In this study, a series of chiral lactams were successfully prepared by one-pot two-step reactions. It included a transamination reaction catalyzed by a ω -transaminase from Bacillus megaterium ( ω -TA Bm ), and subsequent spontaneous cyclisation. The different amine donors were firstly screened with ethyl levulinate as an amine acceptor, and both the excellent conversion and enantiomeric excess ( e.e. p ) of >99% could be achieved with 4-bromophenylethylamine as an amine donor, however, the long reaction time of 24 h was unfavorable for its application. After the amino acid residue Thr120 situated in the small pocket was mutated to Ser, the T120S variant could exhibit 10.6-fold higher activity comparing to that of wild type, and achieve the reaction with both conversion and e.e. p of >99% within 3 h. The T120S variant could also be applied to prepare the other chiral γ- and δ-lactams with excellent conversion and e.e. p . Furthermore, the inclusion bodies of ω -TA Bm and its variants could be aggregated as “native-like structures” to continuously prepare the chiral lactam for 18 h. The aim of the study is to provide a simply and efficiently biocatalytic route for preparing chiral lactams.
The alkaline protease fromBacillus licheniformisstrain 2709 (AprE 2709) is widely used in Chinese industries but faces stability challenges under high-temperature conditions. This study employed molecular modeling and mutagenesis to identify Asn residues at positions 61, 160, and 211 as key sites affecting the stability of AprE 2709. By leveraging the additive and cooperative effects of mutations, the mutant enzyme AprE 2709 (N61G/N160G/N211G) was engineered, exhibiting enhanced thermostability and catalytic activity. The mutant demonstrated a 2.89-fold increase in half-life at 60 °C and a 1.56-fold improvement in catalytic efficiency compared to the wild-type enzyme. Structural analysis revealed that the improved thermostability was due to altered electrostatic interactions and strengthened hydrophobic contacts. Targeting Asn residues prone to deamidation presents a promising strategy for improving protein heat tolerance. These findings not only enhance our understanding of enzyme stability but also lay a foundation for future research aimed at optimizing alkaline proteases for diverse industrial applications, particularly in high-temperature processes.
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