Esterases are crucial biocatalysts in chiral compound synthesis. Herein, a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis. EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester [Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate], producing over 99% yield and 99% enantiomeric excess (e.e.) for (4S, 5R)-monomethyl ester, a crucial chiral intermediate during the synthesis of d-biotin. Notably, the recombinant E. coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain. EstSIT01 displays a preference for short-chain p-NP esters. The optimal temperature and pH were 45 °C and 10.0, with Km and kcat values of 0.147 mmol/L and 5.808 s- 1, respectively. Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure. Collectively, EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.

Methyl (R)-N-(2,6-dimethylphenyl)alaninate ((R)-DMPM) is a key chiral intermediate for the production of (R)-metalaxyl, which is one of the best-selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)-DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)-DMPM. After the 48 h hydrolysis of 256 mM (R,S)-DMPM under the optimized reaction conditions, the enantiomeric excess of product (e.e.p ) was up to 99% and the conversion was nearly 50%. Subsequently, the unhydrolyzed (S)-DMPM was converted to (R,S)-DMPM through the n-butanal-catalyzed racemization. Furthermore, stereoselective hydrolysis of (R,S)-DMPM catalyzed by whole cells of P. asaccharolyticum WZZ003 was scaled up to kilogram-scale, offering (R)-MAP-acid with 98.6% e.e.p and 48.0% yield. Moreover, (R)-metalaxyl was prepared at kilogram scale after subsequent esterification and coupling reactions. Therefore, a practical production process of (R)-DMPM and (R)-metalaxyl with the prospect of industrialization was developed in this study. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:921-928, 2018.

The stereoselective hydrolysis of esmolol in whole blood and in its separated components from rat, rabbit and human was investigated. Blood esterase activities were variable in different species in the order of rat>rabbit>human. Rat plasma showed the high esterase activity and had no stereoselectivity to enantiomers. Rabbit red blood cell (RBC) membrane, RBC cytosol and plasma all hydrolyzed esmolol but with different esterase activity, whereas the hydrolysis in RBC membrane and cytosol showed significant stereoselectivity towards R-(+)-esmolol. Esterase in RBC cytosol from human blood mainly contributed to the esmolol hydrolysis, which was demonstrated with no stereoselctivity. Esterase in human plasma showed a low activity, but a remarkable stereoselectivity with R-(+)-esmolol. In addition, the protein concentration affected the hydrolysis behavior of esmolol in RBC suspension. Protein binding of esmolol enantiomers in human plasma, human serum albumin (HSA) and α1-acid glycoprotein (AGP) revealed that there was a significant difference in bound fractions between two enantiomers, especially for AGP. Our results indicated that the stereoselective protein binding might play a role in the different hydrolysis rates of esmolol enantiomers in human plasma.

A thermostable formamidase from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was revealed to be a novel, thermostable (+)-γ-lactamase. This (+)-γ-lactamase (Sso2810) is composed of only 318 amino acid residues, in contrast to a previously reported (+)-γ-lactamase (Sso2122) with 504 amino acid residues from the same strain. Herein, we demonstrate that a single strain may contain diverse (+)-γ-lactamases. The gene of this thermostable (+)-γ-lactamase was cloned, functionally expressed in Escherichia coli BL21 and purified by a simple yet effective heat treatment method. Sso2810 was biochemically characterized and compared to Sso2122, with phylogenetic analysis indicating different evolutionary histories for the two encoding genes. This newly found thermostable enzyme shows promising properties for industrial applications; specifically, it could be used for the production of chirally pure (-)-γ-lactam for the synthesis of well-known carbocyclic nucleoside antiretroviral agents like Abacavir and Peramivir. The chiral product of the enzyme was purified to >99% enantiomeric excess.

The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1% enantiomeric excess (e.e.) and 97.5% e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9% e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75% sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics.