In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship
- 6 May 2015
- journal article
- research article
- Published by Springer Science and Business Media LLC in Applied Biochemistry and Biotechnology
- Vol. 176 (5), 1303-1314
- https://doi.org/10.1007/s12010-015-1647-6
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.Keywords
This publication has 33 references indexed in Scilit:
- Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applicationsBiotechnology Advances, 2015
- Strategies for the discovery and engineering of enzymes for biocatalysisCurrent Opinion in Chemical Biology, 2013
- Engineering the third wave of biocatalysisNature, 2012
- Introduction – Principles and Historical Landmarks of Enzyme Catalysis in Organic SynthesisPublished by Wiley ,2012
- Rational assignment of key motifs for function guides in silico enzyme identificationNature Chemical Biology, 2010
- Protein function annotation by homology-based inferenceGenome Biology, 2009
- Predicting protein function from sequence and structureNature Reviews Molecular Cell Biology, 2007
- Can sequence determine function?Genome Biology, 2000
- Enzymatic conversion of DL-.ALPHA.-amino-.EPSILON.-caprolactam into L-lysine. III. Bacterial racemization of .ALPHA.-amino-.EPSILON.-caprolactam.Agricultural and Biological Chemistry, 1977
- Enzymatic conversion of DL-.ALPHA.-amino-.EPSILON.-caprolactam into L-lysine. VI. Conversion of D- and DL-.ALPHA.-amino-.EPSILON.-caprolactam into L-lysine using both yeast cells and bacterial cells.Agricultural and Biological Chemistry, 1977