The Research of Two Affinity Mediums for the Purification of a Cholesterol Oxidase(COD) Expression in Escherichia coli

doi:10.3969/j.issn.1673-1689.2012.06.007

Home > Archive>Volume 31, Issue 6, 2012 >599-605. DOI:10.3969/j.issn.1673-1689.2012.06.007

The Research of Two Affinity Mediums for the Purification of a Cholesterol Oxidase(COD) Expression in Escherichia coli
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                        10.3969/j.issn.1673-1689.2012.06.007
                    
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In this study,affinity protocols were developed for the preparation of cholesterol oxidase (COD) from recombinant bacteria,a COD gene from Brevibacterium sp.(DQ345780) was expressed in Escherichia coli BL21(DE3),Riboflavin 5’-phosphate and 7-chroroalloxazine were chosen as the affinity ligands,and they were coupled with Sepharose CL 4B through spacers. After one step of affinity binding with the two mediums,the enzyme could be extracted with high purity.The yields of the enzyme purified with the two mediums were 9.4%and 9.9%,respectively,and the recoveries of typical cholesterol oxidase activity were 85.2%and 93.4%.The purified cholesterol oxidases were 98.0%and 97.5%pure with SDS-PAGE analysis. On SDS-PAGE gel,the enzyme was a single polypeptide with the mass of～50 kDa.The theoretical maximum absorption Qmax were 71.0 and 78.5 mg/g medium;the desorption constant K_d of the two mediums on the mediums were 12.8 and 7.3 g/g medium.

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XIN Yu, ZHANG Ling, ZHANG Yu-ran, CHEN Yi, TONG Yan-jun, WANG Wu. The Research of Two Affinity Mediums for the Purification of a Cholesterol Oxidase(COD) Expression in Escherichia coli[J]. Journal of Food Science and Biotechnology,2012,31(6):599-605.

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