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

Abstract

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 , that is, 76.7% conversion yield in 9.5 h, whereas the wild-type gave 119 g L , that is, 58.2% conversion yield in 9.5 h.

Circular permutation of a bacterial tyrosinase enables efficient polyphenol‐specific oxidation and quantitative preparation of orobol

“…The protein melting temperature (T M ) of purified tWT and circular permutants was measured using the previously mentioned method with slight modification (Hong et al, 2017). In brief, SYPRO Orange (Sigma-Aldrich; 5000×) excitation-emission wavelength was profiled, while raising the temperature (Huynh & Partch, 2015).…”

Development of an integrated process for the production of high‐purity cadaverine from lysine decarboxylase

“…Global warming and the shortage of fossil resources has driven the quest for a new bio‐economy . Cadaverine is a bio‐based platform chemical that plays an indispensable role in industry, medicine and agriculture . In particular, cadaverine is an important polymer monomer for polyamides and polyurethanes.…”

“…[1][2][3] Cadaverine is a bio-based platform chemical that plays an indispensable role in industry, medicine and agriculture. [4][5][6] In particular, cadaverine is an important polymer monomer for polyamides and polyurethanes. Due to the similar structure of cadaverine and 1,6-diaminohexane, cadaverine can replace 1,6-diaminohexane to synthesize bio-based polyamide materials such as nylon 54, nylon 56, nylon 510 and nylon 512.…”

RiSLnet: Rapid identification of smart mutant libraries using protein structure network. Application to thermal stability enhancement

“…CC depicts how a node is "closely" interacting with other nodes. CadA is a potential industrial enzyme that catalyzes the decarboxylation reaction of lysine to generate cadaverine, that is, diamine compound, but in order to increase the purity of the product during its purification, the enzyme reaction at high concentration of the lysine is desirable, which requires high temperature and long reaction time (Hong et al, 2017). Combining these network properties, we can find structurally and functionally important residues in a protein.…”

“…Moreover, we applied our method to increase the thermal stability of lysine decarboxylase (CadA) from Escherichia coli as a model enzyme. CadA is a potential industrial enzyme that catalyzes the decarboxylation reaction of lysine to generate cadaverine, that is, diamine compound, but in order to increase the purity of the product during its purification, the enzyme reaction at high concentration of the lysine is desirable, which requires high temperature and long reaction time (Hong et al, 2017). Thus, increasing the enzyme's stability has been a major issue in its application.…”