Synthesis of α-Ketoisocaproate Through Substrate Coupling Reaction Catalyzed by Leucine Dehydrogenase†

doi:10.7503/cjcu20180698

Abstract

Abstract:

In order to shift the balance of oxidative deamination, the substrate coupling reaction system catalyzed byleucine dehydrogenase(LeuDH) was constructed. The α-KIC and L-2-aminobutyrate with high valuable were synthesized. Meanwhile, the coenzyme was efficiently regenerated. Based on the studies of the substrates specificity and kinetic parameters of LeuDH, the efficiency of substrate coupling reaction was compared using a series of ketonic acids. The result showed that 2-oxobutyrate could be used as the best coupling substrate. The yield of α-KIC was improved from 2.75% of the single-step oxidation reaction to 66.82% of the coupling reaction. The reaction conditions were optimized, including pH values, concentrations of substrate, N $H 4 +$ and coenzyme. The yield of α-KIC and the total turnover number(TTN) of ketonic acid reached 83.25% and 5.88×10⁵, respectively. By adapting the molar ratios of the L-leucine and 2-oxobutyrate, the yield of α-KIC was increased to 92.74%. The substrate coupling reaction system was suggested to be the effective way for the production of α-KIC.

Key words: Leucine dehydrogenase, Substrate coupling, Coenzyme regeneration, α-Ketoisocaprate;, L-2-Aminobutyrate

CLC Number:

O621.25⁺4

TrendMD:

FENG Xian,MU Xiaoqing,NIE Yao,XU Yan. Synthesis of α-Ketoisocaproate Through Substrate Coupling Reaction Catalyzed by Leucine Dehydrogenase^†[J]. Chem. J. Chinese Universities, 2019, 40(4): 698.

Figures/Tables 11

Scheme 1 Schematic illustration of substrate coupling reaction catalyzed by LeuDH for production of α-KIC and L-2-aminobutyrate

Fig.1 Time curves of oxidation of L-leucine and reduction of α-KIC catalyzed by LeuDHAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5). c(α-KIC)=10 mmol/L, c(L-Leucine)=10 mmol/L.

Table 1 Enzymatic oxidation and reduction properties of LeuDH for L-leucine and α-KIC

Substrate	Structural formula	Specific activity/ (U·mg^-1)	V_m/ (mol·min^-1·mg^-1)	K_m/ (mmol·L^-1)	(k_cat/K_m)/ (L·mmol^-1·s^-1)
α-KIC		11.14	695.4	2.38	26.14
L-Leucine		1.66	2.75	24.56	0.012

Fig.2 Effects of coupling substrates to the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising amino acid(10 mmol/L), ketonic acid(10 mmol/L) and NAD+(0.2 mmol/L). a. Trimethylpyruvic acid; b. 3-methyl-2-oxopentanoic acid; c. 2-oxobutyrate; d. pyruvic acid.

Table 2 Enzymatic oxidation and reduction properties of LeuDH for different kinds of amino acids and ketonic acids

Substrate			Yield(%)
2-Oxobutyrate	21.32	64.22	73.73
L-2-aminobutyrate	0.61	0.01	1.14
α-KIC	52.77	143.69	66.82
L-valine	0.71	0.03	2.94
α-Ketoisocaproate	11.14	26.14	70.36
L-leucine	1.66	0.01	2.09
Trimethylpyruvic acid	7.99	1.84	65.46
L-tert-leucine	——	——	——

Fig.3 Effects of initial NAD+ concentration on TTN and the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+.

Fig.4 Effects of NH4+ concentration on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+(0.01 μmol/L).

Fig.5 Effects of pH value on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+(0.01 μmol/L).

Fig.6 Effects of substrate concentration on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine, 2-oxobutyrate and NAD+(0.01 μmol/L).

Fig.7 Time curves of the substrate coupling reaction systemAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(100 mmol/L), 2-oxobutyrate(100 mmol/L) and NAD+ (0.01 μmol/L).

Fig.8 Effects of the molar ratios of L-leucine and 2-oxobutyrate on the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate and NAD+(0.01 μmol/L).

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