三嗪染料修饰曲通X-100亲和分离苹果酸脱氢酶

摘要

三嗪染料(Cibacmn blue F3G-A)的蒽醌部分结构类似于腺嘌呤,可以用于亲和分离以NAD+(NADP+)和FAD为辅酶的脱氢酶.三嗪染料通过亲核取代反应修饰曲通X-100,形成的三嗪染料-曲通X-100与成相聚合物吐温80、磷酸钾盐构成液-固亲和萃取体系.从猪心肌匀浆液纯化苹果酸脱氢酶的条件:三嗪染料-曲通X-100浓度为1.2％(W/V),吐温舳浓度9％(V/V),猪心肌匀浆液0.25 mL(2.5％),pH8.0,加入成相磷酸钾终浓度1.6 mol/L,形成液固两相,固相酶活力平均收得率为81.01％,纯化倍数为4.93.固相30℃保温溶解,加入成相盐磷酸钾,终浓度为0.7mol/L,二次成相.低盐浓度条件下,近80％的酶反萃回液相,简化了苹果酸脱氢酶后续加工工艺.%Due to the structure of anthraquinone of triazine-dye( Cibacron blue F3G-A,short as Cb)similar as adenine.Cb could be used to purify enzymes whose coenzyme was NAD+( NADP+)or FAD with affinity partitioning method. Triton X-100 modified by Cb through nucleophilic substitution reaction,Tween 80 and potassium phosphate constituted the liquid-solid affinity partitioning system. The optimal malate dehydrogenase( short as MDH) purification system from porcine cardiac muscle homogenate condition consists of triton X-100 modified by Cb 1.2% ( W/V) .phase-forming polymer Tween 80 9% ( V/V) .porcine cardiac muscle homogenate 0.25 mL(2.5% ) .and the final concentration of phase-forming potassium phosphate 1.6 mol/L.pH 8.0. The enzyme recovery in solid-state(Tween 80 phase)was 81.01% .and the purification fold was 4.93. After the solid-state dissolved by incubation at 30℃ ,and then the concentration of potassium phosphate adjusted to 0.7 mol/L, the dissolved solid-state re-forms the liquid-solid two phase system with the enzyme recovery of near 80%in salt aqueous phase,which simplified the following processing of MDH.