期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 23, 页码 -出版社
MDPI
DOI: 10.3390/ijms232315318
关键词
monomeric isocitrate dehydrogenase; recombinant protein; hyper-level expression; NADPH-regeneration system
资金
- Marine Biotechnology Program - Ministry of Oceans and Fisheries, Korea [2021C300]
- Ministry of Oceans and Fisheries, Korea
- Commercializations Promotion Agency for R&D Outcomes (COMPA) - Korea government (MSIT)
- National Research Foundation (NRF) of Korea - Ministry of Education, Science and Technology (MEST) of Korea
- [20170305]
- Science & Technology Job Promotion Agency, Republic of Korea [2021C300] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
The enzymatic transformation of chemicals using NADPH-dependent hydroxylase has gained attention, but its industrial application is limited due to high cost. Enzymatic NADPH-regeneration systems are developed as alternatives and have potential in various fields. Two recombinant isocitrate dehydrogenases (IDHs) were compared and found to function well with NADPH consumption systems. These enzymes could be used as alternatives for NADPH regeneration and have potential as fusion partners with NADPH-dependent enzymes.
The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP(+) when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.
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