Engineered disulfide bonds improve thermostability and activity of L‐isoleucine hydroxylase for efficient 4‐HIL production in Bacillus subtilis 168
| Autor: | Xian Zhang, Zhiming Rao, Meijuan Xu, Yang Taowei, Zhina Qiao, Shang-Tian Yang, Youxi Zhao, Shao Minglong, Mengfei Long, Hideki Nakanishi |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Environmental Engineering Mutant Bioengineering Bacillus subtilis 01 natural sciences law.invention 03 medical and health sciences 4‐hydroxyisoleucine Biotransformation law Bacillus subtilis 168 010608 biotechnology Research Articles 030304 developmental biology Thermostability 0303 health sciences biology Chemistry Rational design Wild type Protein engineering biology.organism_classification l‐isoleucine hydroxylase molecular dynamics simulation Biochemistry Recombinant DNA disulfide bond Research Article Biotechnology |
| Zdroj: | Engineering in Life Sciences |
| ISSN: | 1618-2863 1618-0240 |
| DOI: | 10.1002/elsc.201900090 |
| Popis: | 4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l‐Isoleucine hydroxylase specifically converts l‐Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐ido T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases. |
| Databáze: | OpenAIRE |
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