Mihara Y, Utagawa T, Yamada H, Asano Y
Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki-shi 210-8681, Japan.
Appl Environ Microbiol. 2000 Jul;66(7):2811-6. doi: 10.1128/AEM.66.7.2811-2816.2000.
A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5'-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5'-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased K(m) value for inosine was responsible for the increased productivity.
研究了一种以食品添加剂焦磷酸作为磷源的新型核苷磷酸化过程。克隆了摩根氏摩根菌中编码选择性核苷焦磷酸磷酸转移酶的基因。它与摩根氏摩根菌的PhoC酸性磷酸酶基因相同。通过易错PCR对该基因进行体外连续随机诱变以构建突变体文库。将突变体文库导入大肠杆菌，并筛选转化体以检测5'-肌苷酸（5'-IMP）的产生。获得了一种磷酸转移酶反应产率提高的突变酸性磷酸酶。在过量表达突变酸性磷酸酶的大肠杆菌中，从肌苷合成了每升101克（192毫摩尔）的5'-IMP，摩尔产率为88%。这种改进是通过两个突变实现的，即第92位的甘氨酸突变为天冬氨酸以及第171位的异亮氨酸突变为苏氨酸。肌苷的米氏常数（K(m)）值降低导致了生产率的提高。
