신종식 교수

Inspired by kinesin movement along a microtubule, we demonstrate a processive bipedal DNA walker. Powered by externally controlled DNA fuel strands, the walker locomotes with a 5 nm stride by advancing the trailing foot to the lead at each step. Real-time monitoring of specific bidirectional walker movement is achieved via multiplexed fluorescence quenching.

The asymmetric synthesis of chiral amines using prochiral ketones was carried out with (S)-specific ω-transaminase (ω-TA) from Vibrio fluvialis JS17. This reaction is inhibited severely by both products, (S)-amine and deaminated ketone. In addition, thermodynamic equilibrium strongly favored the reverse reaction. l-Alanine proved to be the best amino donor based on easy removal of the products. Optimal pH of the reactions with both whole cells and cell-free extract was 7. Amino acceptor reactivi

The asymmetric synthesis of chiral amines using prochiral ketones was carried out with (S)-specific omega-transaminase (omega-TA) from Vibrio fluvialis JS17. This reaction is inhibited severely by both products, (S)-amine and deaminated ketone. In addition, thermodynamic equilibrium strongly favored the reverse reaction. L-Alanine proved to be the best amino donor based on easy removal of the products. Optimal pH of the reactions with both whole cells and cell-free extract was 7. Amino acceptor

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of tw

Two microorganisms showing high omicron-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-alpha-methylbenzylamine (alpha-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of omicron-transaminases were investigated. omicron-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of alpha-MBA and showed remarkable stab

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse react

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using

Isopropylamine is an ideal amino donor for reductive amination of carbonyl compounds by ω-transaminase (ω-TA) owing to its cheapness and high volatility of a ketone product. Here we developed asymmetric synthesis of unnatural amino acids via ω-TA-catalyzed amino group transfer between α-keto acids and isopropylamine.

Abstract A novel biocatalytic process for production of L ‐homoalanine from L ‐threonine has been developed using coupled enzyme reactions consisting of a threonine deaminase (TD) and an ω‐transaminase (ω‐TA). TD catalyzes the dehydration/deamination of L ‐threonine, leading to the generation of 2‐oxobutyrate which is asymmetrically converted to L ‐homoalanine via transamination with benzylamine executed by ω‐TA. To make up the coupled reaction system, we cloned and overexpressed a TD from Esche

Abstract Asymmetric reductive amination of ketones using ω‐transaminases (ω‐TAs) offers a promising alternative to the chemocatalytic synthesis of chiral amines. One fundamental challenge to the biocatalytic strategy is the very low enzyme activities for most ketones compared with native substrates (i.e., <1% relative to pyruvate). Here we have demonstrated that a single point mutation in the active site of the ( S )‐selective ω‐TA from Ochrobactrum anthropi could induce a remarkable accelera

Creation of enzyme variants displaying desirable catalytic performance usually necessitates tedious and time-consuming procedures for library generation and selection, which may be circumvented by a computational method based on a precise understanding of the reaction mechanism in the context of active site environment. Despite the great potential of ω-transaminases (ω-TAs) for asymmetric synthesis of chiral amines from ketones, it remains elusive why ω-TAs exhibit marginal activities for most k

Substrate inhibition is a common phenomenon in enzyme chemistry, which is observed only with a fast-reacting substrate enantiomer. We report here for the first time substrate inhibition of an enantioselective enzyme by both substrate enantiomers. The enantioselective substrate inhibition, i.e., different mode of inhibition by each substrate enantiomer, of (S)-specific omega-transaminase was found with various chiral amines. A kinetic model based on ping-pong bi-bi mechanism has been developed an

Fabricating a nanostructure capable of reversibly patterning molecules is a fundamental goal within nanotechnology, underlying diverse processes such as information storage, scaffold-assisted assembly, and molecular transport. Here, we describe a DNA scaffold supporting a one-dimensional array of independently and reversibly addressable sites at 7 nm spacing. As a proof-of-concept, we demonstrate robust functioning of the device as rewritable memory. The bit state of each address is controlled b