Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii.
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.
Mihara
Y
Y
Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki-shi 210-8681, Japan. yasuhiro_mihara@ajinomoto.com
Utagawa
T
T
Yamada
H
H
Asano
Y
Y
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D007288:Inosine / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D020613:Morganella morganii / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism*; D010766:Phosphorylation; D017422:Sequence Analysis, DNA
D009705:Nucleosides; D007291:Inosine Monophosphate; D007288:Inosine; D000135:Acid Phosphatase