NOVEL D-HYDANTOINASE SEPARATED FROM BACILLUS STEAROTHERMOPHILUS SD-1

摘要

PURPOSE: Provided is a gene of D-hydantoinase from Bacillus stearothermophilus SD-1 (KCTC 8551P) to manufacture proteins in a high yield. The D-hydantoinase is very useful in manufacturing D-amino acid used in drugs and agricultural chemicals etc. CONSTITUTION: A gene of D-hydantoinase is obtained by the following steps of: i) separating a gene that codes D-hydantoinase from Bacillus stearothermophilus SD-1 KCTC 8551P and cloning the gene with plasmid pBR32; ii) inserting a 3.1kb gene segment that codes D-hydantoinase into plasmid pUC18 to make recombinant plasmid pHBU183 and transforming E. coli with the plasmid to obtain fused protein; iii) to analyze the D-hydantoinase coding gene, cutting 3.1kb gene segment inserted into plasmid pHBU183 with restriction enzymes EcoRI and SacI, and linking new segments with plasmid pBluescript SK to make plasmid pHBMT183; iv) removing unnecessary DNA segments, cutting plasmid pHBMT183 with restriction enzyme EcoRI, and recovering DNA segments with Exonuclease III; v) linking recovered segments with pBluescript SK and inserting the plasmid into E. coli XL1-Blue for base analysis; vi) using the plasmid as a template to determine base sequences with primers T3 and T7.