Functional Characterization of a Novel Dactylosporangium Esterase and Its Utilization in the Asymmetric Synthesis of (R)-Methyl Mandelate

Appl Biochem Biotechnol DOI 10.1007/s12010-016-2095-7

Dun Deng1,2,3 & Yun Zhang 1,3 & Aijun Sun1,3 & Yunfeng Hu 1,3,4

Received: 20 January 2016 /Accepted: 20 April 2016 # Springer Science+Business Media New York 2016

Abstract One novel esterase DAEst6 was identified from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. DAEst6 was further characterized to be an esterase which exhibited high resistance to high pH values. Esterase DAEst6 could resolve racemic methyl mandelate and generate (R)-methyl mandelate, one key drug intermediate, with an enantiomeric excess and a conversion of 99 and 49 %, respectively, after process optimization. The optimal working condition for the preparation of (R)- methyl mandelate through DAEst6 was found to be 10-mM racemic methyl mandelate, no organic co-solvents, pH 7.5, and 40 °C, for 5 h. Our work was the first report about the functional characterization of one novel Dactylosporangium esterase and the utilization of one Dactylosporangium esterase in kinetic resolution. Dactylosporangium esterases represented by DAEst6 possess great potential in the generation of valuable chiral drug intermediates and chemicals.

Keywords Novel esterase . Dactylosporangium . (R)-methyl mandelate . Kinetic resolution . High enantiomeric excess

* Yunfeng Hu [email protected]

1 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China 2 University of Chinese Academy of Sciences, Beijing 100049, People’sRepublicofChina 3 Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China 4 South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou, People’sRepublicofChina Appl Biochem Biotechnol

Introduction

Chiral mandelic acids are one class of α-hydroxy carboxylic acids featured by the existence of two functional groups, one carboxylic acid group and one hydroxyl group at the α-position [1, 2]. Optical pure mandelic acids and their ester derivatives are crucial chemicals and intermediates in the synthesis of many valuable drugs [3]. For example, (R)-mandelic acid has been widely used as a key chiral intermediate for the synthesis of many clinically important pharmaceuticals such as semi-synthetic cephalosporin, penicillin, and anti-obesity agents [4–6]. Other chiral mandelic acids and their derivatives have been extensively utilized as important chemicals used either in the synthesis of many chiral products or in the resolution of racemic compounds in industry [7]. Due to high values of optical pure mandelic acids and their ester derivatives, the preparation of optical pure mandelic acids and their ester derivatives has been a focus of research in industry [8–11]. Traditionally, chiral chemicals could be synthesized using organometallic chemistry. However, synthesis through organometallic chemistry generally requires harsh working conditions and needs toxic metal catalysts, which can cause great pollution to our environments [12]. Another disadvantage of synthesizing chiral chemicals through organometallic chemistry is that the optical purities of final products were not high enough to meet the requirements of following synthetic steps. Another method for the preparation of optical pure mandelic acids and their ester derivatives is through biocatalysis methods. Due to the existence of one carboxylic acid group, optically pure mandelic acids and their ester derivatives should be able to be prepared by kinetic resolution catalyzed by esterases or lipases, which have been proven to be useful biocatalysts in recognizing chirality and exhibit excellent stereo-selectivity. The utilization of esterases/ lipases in the synthesis of optical chemicals requires no expensive co-factors, and the enzymatic kinetic resolution reactions can be carried out under mild conditions. Thus, esterases and lipases have been widely utilized in the synthesis of many valuable chemicals and drug intermediates. Microorganisms have been a great source for the identification of novel genes and novel enzymes. Enzymes isolated from microorganisms are generally more stable than those enzymes identified from plants and animals. Due to relatively inexpensive sequencing technique of microbial genomes, it is much easier to identify novel enzymes from microbial genomes, characterize the functions of novel enzymes, and use novel enzymes as biocatalysts in the synthesis of a great variety of valuable chiral chemicals and chiral drug intermediates [13, 14]. Herein, we identified one novel esterase from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085, characterized the enzymatic function of this esterase, and further utilized this esterase in the synthesis of (R)-methyl mandelate. Both the enantiomeric excess and the conversion of final chiral product (R)-methyl mandelate were quite satisfying after process optimization.

Materials and Methods

Strains and Plasmids

The strain D. aurantiacum subsp. Hamdenensis NRRL 18085 was provided by Agricultural Research Service Culture Collection (NRRL). The plasmid pET-28a (+) (Novagen, USA) was used as the vector for heterologous protein expression. The strains Escherichia coli DH5α and E. coli BL21 (DE3) were used for routine cloning and gene expression, respectively. Appl Biochem Biotechnol

Cloning and Construction of Protein Expression Vector

The sequencing of the genome of D. aurantiacum subsp. Hamdenensis NRRL 18085 was completed by Majorbio LLC, Shanghai. Based on the coding DNA sequence of a putative esterase (here labeled as DAEst6) from the genome of D. aurantiacum subsp. Hamdenensis NRRL 18085, two degenerate primers, DAEst6F (5′-CACCACGAATTCGTGACCT TCCATCCCGTGCCAG-3′) and DAEst6R (5′-CCCAAGCTTTTAGCGC AGGACGTCGTCGAG-3′), for the amplification of DAEst6 were designed, with two restric- tion sites EcoR IandHind III underlined, respectively. PCR products of DAEst6 were digested using EcoR I/Hind III and cloned into vector pET28a(+) to generate the expression plasmid pET28a-DAEst6. Plasmid pET28a-DAEst6 was further transformed into E. coli BL21 (DE3) for protein expression.

Sequence Analysis

Open reading frames (ORFs) were analyzed by http://nocardia.nih.go.jp/fp4/. Deduced amino acid sequences were analyzed using the BLASTP program (http://blast.ncbi.nlm.nih.gov/). Three-dimensional model of enzymes was built and analyzed by SWISS-MODEL server. Phylogenetic and molecular evolutionary analysis was conducted by MEGA software version 5. Signal peptide was predicted by the SignalP 4.0 prediction tool from http://www.cbs.dtu.dk/ services/SignalP/. The theoretical values of protein molecular weight and pI were estimated by ExPASy from http://web.expasy.org/compute_pi/.

Overexpression and Purification of Recombinant DAEst6

The strain E. coli BL21(DE3) containing expression plasmid pET28a-DAEst6 was cultivated in Luria–Bertani medium harboring 50 μg/mL kanamycin at 37 °C until the OD600 reached 0.6. Then, the cells were induced at 20 °C with 0.5 mM IPTG for 16 h and harvested by centrifugation at 4000 rpm for 20 min at 4 °C. The cells were washed twice with PBS buffer (pH 7.4), resuspended by 50 mM Tris-HCl buffer (pH 8.0), and disrupted by sonication on ice for 15 min. Cell debris was removed by centrifugation at 12,000g and 4 °C for 20 min. Recombinant DAEst6 was purified using Ni-NTA affinity chromatography column as follows: first, the column was washed with buffer A containing 50 mM Tris-HCl buffer (pH 7.5) and 300 mM NaCl; then, the column was washed with buffer B containing 40 mM imidazole and 300 mM NaCl; and afterward, target enzyme was washed with elution buffer containing 300 mM imidazole and 300 mM NaCl. Both imidazole and ions were removed by GE PD-10 desalting column (GE Healthcare Life Sciences). The homogeneity of DAEst6 was determined by SDS-PAGE [15], and protein concentrations were assayed using the Bradford method with bovine serum albumin as the standard [16].

Biochemical Characterization of DAEst6

Substrate Specificity of DAEst6

The hydrolytic activity of DAEst6 was measured using 10 mM pNP esters with acyl chains of different lengths (C2–C16) as the substrates [17]. The enzymatic reactions that contained 455 μL 50 mM Tris-HCl buffer (pH 7.5), 5 μLof10mMpNP ester substrates, 20 μLof Appl Biochem Biotechnol ethanol, and 20 μL purified esterase (0.2 μg) were incubated at 40 °C for 5 min. The enzymatic reactions were terminated by using an equal volume of isopropanol and measured immediately at 405 nm. One unit of esterase activity was defined as the amount of enzyme needed to release 1 μmol of p-nitrophenol per minute.

Effect of pH on the Activity and Stability of DAEst6

The effect of pH on the hydrolytic activity of DAEst6 was studied by incubating DAEst6 in reaction systems containing 0.1 mM pNP hexanoate (C6), 20 μLofpurifiedDAEst6(0.2μg), and 50 mM buffers of different pH: acetic acid/sodium acetate (pH 5.0 to 6.0), potassium phosphate (pH 6.5 to 8.0), Tris-HCl (pH 7.5 to 9.0), and glycine/NaOH (pH 9.0 to 10.0) at 40 °C. To determine the pH stability of DAEst6, 20 μLofpurifiedDAEst6(0.2μg) was incubated for 12 h at 4 °C in 40 μL 50 mM buffers of different pH. The activities of DAEst6 were measured afterward to determine the effect of pH on the activity of DAEst6 and pH stability of DAEst6.

Effect of Temperature on the Activity and Stability of DAEst6

The optimal temperature of DAEst6 was determined by incubating 0.2 μgDAEst6inreactions containing 0.1 mM C6 at temperatures ranging from 10 to 60 °C in 50 mM Tris-HCl buffer (pH 7.5). To investigate the thermo-stability of DAEst6, 20 μL of purified DAEst6 (0.2 μg) was incubated in 50 mM Tris-HCl (pH 7.5) for 1 h at temperatures ranging from 30 to 60 °C, and then, the residual activities of DAEst6 were measured in enzymatic reactions containing 0.1 mM C6 in 50 mM Tris-HCl buffer (pH 7.5) at 45 °C as previously described. Enzymatic reactions with untreated DAEst6 were utilized as control experiments.

Effect of Metal Ions on the Stability of DAEst6

Of the purified esterase DAEst6 (0.2 μg), 20 μL was incubated in the presence of 10 different metal ions at a final concentration of 5 mM for 12 h at 4 °C. Afterward, the residual activities of DAEst6 were measured in enzymatic reactions containing 0.1 mM C6 in 50 mM Tris-HCl (pH 7.5) buffer at 45 °C to determine the effect of diverse metal ions on the hydrolytic activity of DAEst6. Enzymatic reactions with untreated DAEst6 were utilized as control experiments.

Effect of Organic Solvents on the Stability of DAEst6

Of purified esterase (0.2 μg), 20 μL was pre-incubated with 10 different organic solvents at a final concentration of 10 % (v/v) for 12 h at 4 °C. The residual activities of DAEst6 were measured in enzymatic reactions containing 0.1 mM C6 in 50 mM Tris-HCl (pH 7.5) buffer at 45 °C as previously described. Enzymatic reactions with untreated DAEst6 were utilized as control experiments to study the effect of diverse organic solvents on the hydrolytic activity of DAEst6.

Effect of Surfactants and Detergents on the Stability of DAEst6

Of purified esterase (0.2 μg), 20 μL was incubated with various surfactants (Tween-20, Tween-80, and Triton-100) at a final concentration of 0.02 % (v/v) or detergents (guanidinium Appl Biochem Biotechnol chloride, urea, SDS, and EDTA) at different concentrations for 12 h at 4 °C. The residual activities of DAEst6 were measured in enzymatic reactions containing 0.1 mM C6 in 50 mM Tris-HCl (pH 7.5) buffer at 45 °C as previously described. Enzymatic reactions with untreated DAEst6 were utilized as control experiments to study the effect of diverse surfactants and inhibitors on the hydrolytic activity of DAEst6.

Kinetic Analysis of DAEst6

The kinetic parameters of DAEst6 were calculated at 45 °C, pH 7.5, and 50 mM Tris-HCl buffer containing C6 with concentrations ranging from 0.01 to 2 mM. Km and Vmax were obtained using a Lineweaver–Burk plot under the optimal reaction conditions.

Process Optimization for the Preparation of (R)-Methyl Mandelate by DAEst6 Through Kinetic Resolution

Effect of pH on the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

The effects of pH on kinetic resolution of (±)-methyl mandelate by DAEst6 were studied by adjusting the pH of enzymatic reactions. A standard 500-μL reaction system contained 50 μg of purified DAEst6, 50 μL 50 mM (±)-methyl mandelate, and 400 μL buffer with pH ranging from 5.0 to 10.0. After incubating the enzymatic reactions for 4 h at 37 °C, the residual methyl mandelate was extracted three times by adding 500 μL ethyl acetate and further analyzed by chiral GC. The enantiomeric excess (e.e.s)of(R)-methyl mandelate, conversion rate (C), and enantiomeric ratio (E) of reaction were calculated by using the equation of Chen et al. [18].

Effect of Temperature on the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

The effect of temperature on the kinetic resolution of (±)-methyl mandelate by DAEst6 was studied by incubating standard enzymatic reactions (50 μg purified DAEst6, 5 mM (±)-methyl mandelate, and 50 mM Tris-HCl buffer, pH 7.5) at temperature ranging from 20 to 50 °C. After the enzymatic reactions were incubated at 37 °C for 4 h, 500 μL ethyl acetate was added to extract residual methyl mandelate and the residual methyl mandelate was further analyzed by chiral GC.

Effect of Co-Solvents on the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

The effect of different co-solvents on the kinetic resolution of (±)-methyl mandelate by DAEst6 was analyzed by adding 10 % (v/v) different organic solvents into standard enzymatic reactions as mentioned above. After the enzymatic reactions were incubated for 4 h at 37 °C, 500 μL ethyl acetate was used to extract the residual methyl mandelate from the enzymatic reactions and the residual methyl mandelate product was further analyzed by chiral GC.

Effect of Substrate Concentration on the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

The effect of substrate concentration on the kinetic resolution of (±)-methyl mandelate catalyzed by DAEst6 was studied by adding substrate (±)-methyl mandelate of different Appl Biochem Biotechnol concentrations (ranging from 5 to 100 mM) into standard enzymatic reactions. After the enzymatic reactions were incubated at 37 °C for 4 h, 500 μL ethyl acetate was utilized to extract the residual methyl mandelate from the enzymatic reactions and the residual (±)-methyl mandelate was further analyzed by chiral GC.

Effect of Reaction Time on the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

The effect of reaction time on the kinetic resolution of (±)-methyl mandelate was studied by incubating standard enzymatic reactions (containing 50 μg DAEst6, 10 mM (±)-methyl mandelate in pH 7.5 Tris-HCl buffer) at 40 °C. Time course of the reactions was monitored by extracting the residual methyl mandelate from an enzymatic reaction every 30 min using 500 μL ethyl acetate. The extracted methyl mandelate was analyzed by chiral GC.

Gas Chromatograph Analysis

The residual (±)-methyl mandelate in reaction system was analyzed via gas chromatograph (FULI GC-9790 II) using an equipped 112–6632 CYCLOSIL-B chiral capillary column

(30 m × 0.25 mm ID, 0.25 μm df). The temperatures of H2 flame ionization detector and injector were set at 250 and 280 °C, respectively. Nitrogen served as carrier gas at a split flow rate of 1.20 mL/min. The oven temperature was held at 100 °C for 1 min, then increased at 10 °C/min to 220 °C, and held for 3 min.

Statistical Analysis

Statistical analyses including mean and standard deviation were calculated using SPSS19.0 software, with three experiments carried out in parallel.

Results and Discussion

Sequence Analysis

Through bioinformatics analysis, an ORF, DAEst6, of 795 bp and coding for a putative esterase of 264 amino acid residues was identified from the genome of D. aurantiacum subsp. Hamdenensis NRRL 18085. DAEst6 shares 85 % identity with one hypothetical protein (WP_020517211.1) from Actinoplanes globisporus, 80 % identity with one hypothetical protein (WP_033361930.1) from D. aurantiacum, and 61 % identity with one hypothetical protein (WP_027344279) from Hamadaea tsunoensis. The functions of above hypothetical proteins have not been well characterized before. At current stage, bacterial lipolytic enzymes can be classified into 15 families [19]. Through protein sequence analysis and BLAST search, a phylogenetic tree to analyze DAEst6 and present 15 families of bacterial lipolytic enzymes was constructed as shown in Fig. 1. Protein sequence analysis shows that DAEst6 contains a catalytic triad formed by Ser124, Asp207, and His239 and a conserved GESAG motif of family VII lipolytic enzymes [20](Fig.2). However, DAEst6, an esterase with 264 amino acid residues, was much shorter than family VII lipolytic enzymes, which generally harbor 500 amino acid residues. Thus, it is possible that DAEst6 belongs to a new branch in family VII lipolytic enzymes. Appl Biochem Biotechnol

Fig. 1 Phylogenetic tree analysis of DAEst6 and present 15 families of bacterial lipolytic enzymes Appl Biochem Biotechnol

Fig. 2 Multiple sequence alignment of DAEst6 with some homologous proteins and family VII esterases. The protein sequences of lipolytic enzymes were retrieved from GenBank (http://www.ncbi.nlm.nih.gov), one microbial esterases (PDB:4XVC) from metagenomic library, one hypothetical protein (WP_020517211.1) from Actinoplanes globisporus, one hypothetical protein (WP_033361930.1) from Dactylosporangium aurantiacum, and one hypothetical protein (WP_027344279) from Hamadaea tsunoensis and three previously identified family VII esterases from Bacillus subtilis (P37967.2), Arthrobacter oxydans (Q01470.1), and Streptomyces coelicolor A3(2) (CAA22794.1). Solid circles represent residues belonging to the catalytic triad of some homologous proteins of DAEst6. Filled triangles indicate the catalytic residues of family VII esterases. Underline indicates the conserved HGG motif Appl Biochem Biotechnol

Phylogenetic Analysis of D. aurantiacum Subsp. Hamdenensis NRRL 18085

An unrooted neighbor-joining phylogenetic tree of D. aurantiacum subsp. Hamdenensis NRRL 18085 and some other strains producing family VII lipolytic enzymes was constructed by using 16S rRNA sequence (Fig. 3). D. aurantiacum subsp. Hamdenensis NRRL 18085 exhibits 99 % identity with D. maewongense strain MW2–25 (NR_112908) and 99 % with D. aurantiacum strain IFO 12592 (NR_115636). However, D. aurantiacum subsp. Hamdenensis NRRL 18085 exhibits relatively low-sequence identities with some other strains that also produce family VII lipolytic enzymes, such as Streptomyces coelicolor, Arthrobacter oxydans,andBacillus subtilis.

Protein Expression

The theoretical molecular weight of DAEst6 was calculated to be 28.2 kDa, and the pI of DAEst6 was calculated to be 5.1. The recombinant DAEst6 was successfully expressed in E. coli BL21 (DE3) and further purified using Ni-NTA affinity chromatography. A single- protein band consistent with the expected theoretical molecular weight could be observed on SDS-PAGE gel as shown in Fig. 4.PurifiedDAEst6fromE. coli was utilized for further functional characterization and kinetic resolution of (±)-methyl mandelate.

Substrate Specificity

The substrate specificity of DAEst6 was analyzed by using p-nitrophenyl esters with various acyl chain lengths (C2–C14) as the substrates. As shown in Fig. 5, p-nitrophenyl hexanoate (C6) was the preferred substrate for DAEst6, and the hydrolysis activities of DAEst6 toward long-chain p-nitrophenyl esters were much lower, indicating that DAEst6 characterized in our study was an esterase.

Fig. 3 Unrooted neighbor-joining phylogenetic tree of D. aurantiacum subsp. Hamdenensis NRRL 18085 with close relatives and some bacteria producing family VII lipolytic enzymes Appl Biochem Biotechnol

Fig. 4 Expression and purification of DAEst6 from E. coli BL21(DE3). M protein standard markers. Lane 1 total proteins from E. coli BL21(DE3). Lane 2 total proteins from E. coli BL21(DE3)-pET28a-DAEst6 before IPTG induction. Lane 3 total proteins from E. coli BL21(DE3)-pET28a-DAEst6 after IPTG induction. Lane 4 purified DAEst6

Effect of pH on the Hydrolytic Activity and Stability of DAEst6

The effect of pH on the hydrolytic activity of DAEst6 was studied by using C6 as the substrate. Standard enzymatic reactions were incubated at various pH and at 40 °C for 12 h. As shown in Fig. 6a, the maximum hydrolytic activity of DAEst6 was detected at pH 7.5 and DAEst6 exhibited relatively lower hydrolytic activities when the pH was lower than 7.0 or higher than

Fig. 5 Hydrolytic activity of 120 DAEst6 toward p-nitrophenyl esters with various acyl chain lengths 100 (C2–C14) 80

Relativie activity (%) activity Relativie 20

0 2 4 6 8 C C C C 10 12 14 C C C p-NP p-NP p-NP p-NP p-NP p-NP p-NP Appl Biochem Biotechnol

a) Fig. 6 Effect of pH on the 120 hydrolytic activity and stability of DAEst6. a Effect of pH on the 100 hydrolytic activity of DAEst6. b pH stability of DAEst6 80 60

Relativie activity (%) activity Relativie 20

0 4 5 6 7 8 9 10 11 pH b) 120

100

Relativie activity (%) activity Relativie 20

0 4 5 6 7 8 9 10 11 pH

8.0. Thus, the optimal pH for the hydrolytic activity of DAEst6 was found to be pH 7.5. The effect of pH on the stability of esterase was examined by pre-incubating the enzyme at different pH (Fig. 6b). DAEst6 was found to be quite stable under alkaline pH ranging from 7.5 to 10.0, indicating that DAEst6 was a novel esterase with high resistance to high pH values.

Effect of Temperature on the Hydrolytic Activity and Stability of DAEst6

The influence of temperature on the hydrolytic activity of DAEst6 was performed at different temperatures ranging from 20 to 50 °C. As shown in Fig. 7a, DAEst6 exhibited relatively high activities at temperatures ranging from 30 to 50 °C. The thermo-stability of DAEst6 was studied by pre-incubating DAEst6 at temperatures ranging from 20 to 60 °C, and the hydrolysis activities of DAEst6 were measured afterward (Fig. 7b). DAEst6 was found to be quite stable at temperatures ranging from 20 to 40 °C, and DAEst6 was quite unstable at temperatures higher than 50 °C.

Effect of Metal Ions on the Hydrolytic Activity of DAEst6

The effect of different metal ions on the hydrolytic activity of DAEst6 was studied by adding 5-mM various metal ions into the standard enzymatic reactions (Table 1). Among the ten tested metal ions, Fe2+,Mn2+,Li2+,Ca2+,Co2+, and Mg2+ basically did not quite affect the hydrolytic activity of DAEst6. Cu2+,Zn2+, and Ba2+ were found to have negative effect on the hydrolytic activity of DAEst6. Appl Biochem Biotechnol

a) Fig. 7 Effect of temperature on 120 the hydrolytic activity and stability of DAEst6. a Effect of temperature )%(ytivitcaeiv 100 on the hydrolytic activity of 80 DAEst6. b Temperature stability of DAEst6 60

i 40 taleR 20

0 10 20 30 40 50 60 70 80 Temperature( ) b) 120

)%(ytivitcaeiv 100

i 40 taleR 20

0 20 30 40 50 60 70 Temperature( )

Effect of Organic Solvents on the Hydrolytic Activity of DAEst6

The effect of organic solvents on the hydrolytic activity of DAEst6 was studied by adding 20 % different organic solvents into standard enzymatic reactions. As shown in Table 2,ofall the ten tested organic solvents, simply the addition of methanol could not affect the hydrolytic activity of DAEst6 and all the other nine organic solvents could affect the hydrolytic activity of DAEst6 to some extent.

Table 1 Effect of metal ions on the hydrolytic activity of DAEst6 Metal ions Concentration Relative activity (%)

Control 100 Fe2+ 5mM 105±2 Mn2+ 5mM 96±2 Ni2+ 5mM 76±2 Li2+ 5mM 110±2 Ca2+ 5 mM 111 ± 2 Cu2+ 5mM 15±1 Co2+ 5mM 92±2 Zn2+ 5mM 19±1 Ba2+ 5mM 36±1 Mg2+ 5mM 100±2 Appl Biochem Biotechnol

Table 2 Effect of various organic solvents on the hydrolytic activity Organic solvents Concentration (v/v) Relative activity (%) of DAEst6 Control 100 Cyclohexane 20 % 83 ± 1 Dichloromethane 20 % 75 ± 7 n-heptane 20 % 80 ± 3 Butanol 20 % 6 ± 1 n-hexane 20 % 82 ± 2 Ethyl acetate 20 % 71 ± 3 Ethanol 20 % 91 ± 4 1,4-dioxane 20 % 6 ± 1 Acetonitrile 20 % 38 ± 2 Methanol 20 % 102 ± 1

Effect of Surfactants and Detergents on the Hydrolytic Activity of DAEst6

The effect of surfactants and inhibitors on the hydrolytic activity of DAEst6 was studied by adding different surfactants and detergents into standard enzymatic reactions. As shown in Table 3, the addition of Triton-100, Tween-20, Tween-80, and EDTA basically did not affect the hydrolytic activity of DAEst6. But, the hydrolytic activity of DAEst6 could be strongly inhibited by SDS, urea, and guanidinium chloride.

Kinetic Analysis of DAEst6

The kinetic parameters of esterase DAEst6 were shown in Table 4. The specific activity of purified DAEst6 was 317 U/mg using C6 as substrate, with Km and Kcat being 365 μMand 4362 s−1, respectively. Compared with the kinetic data of other bacteria esterases belonging to family VII lipolytic enzymes, both the specific activity and the catalytic efficiency (Kcat/Km)of DAEst6 were much higher, indicating that DAEst6 was a novel esterase with much higher hydrolytic activity.

Process Optimization for the Kinetic Resolution of (±)-Methyl Mandelate by DAEst6

Effect of pH on the Kinetic Resolution of (±)-Methyl Mandelate

The variation of pH could alter the conformation of enzymes. The ionic states of the enzymes may also change with pH. So, pH might greatly affect stereo-selectivity during enzymatic kinetic resolution. The effect of pH on the kinetic resolution of (±)-methyl mandelate catalyzed by DAEst6 was studied by adjusting the pH of enzymatic reactions for the kinetic resolution. As shown in Fig. 8, the best enantiomeric excess and conversion could be observed when the kinetic resolution of (±)-methyl mandelate was carried out under pH 7.5 and generate (R)- methyl mandelate, indicating that the optimal pH for the kinetic resolution of (±)-methyl mandelate by DAEst6 was 7.5. Under pH 7.5, the enantiomeric excess and conversion could reach 73 and 36 %, respectively. Appl Biochem Biotechnol

Table 3 Effect of surfactants and inhibitors on the hydrolytic activity Surfactants and detergents Concentration Relative activity (%) of DAEst6 Control 100 T100 0.02 % (v/v)86±1 T80 0.02 % (v/v)90±2 T20 0.02 % (v/v)89±2 SDS 5 mM 3 ± 1 EDTA 5 mM 78 ± 2 Urea 2M 8±1 Guanidinium chloride 2 M 10 ± 1

Effect of Temperature on the Kinetic Resolution of (±)-Methyl Mandelate

The influence of temperature on the kinetic resolution of (±)-methyl mandelate was studied by carrying out standard enzymatic resolutions at different temperatures. As shown in Fig. 9,the best enantiomeric excess and conversion could reach 75 and 37 %, respectively, at 40 °C, indicating that the optimal temperature for the kinetic resolution of (±)-methyl mandelate by DAEst6 was 40 °C.

Effect of Organic Co-Solvents on the Kinetic Resolution of (±)-Methyl Mandelate

Generally, organic solvents could strongly affect the stereo-selectivity of enzymes during kinetic resolution. The effect of organic solvents on the kinetic resolution of (±)-methyl mandelate was studied by adding ten different organic solvents (10 %) into standard kinetic resolution reactions under pH 7.5 and at 40 °C. Although there were many reports about increasing enantiomeric excess and conversion by adding organic solvents, the addition of different organic solvents could not increase the enantiomeric excess and conversion in the kinetic resolution of (±)-methyl mandelate by DAEst6 (Table 5). So, the process optimization of the kinetic resolution of (±)-methyl mandelate by DAEst6 was carried out in enzymatic reactions without any organic solvents.

Effect of Substrate Concentration on the Kinetic Resolution of (±)-Methyl Mandelate

The effect of substrate concentration on the kinetic resolution of (±)-methyl mandelate by DAEst6 was studied by adding racemic substrate of different concentrations ranging from 5 to 100 mM. As shown in Fig. 10, the highest enantiomeric excess and conversion could reach 79 and 36 %, respectively, when 10 mM (±)-methyl mandelate was added to the enzymatic reaction, meaning that 10 mM was the optimal substrate concentration for the kinetic resolution of racemic methyl mandelate.

Time Course on the Kinetic Resolution of (±)-Methyl Mandelate

The effect of time course on the kinetic resolution of (±)-methyl mandelate was also studied by changing the reaction time of standard enzymatic reactions containing 10 mM (±)-methyl mandelate under pH 7.5 and at 40 °C. As shown in Fig. 11, the highest enantiomeric excess and conversion could reach 99 and 49 %, respectively, at 5 h, indicating that the optimal reaction plBohmBiotechnol Biochem Appl

Table 4 Biochemical properties of DAEst6 and some other family VII esterases

−1 Source of esterase Molecular Optimal pH Optimal Optimal substrate/p-NP esters Specific activity Km (μM) Kcat (s )Ref weight/kDa temperature(°C) (U/mg)

Dactylosporangium aurantiacum subsp. 28.2 7.5 45 C6 317±8 365±8 4362±23 This study Hamdenensis NRRL 18085 Thermoanaerobacter tengcongensis 47.1 9.5 70 C6 150 70.2 108 [20] Soil metagenomic library 59 8.0 40 C4 161 1300 229.3 [21] Arthrobacter oxydans 55 - - C4 - 25 - [22] Metagenomic library 63 9.0 55 C6 - --[23]

B–^ means no report Appl Biochem Biotechnol

Fig. 8 Effect of pH on the kinetic 120 120 resolution of (±)-methyl mandelate by DAEst6 Enantiomeric excess 100 100 Conversion

80 80

60 60 Conversion (%) 40 40 Enantiomeric excess (%) excess Enantiomeric

20 20

0 0 4 5 6 7 8 9 10 11 pH time for the kinetic resolution of racemic methyl mandelate by DAEst6 was 5 h. After 5 h, both enantiomeric excess and conversion of product (R)-methyl mandelate remained basically un- changed, meaning that DAEst6 could selectively hydrolyze (S)-methyl mandelate and leave optical pure product (R)-methyl mandelate in the enzymatic reaction after process optimization.

Discussion

Dactylosporangium is a genus that belongs to the phylum Actinobacteria. So far, most research work related to Dactylosporangium was limited to the isolation of

Fig. 9 Effect of temperature on 120 120 the kinetic resolution of (±)-methyl mandelate by DAEst6 Enantiomeric excess 100 100 Conversion

80 80 )%(noisrev

60 60 n oC 40 40 Enantiomeric excess (%) Enantiomeric

20 20

0 0 10 20 30 40 50 60 Temperature( ) Appl Biochem Biotechnol

Table 5 Effects of co-solvents on the enantio-selective hydrolysis of (±)-methyl mandelate by DAEst6

Organic solvents Concentration (v/v) Enantiometric excess (%) Conversion (%)

Control 74 ± 2 36 ± 1 Tert-butyl alcohol 10 % 57 ± 2 31 ± 1 1, 4 dioxane 10 % 18 ± 1 9 ± 1 Methanol 10 % 71 ± 1 36 ± 1 DMSO 10 % 70 ± 1 36 ± 1 Toluene 10 % 28 ± 1 2 ± 1 Cyclohexane 10 % 18 ± 1 10 ± 1 Acetonitrile 10 51 ± 1 26 ± 1 Ethyl alcohol 10 % 4 ± 1 2 ± 1 Ethyl acetate 10 % 2 ± 1 <1.0 Dichloromethane 10 % 3 ± 1 <1.0 Heptane 10 % 54 ± 1 27 ± 1

Dactylosporangium strains and diversity studies [24–26]. In addition, there were a few reports about the isolation of some bioactive nature products, such as dactimicin and tiacumicins from Dactylosporangium strains, and several biosynthetic studies of those natural products [27]. For example, tiacumicin B was a promising anti-infectious natural product isolated from D. aurantiacum subsp. Hamdenensis NRRL 18085 and the biosynthetic gene cluster of tiacumicin B was identified and functionally characterized [28]. However, there were no reports related to developing enzymes identified from Dactylosporangium strains and further utilization of those Dactylosporangium enzymes in the preparation of valuable chemicals. In our work, we identified and cloned a gene DAEst6 encoding a novel esterase from the genome of D. aurantiacum subsp. Hamdenensis NRRL 18085. Based upon protein sequence analysis and phylogenetic analysis, it is possible that this

Fig. 10 Effect of substrate 120 120 concentration on the kinetic resolution of (±)-methyl mandelate Enantiomer excess by DAEst6 100 100 Conversion

80 80

60 60 Conversion (%) 40 40 (%) Enantiomeric

20 20

0 0 0 20 40 60 80 100 Concentration (mM) Appl Biochem Biotechnol

Fig. 11 Effect of time course on 120 120 the kinetic resolution of (±)-methyl mandelate Enantiomeric excess 100 100 Conversion

80 80

60 60 Conversion (%) 40 40 Enantiomeric excess (%) excess Enantiomeric

20 20

0 0 0 1 2 3 4 5 6 Time(h)

novel esterase belong to a new branch of family VII lipolytic enzymes [19]. DAEst6 was heterologously expressed and further functionally characterized to be an esterase that exhibited stable hydrolysis activities under alkalinity conditions and possessed good potential in the hydrolysis of ester products at high pH values. To the best of our knowledge, our work was the first report about the identification and functional characterization of one novel esterase from Dactylosporangium strains. Chiral mandelic acids and their ester derivatives are important building blocks in the synthesis of valuable pharmaceuticals. The development of synthetic methods is of great importance in the preparation of those chiral building blocks. Due to the existence of one carboxylic acid group, chiral mandelic acids and their ester derivatives should be able to be prepared through kinetic resolution by esterases. We utilized the novel esterase DAEst6 identified from D. aurantiacum subsp. Hamdenensis NRRL 18085 in the preparation of chiral mandelic acids and their ester derivatives. In the kinetic resolution of racemic methyl mandelate, esterase DAEst6 could selectively hydrolyze (S)-methyl mandelate and leave (R)-methyl mandelate untouched. The effects of pH, temperature, choice of co-solvents, substrate concentration, and time course on the kinetic resolution of racemic methyl mandelate were studied in details. After process optimization, final chiral product, (R)-methyl mandelate, could be generated by esterase DAEst6 with an enantiomeric excess and a conversion of 99.0 and 49.0 %, respectively. Contrary to some other cases, organic co-solvents did not improve the enantiomeric excess and conversion of desired chiral product and most organic solvents had negative effects on the kinetic resolution. Before our work, there were several reports about the preparation of chiral methyl mandelate through kinetic resolution catalyzed by lipases. For example, both one commer- cialized lipase, Novezym 435, and one uncharacterized extracellular lipase were able to resolve racemic methyl mendelate and give (S)-methyl mandelate [11, 29]. However, esterase DAEst6 characterized in our work could generate (R)-methyl mandelate with high optical purity and exhibited opposite stereo-selectivity. Appl Biochem Biotechnol

Conclusion

In conclusion, DAEst6 was characterized to be a novel esterase identified from one Dactylosporangium strain. DAEst6 was able to resolve racemic methyl mandelate and generate (R)-methyl mandelate, one key drug intermediate, with high enantiomeric excess (99 %) and conversion (49 %) after process optimization. Dactylosporangium esterases represented by DAEst6 possess great potential in the preparation of chiral chemicals and are awaiting further development in industry.

Acknowledgments We are grateful for the financial supports from National Natural Science Foundation of China (No. 21302199), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030404), Guangzhou Science and Technology Plan Projects (201510010012), and Key Project BEngineering High-Performance Microorganisms for Advanced Bio-Based Manufacturing^ from the Chinese Academy of Sciences (KGZD-EW-606). We also would like to thank the constant help from Professor Changsheng Zhang and Professor Jianhua Ju.

References

1. Groger, H. (2001). Enzymatic routes to enantiomerically pure aromatic alpha-hydroxy carboxylic acids: a further example for the diversity of biocatalysis. Advanced Synthesis & Catalysis, 343, 547–558. 2. Zhang, Z. J., Jiang, P., Ma, B. D., & Xu, J. H. (2014). Efficient biocatalytic synthesis of chiral chemicals. Advances in Biochemical Engineering/Biotechnology,1–52. 3. Surivet, J. P., & Vatele, J. M. (1999). Total synthesis of antitumor Goniothalamus styryllactones. Tetrahedron, 55,13011–13028. 4. Savidge, T. A. (1984). In J. V. Erick (Ed.), Biotechnology of industrial antibiotics (drugs & the pharmaceutical sciences). New York: Marcel Dekker. 5. Guo, J. L., Mu, X. Q., & Xu, Y. (2010). Integration of newly isolated biocatalyst and resin-based in situ product removal technique for the asymmetric synthesis of (R)-methyl mandelate. Bioprocess and Biosystems Engineering, 33, 797–804. 6. Yadav, G. D., & Bhagat, R. D. (2004). Synthesis of methyl phenyl glyoxylate via clean oxidation of methyl mandelate over a nanocatalyst based on heteropolyacid supported on clay. Organic Process Research & Development, 8, 879–882. 7. Kinbara, K., Sakai, K., Hashimoto, Y., Nohira, H., & Saigo, K. (1996). Design of resolving reagents: p- substituted mandelic acids as resolving reagents for 1-arylalkylamines. Tetrahedron-Asymmetry, 7, 1539– 1542. 8. Pennacchio, A., Giordano, A., Rossi, M., & Raia, C. A. (2010). Effective synthesis of methyl (R)-mandelate by asymmetric reduction with a thermophilic NADH-dependent alcohol dehydrogenase. Journal of Biotechnology, 150, 525–525. 9. Wei, H. N., & Wu, B. (2008). Screening and immobilization Burkholderia sp. GXU56 lipase for enantioselective resolution of (R,S)-methyl mandelate. Applied Biochemistry and Biotechnology, 149,79– 88. 10. Pifissao, C., & Nascimento, M. D. (2006). Effects of organic solvents and ionic liquids on the aminolysis of (RS)-methyl mandelate catalyzed by lipases. Tetrahedron-Asymmetry, 17,428–433. 11. Yadav, G. D., & Sivakumar, P. (2004). Enzyme-catalysed optical resolution of mandelic acid via RS(−/+)- methyl mandelate in non-aqueous media. Biochemical Engineering Journal, 19,101–107. 12. Noyori, R., & Kitamura, M. (1991). Enantioselective addition of organometallic reagents to carbonyl- compounds—chirality transfer, multiplication, and amplification. Angewandte Chemie International Edition in English, 30,49–69. 13. Patel, R. N. (2003). Microbial/enzymatic synthesis of chiral pharmaceutical intermediates. Current Opinion in Drug Discovery & Development, 6, 902–920. 14. Patel, R. N. (2006). Biocatalysis: synthesis of chiral intermediates for drugs. Current Opinion in Drug Discovery & Development, 9,741–764. 15. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature, 227, 680–685. Appl Biochem Biotechnol

16. Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry, 72,248–254. 17. Winkler, U. K., & Stuckmann, M. (1979). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138,663–670. 18. Chen, C. S., Fujimoto, Y., Girdaukas, G., & Sih, C. J. (1982). Quantitative analyses of biochemical kinetic resolutions of enantiomers. Journal of the American Chemical Society, 104, 7294–7299. 19. Fang, Y. W., Wang, S. J., Liu, S., & Jiao, Y. L. (2015). Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining. International Journal of Biological Macromolecules, 80,334–340. 20. Rao, L., Xue, Y. F., Zhou, C., Tao, J., Li, G., Lu, J. R., et al. (2011). A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes. Biochimica Et Biophysica Acta-Proteins and Proteomics, 1814,1695–1702. 21. Tao, W., Lee, M. H., Wu, J., Kim, N. H., & Lee, S. W. (2011). Isolation and characterization of a family VII esterase derived from alluvial soil metagenomic library. Journal of Microbiology, 49,178–185. 22. Pohlenz, H. D., Boidol, W., Schuttke, I., & Streber, W. R. (1992). Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide- sequence of the corresponding gene. Journal of Bacteriology, 174, 6600–6607. 23. Kang, C. H., Oh, K. H., Lee, M. H., Oh, T. K., Kim, B. H., & Yoon, J. H. (2011). A novel family VII esterase with industrial potential from compost metagenomic library. Microbial Cell Factories, 10,1–8. 24. Phongsopitanun, W., Kudo, T., Ohkuma, M., Suwanborirux, K., & Tanasupawat, S. (2015). Dactylosporangium sucinum sp nov., isolated from Thai peat swamp forest soil. Journal of Antibiotics, 68, 379–384. 25. Thawai, C., & Suriyachadkun, C. (2013). Dactylosporangium siamense sp nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology, 63, 4033–4038. 26. Seo, S. H., & Lee, S. D. (2010). Dactylosporangium darangshiense sp nov., isolated from rock soil. International Journal of Systematic and Evolutionary Microbiology, 60,1256–1260. 27. Kim, B. Y., Kshetrimayum, J. D., & Goodfellow, M. (2011). Detection, selective isolation and characteri- sation of Dactylosporangium strains from diverse environmental samples. Systematic and Applied Microbiology, 34,606–616. 28. Xiao, Y., Li, S. M., Niu, S. W., Ma, L. A., Zhang, G. T., Zhang, H. B., et al. (2011). Characterization of tiacumicin B biosynthetic gene cluster affording diversified tiacumicin analogues and revealing a tailoring dihalogenase. Journal of the American Chemical Society, 133, 1092–1105. 29. Yadav, G. D., Sajgure, A. D., & Dhoot, S. B. (2008). Insight into microwave irradiation and enzyme catalysis in enantioselective resolution of RS-(+/−)-methyl mandelate. Journal of Chemical Technology and Biotechnology, 83, 1145 –1153.