Scherrer S, Robas N, Zouheiry H, Branlant G, Branlant C
Laboratoire d'Enzymologie et de Génie Génétique, Université de Nancy I, URA CNRS 457, Vandoeuvre-Les-Nancy, France.
Appl Microbiol Biotechnol. 1994 Oct;42(1):85-91. doi: 10.1007/BF00170229.
The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
大肠杆菌青霉素G酰胺酶（PGA）是青霉素G衍生物生产中的关键酶，它通过一种不寻常的内部成熟过程从前体多肽产生。我们观察到，在26℃下生长的重组大肠杆菌JM109细胞超声处理后回收的不溶性物质中积累了PGA前体多肽。在溶解试验中使用去污剂和离液剂证明了积累分子的聚集性质。通过对原生质球部分进行胰蛋白酶可及性实验表明聚集体位于周质。最后，我们表明在培养基中添加蔗糖或甘油可强烈减少这种周质聚集，结果PGA产量大幅增加。因此，PGA前体多肽的周质聚集限制了重组大肠杆菌的PGA产量，并且通过在培养基中添加不可代谢的糖（如蔗糖）或甘油可以克服这种限制。
