Kadima T A, Jensen S E, Pickard M A
Department of Microbiology, University of Alberta, Edmonton, Canada.
J Ind Microbiol. 1995 Jan;14(1):35-40. doi: 10.1007/BF01570064.
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV)-synthetase from Streptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45-65% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g-1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.
在能够重复使用酶的条件下，对来自棒状链霉菌的δ-（L-α-氨基己二酰基）-L-半胱氨酰-D-缬氨酸（ACV）合成酶进行了研究。将ACV合成酶与DEAE-三丙烯酸树脂和氨丙基玻璃偶联，得到的固定化酶产物几乎没有或没有催化活性。然而，通过将部分纯化的ACV合成酶物理限制在超滤池中设计了一个酶反应器。该系统在较低浓度的ATP下受到磷酸烯醇丙酮酸的刺激，而纯化酶未观察到这种效应。在半连续条件下，限制性底物半胱氨酸向ACV的转化率高达30%。对反应产物作为潜在抑制剂进行了研究：AMP的抑制作用最强，但只有在使用浓度超过反应混合物中产生的浓度时才会出现这种情况。在氮气气氛下，产物和酶的稳定性都大大提高，在室温下24小时内使用五次后，酶保留了其初始活性的45-65%。根据这些数据推断，在此期间，1.3克部分纯化的酶（0.13 U g-1）在1升反应混合物中能够产生411毫克的ACV。
