High Purity Human Insulin Preparation By Enzymeassisted Semisynthesis, Separation And Purification

Insulin is the main hormone reducing blood glucose concentrations in vivo. It is a specific drug for treating Diabetes Mellitus(DM). The immunogenicity of insulin is closely related with its purity and source. The immunogenicity can result in a series of side effects. It has some disadvantages to the control of DM and the occuration, development and prognosis of its syndrome. In order to eliminate the immunogenicity of insulin radically, high purity human insulin needs offer. Biosynthesis and semisynthesis are the two feasible ways for human insulin preparation, and the latter is a convenient, effective and economic way. In this paper, a study on preparation and purification of high purity human insulin by enzyme-assisted transpeptidation reaction is reported.The only difference between porcine insulin and human insulin is the 30~th amino acid of B-chain, porcine insulin is alanine, and the latter is threonine. Porcine insulin was converted into human insulin ester with threonine-ester through transpeptidation catalyzed by enzyme. Then human insulin ester was separated and purified through negative ion column. Human insulin could be get after ester protection was wiped away. This human insulin was identified by the following methods: PAGE, SDS-PAGE showed the single lane homogeneous with standard human insulin ; Analysis human insulin ester and human insulin of C-terminal showed the corresponding amino acid was threonine-ester and threonine; The product was found to be homogeneous with standard human insulin by the analysis of HPLC. Those results proved this protein was human insulin, the yield was 38.4%, and the purity reached to 97%. In addition immobilized enzyme was prepared, and we studied the immobilized enzyme-catalyzed transpeptidation. Utilizing the difference hydrophobility, hydrophobic column was firstly used to separating human insulin ester, it was effective. At last potency of the human insulin sample was measured by rat blood glucose concentration method. The value was 20.6u/mg.