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

甲醇被普遍认为是一种很有前途的碳源和能源，在不久的将来将作为发酵介质成分。目前，L丝氨酸是由甘氨酸抗性异养菌以甘氨酸为原料工业化生产的。然而，L丝氨酸的产量仍然不足。在利用丝氨酸途径的营养甲醇菌生产L丝氨酸的过程中，即利用甲醇甲氢酶和丝氨酸羟甲基转移酶两种酶的反应，从甲醇和甘氨酸到丝氨酸的转化率很高。从甲醇和甘氨酸到丝氨酸的高转化率是不可能的。到目前为止，作者已经研究了利用一种甲基营养细菌的休眠细胞来酶法生产丝氨酸。尽管丝氨酸途径是微生物中C1类化合物的特征代谢之一，但对丝氨酸途径的酶学研究从来都不是…较多的EN报道主要是由于缺乏在该途径上具有较高酶活性的微生物。相反，发现甲氧华氏菌表现出非常高和稳定的酶活性。因此，本研究旨在阐明包括SHMT在内的丝氨酸途径中酶的酶学和蛋白质化学性质。此外，本研究还利用本研究所获得的信息，进一步阐明了酶反应的调控系统，促进了L丝氨酸的合成。首次将甲氧丝氨酸丝氨酸途径的关键酶--丝氨酸转氨酶、丝氨酸乙氧基氨基转移酶和羟基丙酮酸还原酶纯化到均一或结晶状态。从酶学和蛋白质化学方面对纯化的酶进行了鉴定，结果表明：(1)底物专一性很高；(2)SHMT在免疫学和酶动力学上与其他来源的酶有很大不同，不仅不同于其他来源的酶，也不同于其他丝氨酸途径的亚甲基营养素。利用酶纯化的高底物专一性和高产率和易用性，建立了一种简单、快速和可重复的SGAT和HPR酶分析方法甘油酸激酶(GK)和磷酸烯醇式丙酮酸羧化酶(PEPC)也是首次纯化为同源的C1型微生物，并对其进行了酶学和蛋白质化学研究。这五种酶的研究结果为进一步研究C1-微生物的酶学、生物化学和代谢以及微生物学奠定了基础。最后，在多种兼性甲基化亚甲基亚甲基微生物中，进行了丝氨酸高产菌株的筛选，得到了一株产丝氨酸能力较强的亚甲基亚甲基微生物。NCIB 10099菌株表现出较强的产酶能力。在此基础上，对该菌的发酵条件进行了优化(L丝氨酸的产量为52 mg/ml，相对于甘氨酸的产率为35%)。较少

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