title: In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship.
abstract: In silico identification for enzymes having desired functions is attractive because there is a possibility that numerous desirable enzymes have been deposited in databases. In this study, α-amino-ε-caprolactam (ACL) racemases were searched from the NCBI protein database. Four hundred thirteen fold-type I pyridoxal 5'-phosphate-dependent enzymes which are considered to contain sequences of ACL racemase were firstly obtained by submitting the sequence of ACL racemase from Achromobacter obae to the database. By identifying Lys241 as a key amino acid residue, 13 candidates for ACL racemase were selected. Then, putative ACL racemase genes were synthesized as codon-optimized sequences for expression in Escherichia coli. They were subcloned and expressed in E. coli BL21 and underwent His-tag purification. ACL and amino acid amide racemizing activities were detected among ten of the candidates. The locus tags Oant_4493, Smed_5339, and CSE45_2055 derived from Ochrobactrum anthropi ATCC49188, Sinorhizobium medicae WSM 419, and Citreicella sp. SE45, respectively, showed higher racemization activity against D- and L-ACLs rather than that of ACL racemase from A. obae. Our results demonstrate that the newly discovered ACL racemases were unique from ACL racemase from A. obae and might be useful for applications in dynamic kinetic resolution for D- or L-amino acid production.
authors.affiliation: Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat-Yai, 90112, Thailand.
authors.forename: Wisarut
authors.initials: W
authors.lastname: Payoungkiattikun
authors.forename: Seiji
authors.initials: S
authors.lastname: Okazaki
authors.forename: Shogo
authors.initials: S
authors.lastname: Nakano
authors.forename: Atsutoshi
authors.initials: A
authors.lastname: Ina
authors.forename: Aran
authors.initials: A
authors.lastname: H-Kittikun
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D020134:Catalytic Domain; D003198:Computer Simulation; D008969:Molecular Sequence Data; D020621:Ochrobactrum anthropi / Q000201:enzymology; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D000591:Amino Acid Isomerases; C036861:aminocaprolactam racemase