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

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