High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces clavuligerus.
High performance liquid chromatographic assay of cyclization activity in cell-free systems from Streptomyces clavuligerus.
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棒状链霉菌无细胞系统环化活性的高效液相色谱测定。
DOI：
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发表时间：
1982
期刊：
影响因子：
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通讯作者：
Saul Wolfe
中科院分区：
文献类型：
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作者：
Susan E. Jensen;Donald W. S. Westlake;Saul Wolfe
十三倍过量的二硫苏糖醇在使用棒状链霉菌的ACV环化测定系统中通常遇到的条件下将Δ-（L-α-氨基己二酰基）-L-半胱氨酰-D-缬氨酸（ACV）保持在其单体形式。本文建立了一种反相高效液相色谱（HPLC）分离ACV单体与异青霉素N、青霉素N及其它环化组分的方法，移动的相为5%甲醇-95%KH_2 PO_4（0.05 M，用浓H3 PO 4调节至pH 4.0;固定相：muBondapak-C18;流速：2 ml/分钟，持续5分钟，其余为3 ml/分钟;检测：220 nm）。在这些条件下，异青霉素N和青霉素N的真实样品的保留时间为5.25分钟，这与在环化反应混合物中观察到的新形成的物质的峰一致。环化反应混合物中异青霉素N和青霉素N[（异）青霉素N]的合并浓度与ACV单体的伴随减少密切相关。环化反应混合物，其中S. clavuligerus NRRL 3585是酶源，在1小时孵育期后含有浓度为43.3微克/ml的（异）青霉素N。环化反应混合物，其中盐沉淀的无细胞提取物从S。棒杆菌为酶源，含青霉素N 39.0 μ g/ml（异）。
A thirteen-fold excess of dithiothreitol maintains delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) in its monomeric form under the conditions normally encountered in an ACV cyclization assay system, using Streptomyces clavuligerus. A reversed phase high performance liquid chromatographic (HPLC) system which separates ACV monomer from isopenicillin N, penicillin N and from other cyclization assay components has been developed as follows; mobile phase: 5% methanol-95% KH2PO4 (0.05 M adjusted to pH 4.0 with concentrated H3PO4; stationary phase: muBondapak-C18; flow rate: 2 ml/minute for 5 minutes, 3 ml/minute for the remainder; detection: 220 nm). Under these conditions, authentic samples of isopenicillin N and penicillin N elute with a retention time of 5.25 minutes, which coincides with a peak of newly-formed material observed in cyclization reaction mixtures. The combined concentration of isopenicillin N and penicillin N[(iso)penicillin N] in cyclization reaction mixtures corresponds closely to the concomitant decrease in the ACV monomer. Cyclization reaction mixtures, in which crude cell-free extract from S. clavuligerus NRRL 3585 is the enzyme source, contain (iso)penicillin N at a concentration of 43.3 micrograms/ml after a 1-hour incubation period. Cyclization reaction mixtures, in which salt-precipitated cell-free extract from S. clavuligerus is the enzyme source, contain 39.0 micrograms/ml (iso)penicillin N.