Efficient Production of Enantiopure d-Lysine from l-Lysine by a Two-Enzyme Cascade System

Abstract

Abstract:The microbial production of D-lysine has been of great interest as a medicinal raw material. Here, a two-step process for D-lysine production from L-lysine by the successive microbial racemization and asymmetric degradation with lysine racemase and decarboxylase was developed. The whole-cell activities of engineered Escherichia coli expressing racemases from the strains Proteus mirabilis (LYR) and Lactobacillus paracasei (AAR) were first investigated comparatively. When the strain BL21-LYR with higher racemization activity was employed, L-lysine was rapidly racemized to give DL-lysine, and the D-lysine yield was approximately 48% after 0.5 h. Next, L-lysine was selectively catabolized to generate cadaverine by lysine decarboxylase. The comparative analysis of the decarboxylation activities of resting whole cells, permeabilized cells, and crude enzyme revealed that the crude enzyme was the best biocatalyst for enantiopure D-lysine production. The reaction temperature, pH, metal ion additive, and pyridoxal 5 -phosphate content of this two-step production process were subsequently optimized. Under optimal conditions, 750.7 mmol/L D-lysine was finally obtained from 1710 mmol/L L-lysine after 1 h of racemization reaction and 0.5 h of decarboxylation reaction. D-lysine yield could reach 48.8% with enantiomeric excess (ee) ≥ 99%.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

“…S3 ). The observation that this activity rate is 30 times smaller than that of E. coli LdcI at the same conditions 31 , 32 may indicate that, similarly to LdcI and related enzymes 33 , 34 , optimal LdcF activity is pH, salt, and temperature-dependent. We were able to determine the structure of F. novicida LdcF from X-ray diffraction data collected to a resolution of 3.4 Å (Supplementary Table S1 and Supplementary Fig.…”

Sustainable and Continuous Synthesis of Enantiopure l‐Amino Acids by Using a Versatile Immobilised Multienzyme System

“…So far, this analysis has not assessed biocatalyst fabrication, downstream processing, or economic issues that may further limit industrial implementation. Among the systems described in Table , the deracemisation of d,l ‐alanine proposed by the group of Asano and the stereoinversion of l ‐lysine described by Wang et al., result in the lowest E factors. Additionally, the strategy reported by Asano et al.…”

Enzymatic cascade systems for D-amino acid synthesis: progress and perspectives

“…Since many enzymes that have been reported to directly transfer D,L-amino acids to D-amino acids are rarely active towards lysine [49], Wang et al developed a strategy that employs enzymes that selectively degrade L-lysine from a mixture of D,L-lysine [30]. Because of the overcapacity of L-lysine in industry today wherein the annual output exceeds two million tons, L-lysine is promising as a starting material to produce D-amino acids.…”

“…More researchers have chosen to couple two or more enzymes, or even combine chemical catalysts with biocatalysts for the production of targets [26][27][28]. There are many examples of using multi-enzyme systems to synthesize D-amino acids, since the catalytic properties and advantages of various enzymes can be combined to produce D-amino acids with higher efficiency and stereoselectivity [15,[29][30][31].…”