Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 105, Issue 9, Pages 3625-3634Publisher
SPRINGER
DOI: 10.1007/s00253-021-11323-w
Keywords
L-tert-leucine; Leucine dehydrogenase; Iterative saturation mutation; Error-prone PCR; High-throughput screening; Catalytic efficiency; Directed evolution
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Funding
- Postgraduate Research and Practice Innovation Program of Jiangsu Province [KYCX19_1831]
- National Natural Science Foundation of China [21336009, 21176103]
- National High Technology Research and Development Program of China [2015AA021004]
- Overseas Expertise Introduction Project for Discipline Innovation, China [111-2-06]
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The study focused on improving the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus through the screening of mutation libraries. A mutant E24V/E116V with enhanced affinity for TMP and NADH was selected, resulting in significantly increased turnover number and specific space-time conversion during the synthesis of L-tert-leucine.
L-tert-leucine (L-Tle) is widely used as vital chiral intermediate for pharmaceuticals and as chiral auxiliarie for organocatalysis. L-Tle is generally prepared via the asymmetric reduction of trimethylpyruvate (TMP) catalyzed by NAD(+)-dependent leucine dehydrogenase (LeuDH). To improve the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus, mutation libraries constructed by error-prone PCR and iterative saturation mutation were screened by two kinds of high-throughput methods. Compared with the wild type, the affinity of the selected mutant E24V/E116V for TMP and NADH increased by 7.7- and 2.8-fold, respectively. And the k(cat)/K-m of E24V/E116V on TMP was 5.4-fold higher than that of the wild type. A coupled reaction comprising LeuDH with glucose dehydrogenase of Bacillus amyloliquefaciens resulted in substrate inhibition at high TMP concentrations (0.5 M), which was overcome by batch-feeding of the TMP substrate. The total turnover number and specific space-time conversion of 0.57 M substrate increased to 11,400 and 22.8 mmol center dot h(-1)center dot L-1 center dot g(-1), respectively.
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