多级超滤及亲和超滤法分离纯化NADH工艺的研究

Study on the Isolation and Purification of NADH by Multi-ultrafiltration and Affinity Ultrafiltration

研究出一种经济、高效、简便且容易放大的提取、分离和纯化辅酶NADH的工艺条件并进行优化。使用化学渗透法处理啤酒酵母细胞Saccharomyces cerevisiae粗提NADH,多级超滤及亲和超滤法进一步分离和纯化NADH。选用酵母醇脱氢酶(YADH)作为亲和配体,在pH值8.0,离子强度为0.1 mol/L时,用YADH和细胞渗透液中的NADH亲和。使用MWCO=30 000的滤膜超滤。结果:NADH-YADH复合物留在截留液中,从而与其它小分子物质分离。改变pH值和离子强度,NADH-YADH复合物解离,通过MWCO=1 000的滤膜超滤,NADH分离到滤过液中,YADH作为截留而回收,回收率达93%,活性损失15%。试验表明使用化学渗透法处理酵母细胞,用亲和超滤纯化可获得高的NADH产率。和以前的方法相比具有简单、经济、省时、高效等特点,且容易放大。

The purpose is to find and optimize a technique which is simple, inexpensive and high-yield, for isolation and purification of NADH. Saccharomyces cerevisiae cells were treated by chemical permeabilization to release NADH. Isolation and purification of NADH were conducled by multi-ultrafiltration and affinity ultrafiltration. The NADH was separated by binding to YADH at pH 8. 0, and with ionic strength 0. 1 mol/L. The NADH-YADH complex was separated from other molecules by ultrafiltration using membrane of which the molecular weight cut-off （MWCO） was 30000 and the binary complex was retained. The NADH-YADH complex was cleaved by changing pH and ionic strength. Ultrafiltration （MWCO=1000）was carried out then to isolate NADH as ultrafiltrate. YADH was recovered as ultraretention with 95 % recovery and 15 % loss in activity. It was found that treating Saccharomyces cerevisiae cells by chemical permeabilization and purification by affinity ultrafiltration gives NADH with high yields. The technique is simple, inexpensive, and less time-consuming. It＇s easier to be scaled up than other methods, too.