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

甲醇通常被认为是在不久的将来作为发酵培养基成分的有前途的碳和能量来源。目前，L-丝氨酸的工业化生产是由甘氨酸抗性异养细菌从甘氨酸中进行的。然而，L-丝氨酸的生产仍然不足。在使用具有丝氨酸途径的甲基营养菌生产L-丝氨酸的方法的情况下，即，该方法使用两种酶，甲醇脱氢酶（MDH）和丝氨酸羟甲基转移酶（SHMT）的反应，可以预期从甲醇和甘氨酸到丝氨酸的高转化率。可以预期从甲醇和甘氨酸到丝氨酸的高转化率。本文作者对甲基营养菌Hyphomicrobium methylovorum的静息细胞酶法生产丝氨酸进行了研究。虽然丝氨酸途径是微生物中C1化合物的特征代谢之一，但丝氨酸途径的详细酶促研究从未被提及。 ...更多信息 en报道的主要原因是缺乏在该途径上具有高酶活性的微生物。相反，H.发现甲基四氢叶酸菌显示出非常高和稳定的酶活性。因此，本研究的目的是阐明酶的丝氨酸途径，包括SHMT的酶学和蛋白质化学性质。此外，本研究还进一步阐明了酶反应的调控机制，并利用本研究所获得的信息对L-丝氨酸的合成进行了改进。甲基四氢叶酸、SHMT、丝氨酸-乙醛酸氨基转移酶（SGAT）和羟基丙酮酸还原酶（HPR）均首次被纯化至均一或结晶形式。从酶学和蛋白质化学方面对纯化的酶进行了表征，发现其均为β-内酰胺酶：（1）底物特异性很高;（2）SHMT在免疫学和酶动力学上与其他来源的酶有很大的不同，不仅与哺乳动物肝脏和大肠杆菌的酶不同;利用SGAT和HPR的高底物特异性、高产率和易于纯化的特点，建立了一种简便、快速、重复性好的酶法测定方法，并首次将甘油酸激酶（GK）和磷酸烯醇式丙酮酸羧化酶（PEPC）作为C1微生物进行了纯化，并对其进行了酶学和蛋白质化学表征。这五种酶的研究结果对C1微生物的酶学、生物化学和代谢研究以及微生物学研究都有一定的意义。最后，从多种甲基营养型丝状菌中筛选出丝氨酸高产菌株，并从中筛选出产丝氨酸能力较强的丝微菌NCIB 10099菌株。然后，优化细菌的生产率（L-丝氨酸产量，52 mg/ml，相对于所用甘氨酸的产率为35%）。少

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