|
KMID : 1094720150200030439
|
|
Biotechnology and Bioprocess Engineering
2015 Volume.20 No. 3 p.439 ~ p.446
|
|
|
Directed evolution and mutagenesis of lysine decarboxylase from Hafnia alvei AS1.1009 to improve its activity toward efficient cadaverine production
|
|
Wang Chen
Zhang Kai
Zhongjun Chen
Cai Heng
Honggui Wan
Ouyang Pingkai
|
|
|
Abstract
|
|
|
|
Lysine decarboxylase (LDC) exhibits a significant role in cadaverine (1,5-pentanediamine, diaminopentane) production from lysine. In this study, an error-prone PCR and DNA shuffling were performed to improve the activity of LDC from Hafnia alvei AS1.1009 for cadaverine production. A sensitive high-throughput screening strategy based on a pH indicator was established for directed evolution of LDC. Several improved mutants were obtained from directed evolution and LDCV147F/E583G mutant showed highest activity to catalyze lysine to cadaverine. This mutant showed 1.62-fold high LDC activity when compared to wild-type. Further analysis by site-directed mutagenesis reveled that only the mutant E583G was sufficient for higher catalytic activity. Wild type LDC and mutant LDCE583G were purified by an improved method including hydrophobic chromatography. These purified enzymes were characterized and the kinetic parameters were compared between LDCE583G and WT LDC. V max of LDCE583G was 1.32-fold higher than that of WT LDC. Use of LDCE583G mutant showed 1.48-fold improved productivity of cadaverine when compared to wild type. The concentration of cadaverine in E. coli JM109/pTrc99a-ldc2-41 was 63.9 g/L with conversion yield of 93.4% during 5 h. These results indicate that the mutation has positive effects on improving LDC activity and a potential candidate for cadaverine production.
|
|
|
KEYWORD
|
|
|
cadaverine, directed evolution, DNA shuffling, error-prone PCR, lysine decarboxylase
|
|
|
FullTexts / Linksout information
|
|
|
|
|
Listed journal information
|
|
 
|