Enhancement of the thermal and alkaline pH stability of Escherichia coli lysine decarboxylase for efficient cadaverine production
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To enhance the thermal and alkaline pH stability of the lysine decarboxylase from Escherichia coli (CadA) by engineering the decameric interface and explore its potential for industrial applications.
The mutant T88S was designed for improved structural stability by computational analysis. The optimal pH and temperature of T88S were 7.0 and 55 °C (5.5 and 50 °C for wild-type). T88S showed higher thermostability with a 2.9-fold increase in the half-life at 70 °C (from 11 to 32 min) and increased melting temperature (from 76 to 78 °C). Additionally, the specific activity and pH stability (residual activity after 10 h incubation) of T88S at pH 8.0 were increased to 164 U/mg and 78% (58 U/mg and 57% for wild-type). The productivity of cadaverine with T88S (284 g l-lysine L−1 and 5 g DCW L−1) was 40 g L−1 h−1, in contrast to 28 g L−1 h−1 with wild-type.
The mutant T88S showed high thermostability, pH stability, and activity at alkaline pH, indicating that this mutant is a promising biocatalyst for industrial production of cadaverine.
KeywordsBiotransformation Cadaverine Lysine decarboxylase Multimeric interface Stability
We are grateful for the financial support from the Tianjin Municipal City, the first “Special Support Plan for Talents Development” and “High-level Innovation and Entrepreneurship Team”, National Natural Science Foundation of China (Nos. 21606251, 31370113, and 31370829), Science and Technology Foundation for Selected Overseas Chinese Scholar of Tianjin (2017), and Science and Technology Project of Tianjin (15PTCYSY00020).
Supplementary Table 1—The values of the folding free energy changes (∆∆G) predicted by iStable and CUPSAT servers.
Supplementary Table 2—Primers used in this study.
Supplementary Fig. 1—Differential scanning calorimeter traces of the purified LDCs (0.2 mg/mL) with 100 mM potassium phosphate buffer (0.1 mM PLP, 100 mM NaCl, pH 7.4) at 1 °C/min. Black curve: wild-type; Red curve: T88S. The melting temperature for wild-type CadA and T88S is 76 and 78 °C, respectively.
Supplementary Fig. 2—SDS-PAGE (12%) of recombinant wild-type and mutant enzymes expressed in E. coli BL21 (DE3). Lane M, protein marker; lane 1, wild-type; lane 2, T88D; lane 3, T88F; lane 4, T88S; lane 5, T88N; lane 6, T88P; lane 7, T88Q. A: soluble fraction; B: insoluble fraction.
Supplementary Fig. 3—Thermal inactivation of CadA wild-type and mutants at 70 °C. The residual activities of crude enzymes were measured every 10 min at pH 8.0. The original activity before pre-incubation was taken to be 100% (95 ± 3.1, 105 ± 5.2, 140 ± 3.5, and 175 ± 6.5 U/mg for wild-type, T88D, T88F, and T88S, respectively). The data are presented as mean ± standard deviation (SD) from three independent experiments.
Supplementary Fig. 4—Crystal structure of CadA wild-type (PDB: 3N75) and predicted structures of variants. A: wild-type; B: T88S; C: T88F; D: T88D. The predicted structures of variants were generated using YASARA software (http://www.yasara.org/) as the following steps: the mutated site was swapped with the specified residue; the side-chain of the mutated residue was optimized using side-chains with rotamer library (SCWRL) method. The adjacent monomers were colored in green or light blue.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Hong EY, Lee SG, Park BJ, Lee JM, Yun H, Kim BG (2017) 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. Biotech J. https://doi.org/10.1002/biot.201700278 Google Scholar