Eun Young Hong

Engineering Transaminase for Stability Enhancement and Site‐Specific Immobilization through Multiple Noncanonical Amino Acids Incorporation

In general, conventional enzyme engineering utilizes 20 canonical amino acids to alter and improve the functional properties of proteins such as stability, and activity. In this study, we utilized the noncanonical amino acid incorporation technique to enhance the functional properties of ω‐transaminase (ω‐TA). Herein, we enhanced the stability of ω‐TA by residue‐specific incorporation of (4R)‐fluoroproline [(4R)‐FP] and successfully immobilized onto chitosan or polystyrene (PS) beads with site‐specifically incorporated L‐3,4‐dihydroxyphenylalanine (DOPA) moiety. The immobilization of ω‐TAdopa and ω‐TAdp[(4R)‐FP] onto PS beads showed excellent reusability for 10 cycles in the kinetic resolution of chiral amines. Compared to the ω‐TAdopa, the ω‐TAdp[(4R)‐FP] immobilized onto PS beads exerted more stability that can serve as suitable biocatalyst for the asymmetric synthesis of chiral amines.

Identification of novel thermostable ω-transaminase and its application for enzymatic synthesis of chiral amines at high temperature

A novel thermostable ω-transaminase from Thermomicrobium roseum which showed broad substrate specificity and high enantioselectivity was identified, expressed and biochemically characterized. The advantage of this enzyme to remove volatile inhibitory by-products was demonstrated by performing asymmetric synthesis and kinetic resolution at high temperature.

Simultaneously enhancing the stability and catalytic activity of multimeric lysine decarboxylase CadA by engineering interface regions for enzymatic production of cadaverine at high concentration of lysine

Cadaverine (1,5-diaminopentane) is a major source of many industrial polyamides such as nylon and chelating agents. Currently, cadaverine is produced by the microbial fermentation of glucose to lysine, which is then decarboxylated by lysine decarboxylase (CadA). However, utilizing CadA for cadaverine production causes enzyme instability. In order to stabilize the CadA homo-decamer structure for in vitro decarboxylation reaction, mutants are designed. Of the four disulfide bond mutants in the multimeric interfacial region, B1 (F14C/K44C) showed a 216-folds increase in the half-life of CadA at 60 °C. On top of B1, another round of mutant screening is performed around F14C and K44C to generate B1/L7M/N8G, which is then examined for cadaverine production (2M lysine and 10% v/v of cell-extract at 50 °C). The reaction pH increased from 4.9 to 8.3, and the final titer of the mutant is 157 g L-1 , that is, 76.7% conversion yield in 9.5 h, whereas the wild-type gave 119 g L-1 , that is, 58.2% conversion yield in 9.5 h.

Rational engineering of ornithine decarboxylase with greater selectivity for ornithine over lysine through protein network analysis

Ornithine decarboxylase (ODC) converts C5 ornithine into C4 putrescine, a monomer for polyamide synthesis. However, ODC also has minor activity towards cell metabolite C6 lysine and yields C5 cadaverine. The accumulation of cadaverine in the reaction solution causes increase in the operational cost of subsequent distillation process for putrescine purification. Here, to increase ODC substrate specificity toward ornithine over lysine, molecular modelling and protein network analysis, specifically k-clique community analysis, around the substrate tunnel were performed. This resulted in a mutant with two-fold increase in substrate specificity (ornithine versus lysine) without losing its original activity towards ornithine (kcat/KM  = 61.5 s-1  mM-1), compared to the native enzyme. When this mutant was used for putrescine synthesis, 31.6 g/L putrescine (based on 51.5 g/L ornithine) titer was achieved, while 0.007 g/L (based on 2.57 g/L lysine) cadaverine was produced. This corresponds to four-fold decrease in cadaverine yield compared to the native ODC.

Circular permutation of a bacterial tyrosinase enables efficient polyphenol-specific oxidation and quantitative preparation of orobol: LEE et al.

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho‐diphenol, and subsequently to ortho‐quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho‐hydroxy‐polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally‐designed CP library, we introduced 18 new termini at the edge of each nine loops that link α‐helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3′‐hydroxy‐genistein) was quantitatively synthesized with 1.48 g/L, which was a 6‐fold higher yield of truncated wild‐type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.