Immobilized metal ion affinity chromatography in enzyme fractionation.

Ribitol dehydrogenase from Mycobacterium butyricum and alpha-mannosidase from Lupinus luteus seedlings were fractionated by the immobilized metal ion (Cu2+ or Zn2+) affinity chromatography (IMAC) on iminodiacetic acid coupled to Sepharose 6B. In a single step, ribitol dehydrogenase was purified 10-12 fold with the recovery above 80% when using Zn(2+)-Sepharose 6B as the sorbent and decreasing linear gradient of pH from 7 to 4. In the same conditions purification of alpha-mannosidase was less effective (2-3 fold, recovery 60-70%).