Enhanced catalytic efficiency and coenzyme affinity of leucine dehydrogenase by comprehensive screening strategy for L-tert-leucine synthesis

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 catal...

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Veröffentlicht in:Applied microbiology and biotechnology 2021-05, Vol.105 (9), p.3625-3634
Hauptverfasser: Zhou, Feng, Mu, Xiaoqing, Nie, Yao, Xu, Yan
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Sprache:eng
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Zusammenfassung: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·h −1 ·L −1 ·g −1 , respectively. Key points • The constructed new high-throughput screening strategy takes into account the two indicators of catalytic efficiency and coenzyme affinity. • A more efficient leucine dehydrogenase (LeuDH) mutant (E24V/E116V) was identified. • E24V/E116V has potential for the industrial synthesis of L-tert-leucine. Graphical abstract
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-021-11323-w