Purification of industrial hydantoinase in one chromatographic step without affinity tag.
Hydantoinase is used in industry as a biocatalyst for the production of optically pure D- or L-amino acids. Previously, homogeneous hydantoinase was obtained by multi-chromatographic purification procedures. Here, we reported a process that contained only a single chromatographic step to purify a recombinant hydantoinase to homogeneity. Hydantoinase from Agrobacterium radiobacter NRRL B11291 was expressed in Escherichia coli. The recombinant enzyme was purified following heat treatments, high concentration alcohol precipitation, and chelating Sephacel chromatography. The recombinant hydantoinase did not contain any affinity tags from the plasmid. This simplified procedure provided a convenient way to obtain hydantoinase in high yield (71%) and high purity. It should be very useful for further industrial application and for the study of the structure-function of hydantoinase.
Huang
Cheng-Yang
CY
Department of Biological Science and Technology, College of Science, National Chiao Tung University, 75 Po-Ai Street, Hsinchu 30050, Taiwan, ROC.
Chao
Yun-Peng
YP
Yang
Yuh-Shyong
YS
D000438:Alcohols; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D000438:Alcohols; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase