title: New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates.
abstract: To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Guangyue
authors.initials: G
authors.lastname: Li
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Peiyuan
authors.initials: P
authors.lastname: Yao
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Peiqian
authors.initials: P
authors.lastname: Cong
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Ren
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.|Departments of Chemistry and Microbiology &Immunology, McGill University, Montreal, Quebec, H3A2B4, Canada.
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308, PR China.
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D000588:Amines / Q000378:metabolism*; D007536:Isomerism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000588:Amines; D050505:Mutant Proteins; D011994:Recombinant Proteins; C019962:cyclohexylamine oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors