Premium
Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli
Author(s) -
Tong Yanjun,
Xin Yu,
Yang Hailin,
Zhang Ling,
Tao Xiumei,
Xu Hui,
Wang Wu
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300302
Subject(s) - chemistry , chromatography , affinity chromatography , escherichia coli , sarcosine , d amino acid oxidase , sepharose , monomer , ethylenediamine , urate oxidase , enzyme , oxidase test , biochemistry , amino acid , glycine , organic chemistry , gene , polymer
An efficient affinity‐purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4‐Aminopyrrole‐2‐carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C 8 column and reducing SDS‐PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS‐PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ∼46 kDa. The desorption constant K d and theoretical maximum absorption Q max were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.