Research On The Purification And Characteristics Of The C-N Lyase From A Mold

3-Ketovalidoxylamine A C-N lyase is one of three key enzymes in the production of valienamine,which is an important pharmaceutical intermediate with broad use in the synthesis of some strongerα-glucosidase inhibitors.In this research,a validamycin A-degrading fungi strain,was isolated and identified as PenicilliumBase on morphology and 18S rDNA.In this study,conditions were varied to investigate valienamine production using this strain.We found the optimized conditions included （1）use of validamycin A as the sole carbon source,（2）use of（NH₄）₂SO₄ as the sole nitrogen source,（3）pH 7.0,（4）the optimal temperature is 30℃.Under these optimal conditions,an average of 0.85 g L-1 valienamine was obtained after fermentation for 4 d. 3-Ketovalidoxylamine A C-N lyase was purified to homogeneity by applying hydrophobic interaction and anion exchange,giving a yield of 32.2%and a specific activity of 12.89 U/mg.According to the results of SDS-PAGE,the purified enzyme has an estimated molecular mass of 21.6 kDa.We investigated the characteristic of the purified 3-Ketovalidoxylamine A C-N lyase.The purified enzyme exhibited a pH optimum of pH 7.5 and a temperature of 40℃.The result showed that the enzyme was poor in thermostabilization.After keeping in 50℃for 100min,the enzyme lost almost its activity.And the enzyme was seemed to be stable in pH range of 7.0-8.0.Inhibition of enzyme activity was observed in the presence of metal ions,such as Hg²⁺,Ni⁺,Pb²⁺and Sn²⁺, whereas Ca²⁺could promate it’s activity.