Preparative 2'-reduction of ATP catalyzed by ribonucleotide reductase purified by liquid-liquid extraction.

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摘要：

Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction. A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2â²-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2â²-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.

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关键词：

liquid-liquid extraction enzymatic synthesis ribonucleotide reductase ATP 2â²-deoxyadenosine-5â²-triphosphate

DOI：

10.1271/bbb.64.1836

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年份：

2000