Feng-li Zhou, Xiaoqing Mu, Y. Nie
Apr 30, 2021
Citations
0
Influential Citations
11
Citations
Journal
Applied Microbiology and Biotechnology
Abstract
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 kcat/Km 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.