Studies on the Characterization of Metabolic Properties of C_1-microorganisms and Its Application to Serine Synthesis

点击翻译按钮获取中文摘要

Methanol is generally considered to be a promising source of carbon and energy as the fermentation medium ingredient in the near future. At present, L-serine is industrially produced from glycine by glycine-resistant heterotrophic bacteria. However, the production of L-serine is still insufficient. In the case of the process of L-serine production using methylotrophs with the serine pathway, i.e., the process using the reactions of two enzymes, Methanol Mehydrogenase (MDH) and Serine Hydroxymethyltransferase (SHMT), one can expect a high conversion rate from mathanol and glyceine to serine. O ne can expect a high conversion rate from methanol and glycien to serine. The present author has so far been studied on the enzymatic production of serine using resting cells of a methylotrophic bacterium, Hyphomicrobium methylovorum. Although the serine pathway is one of the characteristic metabolisms of C1 compounds in microorganisms, detcils enzymatic studies of the serine pathway have never be … More en reported mainly because of lack of microorganisms which have high activities of enzymes on the pathway. On the contrary, H. methylovorum was found to show extraordinarily high and stable activities of the enzymes. Therefore, the present study was aimed in elucidating the enzymological and protein-chemical properties of the enzymes on the serine pathway including SHMT. In addition, the study extended to the elucidation of the regulation system of the enzyme reactons and the improvement of L-serine synthesis by making use of the information obtained in this study.The key enzymes of the serine pathway in H. methylovorum, SHMT, Serine-Glyoxylate Aminotransferase (SGAT), and Hydroxypyruvate Reductase (HPR), were all purified to homogeneity or to crystalline form for the first time. The purified enzymes were characterized from the enzymological and protein-chemical aspects, and were found to be all uniqus enzymes : (1) The substrate specificity was very high, (2) the SHMT was immunologically and enzyme-kinetically quite different from the enzymes from other sources, not only mammalian liver and E. coil, but also other methylotrophs with the serine pathway.Making use of the high substrate specificity and high yield and easiness of the enzyme purification, an easy, rapid and reproducible enzymatic assay method using SGAT and HPR was established.Furthermore, Glycerate Kinase (GK) and Phosphoenolpyruvate Carboxylase (PEPC) were also purified to homogeneity for the first time as C1 microorganisms and characterized enzymologically and protein-chemically. These results of the five enzymes surely were considered to contribute to enzymology, biochemistry and metabolic studies of C1-microorganisms as well as microbiology.Finally, among the various facultatively methylotrophicHyphomicroorganisms, screening for high serine producer was carried out, and as a result, Hyphomicrobium sp. NCIB 10099 strain was found to show a higher ability of the production. Then, the productivity of the bacterium was optimized (L-serine production, 52 mg/ml, 35% yield against the glycine used). Less