ω-Transaminase from Ochrobactrum anthropi Is Devoid of Substrate and Product Inhibitions

Abstract

-Transaminases display complicated inhibitions by ketone products and both enantiomers of amine substrates. Here, we report the first example of -transaminase devoid of such inhibitions. Owing to the lack of enzyme inhibitions, the -transaminase from Ochrobactrum anthropi enabled efficient kinetic resolution of ␣-methylbenzylamine (500 mM) even without product removal.O mega-transaminase (-TA) catalyzes reversible transfer of an amino group between primary amines and carbonyl compounds which is mediated by pyridoxal 5=-phosphate (PLP) bound to the enzyme as a prosthetic group (1, 2). During the last decade, -TA has gained increasing attention for chiral amine production owing to a unique enzyme property enabling both oxidative deamination of an amine involving conversion of enzyme-bound PLP to pyridoxamine 5=-phosphate (PMP) and reductive amination of a carbonyl compound accompanied by regeneration of the PLP form of enzyme (E-PLP) from the PMP form of enzyme (E-PMP), as shown in Fig. 1 (3-6). -TAs exhibit broad substrate specificity, a high turnover rate, stringent stereoselectivity, high enzyme stability, and no requirement for an external cofactor, all of which renders the enzyme suitable for industrial process development (2, 7).In the biocatalytic process design, complicated enzyme properties often limit efficient process operation (8, 9). Despite the enzyme properties of -TAs beneficial for industrial applications, severe product inhibition causing drastic reductions in the enzyme activity has been a major challenge to exploit the -TA reactions for industrial processes (3-5, 10-12). For example, -TA from Bacillus thuringiensis JS64 showed only 5% residual activity at 20 mM acetophenone in transamination between ␣-methylbenzylamine (␣-MBA) and pyruvate (10). Therefore, several reaction engineering approaches to alleviate the inhibition by removing the ketone product using solvent extraction were developed (10, 12). The product inhibition has been regarded as a general property of -TAs, and there have been no reports on the discovery of an -TA lacking the product inhibition (3-6, 13). Yun et al. reported that product inhibition of -TA from Vibrio fluvialis JS17 by aliphatic ketones can be attenuated by directed evolution (14). However, it remains challenging to completely eliminate the product inhibition behaviors, although such an inhibition-free enzyme is in high demand for cost-effective processes.In the previous studies, we cloned, overexpressed, and purified (S)-selective -TAs from Ochrobactrum anthropi (15) and Paracoccus denitrificans PD1222 (16). Briefly, Escherichia coli BL21(DE3) cells carrying a pET28a(ϩ) expression vector harboring the O. anthropi -TA or P. denitrificans -TA gene were cultivated in LB medium (typically 1 liter) and the His-tagged -TA was purified using a HisTrap HP column (GE Healthcare) followed by a HiTrap desalting column (GE Healthcare) as described elsewhere (15,16). When necessary, the purified enzyme solution was concentrated by an ultrafiltration kit (Ultracel-30) from Mi...

Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

“…[21] Initial rate data were fitted to aM ichaelis-Menten equation, and the K M and k cat values were calculated from the slopes and yi ntercepts of the double-reciprocal plots.R ate constants were determined from three independent initial rate measurements over the concentration ranges of substrate and cosubstrate listed in the Supporting Information, Table S6. HPLC analytes used in the initial rate measurements are also presented in Table S6.…”

Kinetic resolution of amines by (R)-selective omega-transaminase from Mycobacterium vanbaalenii

“…(i) Use of organic solvent (a biphasic reaction system) to extract ketone from aqueous media, (ii) enzyme reaction under reduced pressure to selectively eradicate the inhibitory volatile ketone, and (iii) enzymatic reduction of inhibitory ketone by dehydrogenase. Even though, an inhibitionfree enzyme is in high demand for cost-effective processes, it continues to remain difficult challenge [25]. Very recently, Shin group firstly reported (S)-selective v-TA from Ochrobactrum anthropi showed no product inhibition by ketone [25].…”

“…Even though, an inhibitionfree enzyme is in high demand for cost-effective processes, it continues to remain difficult challenge [25]. Very recently, Shin group firstly reported (S)-selective v-TA from Ochrobactrum anthropi showed no product inhibition by ketone [25]. v-TAMV is the first example of (R)-selective v-TA showing very low product inhibition by ketone (acetophenone) and this property of the enzyme is really beneficial for the kinetic resolution of high concentration of substrates.…”

Biocatalytic cascade reactions for asymmetric synthesis of aliphatic amino acids in a biphasic reaction system

“…The ketone product from the ω-TA reaction was known to exert a severe inhibition on the ω-TA activity [16]. For example, the product inhibition constant of PDTA for acetophenone was measured to 2.4 mM in the reaction between (S)-α-MBA and pyruvate [24]. Therefore, an organic solvent was overlaid on the aqueous phase where the cascade reaction was occurring, so the inhibitory ketone could be selectively extracted to reduce the product inhibition [16].…”