PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF ACYL COENZYME-A-6-AMINOPENICILLANIC ACID ACYLTRANSFERASE OF PENICILLIUM-CHRYSOGENUM
PURIFICATION TO HOMOGENEITY AND CHARACTERIZATION OF ACYL COENZYME-A-6-AMINOPENICILLANIC ACID ACYLTRANSFERASE OF PENICILLIUM-CHRYSOGENUM
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DOI：
10.1128/aac.31.11.1675
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发表时间：
1987-11-01
影响因子：
4.9
通讯作者：
MARTIN, JF
中科院分区：
文献类型：
--
作者：
ALVAREZ, E;CANTORAL, JM;MARTIN, JF
产黄青霉AS-P-78菌株的酰基辅酶A(CoA)：6-氨基青霉酸(6-APA)酰基转移酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和等电聚焦纯化后，均一。该酶是一种相对分子质量为30,000+/-1,000，等电点约为5.5的单体。最适pH为8.0℃，最适温度为25℃。该酶以苯乙酰辅酶A或苯氧乙酰辅酶A为酰基供体，将6-APA转化为青霉素。该纯酶对6-APA具有较高的特异性和亲和力，不能以青霉素、7-氨基头孢烷酸、头孢菌素C和异头孢菌素C为底物。该酶可将异青霉素N转化为青霉素G，但转化效率低于以6-APA为底物时的转化效率。不显示青霉素G酰基酶活性。酰基CoA：6-APA酰基转移酶需要二硫苏糖醇或其他含硫醇化合物，并由含硫醇试剂保护，以防热失活。几种二价和三价阳离子以及对氯汞苯甲酸酯和N-乙基马来酰亚胺对酰基转移酶有抑制作用。在青霉素生物合成受阻的四个不同突变体中，该活性缺失。
The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.