Cloning And Expression Of A Novel Esterase EstZF172 For The Preparation Of A Chiral Intermediate Of Pregabalin

Pregabalin, a lipophilic derivative of the neurotransmitter γ-aminobutyric acid (GABA), is a new generation of antiepileptic drugs (Lyrica(?)), which has been used for treating several central nervous system disorders, and its domestic and foreign markets demand and global sales are improving significantly. (35)-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester ((S)-cyanodiester, (S)-CNDE) is the key chiral intermediate for the preparation of Pregabalin. Biological resolution is a green and effective method in the route for the preparation of optically pure (S)-CNDE5 with great development potential and good application perspective.A DNA fragment containing the target gene from strain Pseudomonas CGMCC No. 4184 which was isolated with the excellent capacity for the stereo specific hydrolysis of (R)-CNDE, was obtained through genomic library construction and activity screening in the method of lipolytic/esterolytic-reaction on CNDE-supplemented plates with halo-formation coupling with CNDE-resolution detected by GC-detection. ORF analysis and the activity verification by recombinant cells with subcloning genes were conducted to lock the target gene estZF172. The gene was cloned and then transformed into E. coli BL21(DE3), and the recombinant protein was induced to produce in a soluble form. Compared with the catalytic activity of Pseudomonas CGMCC No.4184, the recombinant strain exhibited a 425-fold improvement for CNDE (3,948 U g-1) with the lvalue of 95.Based on the amino acid sequence alignments, esterase EstZF172 was found to contain four conserved motifs.3D structure and the function of several important amino acid residues were analyzed by homology modeling and molecular docking, followed by the site-directed mutagenesis for verification. Along with the results of the phylogenetic tree analysis and the test for the substrate specificity of the purified enzyme, EstZF172 was classified as a carboxylesterase belonging to Family VIII lipolytic enzymes, showing remarkable similarities to β-lactamases and DD-peptidases with the active serine residue located in the motif of S-X-X-K. The enzymatic characteristics was then studied with the purified esterase using p-nitrophenyl hexanoate as the substrate. The optimum temperature and pH were 50℃ and pH 9.0 in Tris-HCl buffer, respectively, and the enzymatic activity was significantly decreased in the presence of some additives, including metal ions of Co2+, Ni2+, Mn2+ and Zn2+, sulfhydryl reagents of AgNO3 andCuSO4, as well as detergents of SDS and Triton X-100.The activity towards CNDE was increased by 21% as a result of the optimization on the conditions of producing soluble enzymes in LB medium. For the chiral hydrolysis of rac-CNDE to obtain (5)-CNDE catalyzed by recombinant cells, the optimum operating temperature and pH were determined to be 35℃ and 8.5, respectively. The substrate and product inhibition were then studied, and some key parameters of the bio catalytic process including whole-cell catalyst loading and substrate loading were also optimized. The optimal substrate/biocatalyst ratio was 38.8 with a substrate loading of 127.5 g L-1 (500 mM). Under the optimal conditions, the ees was over 99.5%℃when the conversion was reaching 52.3%, with the productivity of 34.1 mmol L-1 h-1. What’s more, the addition of appropriate amount of strong-base anion-exchange resin CHA26107 aslo showed a positive effect on the suppression of the product inhibition during the conversion process.