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Microbial Production of L-Serine and Its Related Hydroxy Amino Acids

L-丝氨酸及其相关羟基氨基酸的微生物生产

基本信息

批准号：
60860011
负责人：
YAMADA Hideaki
金额：
$ 5.76万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

项目摘要

L-Serine, which is a non-essential amino acid in the nutritional sense, is used primarily used as dietary supplement in parental nutrition and recently as a substrate for the enzymatic synthesis of other important amino acids such as L-tryptophan. Microbial serine production has so far been reported using heterotrophs and methyltrophs. Although extensive studies habe been done on the process by means of serine hydroxymethyltransferase (SHMT) reaction using as a raw material, the yields and produced amounts of L-serine are still so low. The present project aimed at establishing the fundamental methods for the production of L-serine by a methylotroph having the serine pathway, and also at developing this process to the production of serine related hydroxy amino acids.In the 1985 fiscal year, we studied the improvement of L-serine productivity of the serine-producing methylotroph, Hyphomicrobium methylotroph KM 146 by deriving a glycine-resistant mutant GM2. The mutant was found to produce 34 mg/ml L-serine (1.4-fold more than the parent) in a resting cell system. We also found that H. methylovorum and newly isolated methanol-utilizing bacterium, Pseudomonas sp. KM 193, produced L-threonine and allothreonine from ethanol and glycine by methanol-grown cells.In the 1986 fiscal year, we studied the purification and characterization of methanol dehydrogenase (MDH) and SHMT, enzymes involved in serine production, from H. methylovorum GM2. The enzymes were purified to homogeneity. Both showed a wide substrate specificity and high stability to long preservation. The optimum pH and temperature were 9 and 35゜C, and 8 and 37゜C for MDH and SHMT, respectively. Such basic information about the enzymatic properties of the two enzymes was considered to contribute to the further improvement of L-serine production by H. methylovorum.

L-丝氨酸是营养意义上的非必需氨基酸，主要用作父母营养中的膳食补充剂，最近用作酶促合成其他重要氨基酸如L-色氨酸的底物。迄今为止，已经报道了使用异养生物和甲基营养生物的微生物丝氨酸生产。以丝氨酸羟甲基转移酶（SHMT）为原料，利用SHMT反应生产L-丝氨酸，虽然已经进行了大量的研究，但L-丝氨酸的产率和产量仍然很低。本课题的目的是建立由具有丝氨酸途径的甲基营养菌生产L-丝氨酸的基本方法，并将该方法发展到生产丝氨酸相关的羟基氨基酸。在1985财政年度，我们研究了通过衍生甘氨酸抗性突变体GM 2来提高丝氨酸生产甲基营养菌Hyphomicrobium methylotroph KM 146的L-丝氨酸生产力。发现该突变体在静息细胞系统中产生34 mg/ml L-丝氨酸（比亲本高1.4倍）。我们还发现H. 1986年，我们对嗜甲醇菌（H. methylovorum）和新分离的甲醇利用菌（Pseudomonassp. KM 193）的丝氨酸生产酶甲醇脱氢酶（methanol dehydrogenase，MDH）和丝氨酸代谢酶SHMT的分离纯化和性质进行了研究。食蚜蝇GM 2.将酶纯化至均一。两者均表现出广泛的底物特异性和长期保存的高稳定性。MDH和SHMT的最适pH和温度分别为9和35 ℃，8和37 ℃。这两种酶的酶学性质的基本信息被认为有助于进一步提高H.食甲基菌