Bum-Yeol Hwang

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 ω-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

ABSTRACT A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant ω-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, ω-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using ω-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

Lipase-catalyzed reaction in the packed-bed reactor with continuous extraction column to overcome a product inhibition

Abstract The resolution of rac -α-methyl-β-propiothiolactone ( rac -MPTL) was performed in a packed-bed reactor (PBR) using Pseudomonas cepacia lipase (PCL) in organic media to produce enantiopure ( R )-MPTL. By comparing enzyme stability of three enzyme forms, i.e. commercial PCL powder, Celite-immobilized PCL, and cross-linked enzyme crystals of PCL (CLECs-PCL), Celite-immobilized PCL was chosen for the construction of PBR because of its comparable stability to that of CLEC and easy handling. Owing to the severe product inhibition by 3-mercapto-α-methylpropionic acid (MMPA), the batch reaction system was inappropriate for the hydrolysis with PCL at high concentration of rac -MPTL. To overcome these problems, PBR with a continuous extraction column was used. The product inhibition was successfully overcome by incorporating an aqueous extraction unit. However, the yield of ( R )-MPTL (enantiomeric excess, ee>99%) was only about 20% based upon the initial concentration of rac -MPTL, due to the concomitant partitioning of rac -MPTL to the aqueous phase in the extraction column as well as the subsequent auto-hydrolysis of rac -MPTL to rac -MMPA during extraction. To reduce the undesired partitioning of rac -MPTL to aqueous phase, various salts were screened to modulate the partitioning coefficient of the reaction components. Using 1 M ammonium sulfate solution as the aqueous phase of the extraction column, the yield of ( R )-MPTL (ee>99%) was enhanced to 40%.

Computer-aided molecular modeling of the enantioselectivity of Pseudomonas cepacia lipase toward γ- and δ-lactones

Abstract Computer-aided molecular modeling was performed to investigate the experimentally determined enantioselectivities of Pseudomonas cepacia lipase (PCL) toward various saturated γ- and δ-lactones. Experimental data indicated that PCL preferentially hydrolyzes the ( R )-enantiomers of both types of substrates. Interactions between the non-polar aliphatic alkyl chain of the ( S )-enantiomers and the polar side chain of residue Y29 were identified to mediate enantioselectivity. Upon binding, the tyrosine was displaced, thus initiating a cascade of local geometry changes which led to the breakdown of the essential H-bond network at the active site H286. The lactone ring of the ( S )-δ-enantiomers further added to this process, since it was forced into an unfavorable position by repulsion from Y29, directly affecting the position of H286. In contrast, the respective ( R )-enantiomers fit without distorting side chains essential for catalysis in the binding pocket of PCL. In δ-lactones, the stereocenter was located close to the imidazole ring of H286, suggesting a more intense interaction with H286 as compared to γ-lactones. The length of the aliphatic chain adjacent to the stereocenter also affected the enantiopreference toward hydrolysis of δ-lactones, while for γ-lactones, the enantioselectivity did not significantly change with increasing alkyl chain length. In the cases of ( S )-δ-octa- and ( S )-δ-nonalactone, two alternative possible binding modes were examined, indicating that the respective substrate resolutions led to poor enantioselectivity as compared to the longer-chain δ-lactone substrates.

Pro-antibiotic Substrates for the Identification of Enantioselective Hydrolases

A new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2–phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (Eapp>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

Kinetic Resolution of Racemic α‐Methyl‐β‐propiothiolactone by Lipase‐Catalyzed Hydrolysis

Kinetic resolution of racemic α‐methyl‐β‐propiothiolactone (rac‐MPTL) using lipases in organic solvent was studied. The lipase from Pseudomonas cepacia (PCL) showed the highest (S)‐enantioselectivity (E > 100), and cyclohexane containing 1% (v/v) buffer was identified as the best reaction medium for maintaining high enantioselectivity as well as high reaction rate. While the substrate inhibition was not observed up to 300 mM rac‐MPTL, severe product inhibition was observed even at 50 mM racemic 3‐mercapto‐α‐methyl propionic acid (rac‐MMPA), which made the use of high substrate concentration difficult. To overcome the product inhibition, the products, (R)‐MMPA, were neutralized by addition of a dilute basic solution. Although the resolution reaction proceeded further by the base titration, the enantioselectivity of the reaction decreased as a result of nonenantioselective hydrolysis of rac‐MPTL in the basic solution. Under these conditions, 200 mM rac‐MPTL was successfully resolved to above 95% eeS with 53% conversion.

Computational Selection, Identification and Structural Analysis of ω-Aminotransferases with Various Substrate Specificities from the Genome Sequence of Mesorhizobium loti MAFF303099

ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appreared to be determined by residues positioned at the entrance region of the active site binding pockets.