for mutation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003esubstitute\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF, I, L, M, A, Q, V, W, D, G, P, S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA, I, L, V, W\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA, L, S, V, W\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF, G, L, T, Y\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI, T, Y\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA, F, S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparison of WZest with other esterases hydrolyzing (\u003c/b\u003e \u003cb\u003eR, S\u003c/b\u003e \u003cb\u003e)-1\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo other esterases, EHest [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and PAE07 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] can also hydrolyze (\u003cem\u003eR, S\u003c/em\u003e)-1. Multiple sequence alignment of the two and WZest was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. There were large amino acid sequence differences among them. Pairwise sequence alignment showed that WZest had higher homology with EHest (25.18%), while it had no homology with PAE07 (\u0026lt;\u0026thinsp;20% homology). In Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, the red box showed the consensus motif (GXSXG) of the α/β hydrolase family, which was shared by all three esterases. The symbol ▼ indicated the catalytic triad S111, D241, and H274 of WZest. The catalytic residues S and H of EHest and PAE07 can be found at the aligned positions, but no catalytic residue D (or E) of the two esterases was found.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe kinetic parameters of WZest and PAE07 catalyzing hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 was shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The characteristic catalytic rate (K\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e) of WZest was one order of magnitude higher than that of PAE07. That was mainly due to the much smaller K\u003csub\u003em\u003c/sub\u003e of the former. Because the substrate (\u003cem\u003eR, S\u003c/em\u003e)-1 was a water-insoluble oil, it was emulsified by Tween80 (0.05%) when determining the kinetics parameters of WZest. That meant the reaction mainly occurred at the oil-water interface. The activity of WZest was also measured in a homogeneous solution with 10% acetone as co-solvent. The relationship between the WZest-catalyzed reaction rate and substrate concentration in both kinds of solutions was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. The enzyme was active in both of them, the maximum activity in the emulsion was about 2.8 times higher than that in the homogeneous solution. In general, lipase is active in the emulsion but not in the homogeneous solution. Because there is a hydrophobic \u0026ldquo;lid\u0026rdquo; over its active center, only when the \u0026ldquo;lid\u0026rdquo; opens at the oil-water interface, the substrate can enter. The enzyme WZest had not such a \u0026ldquo;lid\u0026rdquo; (see in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), but it was active in the both solutions. Contrary to lipase, most of the esterases are active only in homogeneous solutions and inactive in the emulsion solution. However, the solubility of the oil substrate is low in homogeneous solutions even if the cosolvent is added, and the cosolvent may inhibit the enzyme activity. In this work, the esterase WZest was active in the emulsion solution, in which the oil substrate was dispersed in the solution by the surfactant. That can avoid the above problems.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe kinetic parameters of hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 catalyzed by WZest and PAE07\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eesterase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e (s\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eK\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e ((mM)\u003csup\u003e-1\u003c/sup\u003es\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eenantioselctivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWZest*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAE07 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1393\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e*recombinant, purified by Ni-NTA affinity column chromatography\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA strain \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 that could hydrolyze (\u003cem\u003eR, S\u003c/em\u003e)-1 was screened from the activated sludge. The intracellular active esterase WZest from the strain had almost no enantioselectivity towards the substrate (\u003cem\u003eR, S\u003c/em\u003e)-1. Its optimal catalytic temperature and pH was 30℃ and pH 8.0, respectively. It was both active in the emulsion with the substrate (\u003cem\u003eR, S\u003c/em\u003e)-1 emulsified with Tween80, and the homogeneous solution with acetone as co-solvent. WZest was successfully heterologous expressed in the recombinant \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)-pET-28a (+)-GE04845. By site-directed mutation, WZest can be changed to be either (\u003cem\u003eR\u003c/em\u003e)-selective or (\u003cem\u003eS\u003c/em\u003e)-selective. The Mutant WZest-W23T had a great improvement in (\u003cem\u003eR\u003c/em\u003e)-enantioselectivity, with the enantiomeric ratio (E) of 85. WZest showed significant sequence differences from the two reported esterases capable of hydrolyzing (\u003cem\u003eR, S\u003c/em\u003e)-1. In the future, the catalytic properties of WZest on oil substrates in emulsions will be further investigated and compared with lipases\u0026rsquo;. In addition, the relationship between mutation and enzyme activity/enantioselectivity will be further investigated, which will help to rapidly improve the enantioselectivity of the enzymes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Publish\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFundings\u003c/h2\u003e \u003cp\u003eThis work was supported by no funds, grants, or other support.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData Availability\u003c/b\u003e All data generated during this study are included in this published article and its supplementary information files.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e \u003cp\u003eRuixue Yang, Jianing Wu and Yunhe Zhang contributed to the study conception and design. The draft of the manuscript was written by Ruixue Yang and Jianing Wu. Zhaohui Zhang designed the research idea, analyzed the data, interpreted the results, and revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZheng, J. Y., Liu, Y. Y., Luo, W. F., Zheng, R. C., Ying, X. X., \u0026amp; Wang Z. (2016). Biocatalytic Resolution of \u003cem\u003eRac\u003c/em\u003e-\u0026alpha;-Ethyl-2-OxoPyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant \u003cem\u003eBacillus cereus\u003c/em\u003e Esterase. \u003cem\u003eApplied Biochemistry and Biotechnology\u003c/em\u003e, 178, 1471\u0026ndash;1480.\u003c/li\u003e\n\u003cli\u003eYoon, S., Kim, S., Park, S., Hong, E., Kim, J., Kim, S., Yoo, T. H.，\u0026amp; Ryu，Y. (2014). 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Improved enantioselectivity of thermostable esterase from \u003cem\u003eArchaeoglobus fulgidus\u003c/em\u003e toward (S)-ketoprofen ethyl easer by directed evolution and characterization of mutant esterases. \u003cem\u003eApplied Microbiology and Biotechnology\u003c/em\u003e, 99, 1-9.\u003c/li\u003e\n\u003cli\u003eKourist, R., Bartsch, S., \u0026amp; Bornscheuer, U. T. (2007). Highly enantioselective synthesis of arylaliphatic tertiary alcohols using mutants of an esterase from \u003cem\u003eBacillus subtilis\u003c/em\u003e. \u003cem\u003eAdvanced Synthesis and Catalysis\u003c/em\u003e, 349(8-9), 1393\u0026ndash;1398.\u003c/li\u003e\n\u003cli\u003eIvancic, M., Valinger, G., Gruber, K., \u0026amp; Schw, H. (2007). Inverting enantioselectivity of \u003cem\u003eBurkholderia gladioli\u003c/em\u003e esterase EstB by directed and designed evolution. \u003cem\u003eJournal of Biotechnology\u003c/em\u003e, 129, 109\u0026ndash;122.\u003c/li\u003e\n\u003cli\u003eYu, S. S., Li, J. L., Yao, P. Y., Feng, J. H., \u0026amp; Zhu, D. M. (2021). Inverting the Enantiopreference of Nitrilase-Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror-Image Strategy. \u003cem\u003eAngewandte\u003c/em\u003e\u003cem\u003eChemie\u003c/em\u003e\u003cem\u003eInternational\u003c/em\u003e\u003cem\u003eEdition\u003c/em\u003e, 60, 3679\u0026ndash; 3684.\u003c/li\u003e\n\u003cli\u003eZhang, H. J., Cheng, Z. G., Wei, L. T., Yu, X. J., Wang, Z., Zhang Y. J. (2022). Semi-rational protein engineering of a novel esterase from Bacillus aryabhattai (BaCE) for resolution of (R,S)-ethyl indoline-2-carboxylate to prepare (S)-indoline-2-carboxylic acid. \u003cem\u003eBioorganic Chemistry\u003c/em\u003e, 120:105602.\u003c/li\u003e\n\u003cli\u003eChoi, G. S., Kim, J. Y., Kim, J. H., Ryu, Y. W., \u0026amp; Kim, G. J. (2003). Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester. \u003cem\u003eProtein Expression and Purification\u003c/em\u003e, 29, 85\u0026ndash;93.\u003c/li\u003e\n\u003cli\u003eXu, Y., Yang, J., Li, W., Song, S., Shi, Y., \u0026amp; Feng, Y. (2022). Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target. \u003cem\u003ePLoS Pathogens\u003c/em\u003e, 18(7), e1010615.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Methyl (R, S)-2, 6-dimethylphenylaminopropionate, (R)-metalaxyl, Esterase, Strain screening, Site-directed mutant, Enantioselectivity","lastPublishedDoi":"10.21203/rs.3.rs-5761267/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5761267/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMethyl (\u003cem\u003eR, S\u003c/em\u003e)-2, 6-dimethylphenylaminopropionate ((\u003cem\u003eR, S\u003c/em\u003e)-1), is an intermediate in the production of the agricultural fungicide (\u003cem\u003eR, S\u003c/em\u003e)-metalaxyl. (\u003cem\u003eR, S\u003c/em\u003e)-1 can be hydrolyzed enantioselectively by some hydrolases to produce (\u003cem\u003eR\u003c/em\u003e)-1, which was used for production of (\u003cem\u003eR\u003c/em\u003e)-metalaxyl. In this work, a strain \u003cem\u003eBurkholderia\u003c/em\u003e sp. YD106 that could hydrolyze (\u003cem\u003eR, S\u003c/em\u003e)-1 was screened from the activated sludge, but it had almost no enantioselectivity. The intracellular active esterase WZest was successfully heterologous expressed in the recombinant \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3)-pET-28a (+)-GE04845. Using the recombinant strain as the parent strain, the mutants were constructed by site-directed mutation. Among all 33 mutants, 7 had altered enantioselectivity, of which 4 mutants were (\u003cem\u003eR\u003c/em\u003e)-enantioselective, and 3 were (\u003cem\u003eS\u003c/em\u003e)-enantioselective. The mutant WZest-W23T had the highest (\u003cem\u003eR\u003c/em\u003e)-enantioselectivity. When it catalyzed hydrolysis of (\u003cem\u003eR, S\u003c/em\u003e)-1 at 44.6% substrate conversion, \u003cem\u003ee.e.\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e reached 94.70% with enantiomeric ratio (E) of 85.0. WZest showed significant amino acid sequence differences from the two reported esterases capable of hydrolyzing (\u003cem\u003eR, S\u003c/em\u003e)-1. It was both active in two kinds of solutions. One was emulsion with the substrate (\u003cem\u003eR, S\u003c/em\u003e)-1 emulsified with Tween80, the other was homogeneous solution with acetone as co-solvent. The activity of WZest in the former was higher than that in the latter.\u003c/p\u003e","manuscriptTitle":"A novel esterase from Burkholderia sp. YD106 capable of hydrolysis of methyl (R, S)-N-(2, 6-dimethylphenyl) alaninate, and its mutation for improving enantioselectivity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 10:08:17","doi":"10.21203/rs.3.rs-5761267/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-27T06:17:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-24T09:15:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Applied Biochemistry and Biotechnology","date":"2025-03-24T08:07:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Applied Biochemistry and Biotechnology","date":"2025-03-23T21:00:39+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept with revisions","date":"2025-03-06T00:50:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"applied-biochemistry-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"abab","sideBox":"Learn more about [Applied Biochemistry and Biotechnology](https://www.springer.com/journal/12010)","snPcode":"12010","submissionUrl":"https://submission.nature.com/new-submission/12010/3","title":"Applied Biochemistry and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"724d8cf2-09f0-472e-86d9-108e20ec37d3","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:06:51+00:00","versionOfRecord":{"articleIdentity":"rs-5761267","link":"https://doi.org/10.1007/s12010-025-05292-3","journal":{"identity":"applied-biochemistry-and-biotechnology","isVorOnly":false,"title":"Applied Biochemistry and Biotechnology"},"publishedOn":"2025-06-18 15:57:33","publishedOnDateReadable":"June 18th, 2025"},"versionCreatedAt":"2025-03-25 10:08:17","video":"","vorDoi":"10.1007/s12010-025-05292-3","vorDoiUrl":"https://doi.org/10.1007/s12010-025-05292-3","workflowStages":[]},"version":"v1","identity":"rs-5761267","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5761267","identity":"rs-5761267","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}