The effective synthesis ofN-benzyl cyclo-tertiary amines using imine reductase, key components in natural products and pharmaceutical synthesis, is a green approach. Traditional methods faced challenges with enzyme activity and selectivity. This study focused on enhancingMesorhizobiumimine reductase (MesIRED) for betterN-benzyl cyclo-tertiary amine production. Through alanine scanning and consensus mutation, 12 single-siteMesIRED mutants were identified from 23 candidates, showing improved conversion ofN-benzylpyrrolidine andN-benzylpiperidine. Notably, mutants from I177, V212, I213, and A241 significantly boosted conversions. The best-performing mutant forN-benzylpyrrolidine,MesIREDV212A/I213V(M1), increased conversion from 23.7% to 74.3%. ForN-benzylpiperidine,MesIREDV212A/I177A/A241I(M2) enhanced conversion from 22.8% to 66.8%. Tunnel analysis revealed M1 and M2 have more efficient tunnels for larger product movement compared to wild-typeMesIRED. Using recombinantE. colicoexpressingMesIRED and glucose dehydrogenase (GDH), high conversions were achieved: 75.1% forN-benzylpyrrolidine (M1) and 88.8% forN-benzylpiperidine (M2). A preparative experiment resulted in 86.2% conversion and 60.2% yield forN-benzylpiperidine. This research offers an efficient method for engineering IRED, significantly improving conversion and selectivity forN-benzyl cyclo-tertiary amines, aiding drug synthesis and providing insights into rational design of other enzymes.Keywords:imine reductase;N-benzyl cyclo-tertiary amines;alanine scanning;consensus mutation;whole-cell biocatalysis;coexpression
Kemin Lv,Xiaozhou Li,Kequan Chen,Bin Wu,Bingfang He,Gerhard Schenk
Processive endoglucanase is a typical bifunctional biocatalyst for cellulose degradation. A GH5 processive endoglucanase from Bacillus subtilis BS-5 was previously identified and shown to exhibit highly efficient catalytic performance. To further augment its catalytic efficiency, both consensus mutagenesis and loop engineering were applied. Compared to the wild-type enzyme, a variant (M3-1) with the four point mutations, i.e., K91I, A198T, Q237D, and V240P, exhibits an 8.5- and 4.8-fold increase in catalytic efficiency toward the soluble substrate carboxymethyl cellulose-Na (CMC) and the insoluble phosphoric acid-swollen cellulose (PASC), respectively. Molecular dynamics simulations were employed to elucidate the conformational changes that led to the enhanced catalytic efficiency. Structural superpositions suggest that the mutations cause a swing in loop 230–241, which in turn affects enzyme–substrate affinity and recognition. Residues K91 and A198 are located distal from the active site. The mutations K91I and A198T influence key amino acids within the active pocket through residue interaction networks in the protein. Furthermore, dynamic cross-correlation matrices (DCCMs) indicate that variant M3-1 possesses a conformation that is more favorable than that of the wild-type enzyme, promoting an increased frequency of interactions between active site residues and substrate molecules and thereby enhancing catalytic efficiency. The combined results provide valuable insights into the rational design and engineering of processive endoglucanases and other glycoside hydrolases, enabling the development of improved enzymatic catalysts for biotechnology applications.
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