Ljubijankić G, Storici F, Glisin V, Bruschi C V
Microbiology Group, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012, Trieste, Italy.
Gene. 1999 Mar 4;228(1-2):225-32. doi: 10.1016/s0378-1119(98)00584-8.
The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.
编码异源二聚体青霉素G酰胺酶（PAC）的雷氏普罗威登斯菌和大肠杆菌pac基因在酿酒酵母中成功表达。此外，这些重组酶从酵母细胞分泌到培养基中，这与细菌宿主不同，在细菌宿主中，酶保留在周质中。与编码雷氏普罗威登斯菌PAC的基因相反，大肠杆菌pac在酵母中的表达较差。使用多拷贝质粒获得了最高产量的雷氏普罗威登斯菌PAC，产量为每升1500单位。该产量比在最佳重组细菌表达系统中获得的产量高一个数量级。重组雷氏普罗威登斯菌酶仅部分且选择性地进行O-糖基化。仅每隔第六个或第七个α亚基被糖基化，而β亚基根本不被糖基化。未检测到N-糖基化。
