Development of production process for chiral β-hydroxy-α-amino acids by aldolase reaction

醛缩酶反应制备手性β-羟基-α-氨基酸工艺的开发

• 批准号: 12556013
• 负责人: KATAOKA Michihiko
• 金额: $ 8万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556013/
• 关键词: Chiral building block; β-Hydroxy-α-amino acid; Aldolase; Threonine

Threonine aldolase (TA) catalyzes reversible degradation of β-hydroxy-α-amino acids to glycine and corresponding aldehydes. We found and characterized several microbial TAs showing stereospecificity, and cloned genes encoding these enzymes. By using recombinant TAs, an enzymatic resolution process was established for the production of optically pure β-hydroxy-α-amino acids. In addition, the TA-catalyzed direct synthesis of β-hydroxy-α-amino acid from aldehyde and glycine was investigated.Low-specificity L-TAs (LSLTAs) from Pseudomonas sp. NCIMB 10558, Saccharomyces cerevisiae and Escherichia coli cleave both L- and L-allo-Thr, while L-allo-TA (LATA) of Aeromonas jandaei almost specifically acts on the L-allo-isomer (L-allo-/L- ratio > 100) In spite of these different stereospecificities, LATA shows significant homology to LSLTAs in primary structure. Introduction of random point mutagenesis into the LATA gene by error-prone PCR was carried out in order to alter its stereospecificity toward Thr isomers. Among 800 mutants screened, strain F48 showed TA activity toward not only L-allo-Thr but also L-Thr (L-allo-/L- ratio 【approximately equal】 10). In the DNA sequence analysis of a variant F48, two substitutions (T at positions 765 and 876 to C and A, respectively) were found. The former substitution was silent with respect to amino acid sequence, while the latter changed Ser at the position 292 to Arg. From cell-free extract of the mutant strain F48, two TAs with different molecular mass were isolated. These two enzymes consisted of 4 or 8 identical subunits with molecular mass of 38,000, which showed same N-terminal amino acid sequence. Tetramer structure is the native state, and octamer might be formed by combination of tetramer. The tetramer enzyme showed higher activity than the octamer form.

苏氨酸醛缩酶（TA）催化β-羟基-α-氨基酸可逆降解为甘氨酸和相应的醛。我们发现并表征了几种表现出立体特异性的微生物TA，并克隆了编码这些酶的基因。利用重组TA，建立了一种酶法拆分β-羟基-α-氨基酸的方法。此外，还研究了TA催化乙醛和甘氨酸直接合成β-羟基-α-氨基酸的反应，假单胞菌NCIMB 10558、酿酒酵母和大肠杆菌的低特异性L-TA（LSLTA）能切割L-和L-异苏氨酸，而詹氏气单胞菌的L-allo-TA（LATA）几乎特异性地作用于L-allo-异构体（L-allo-/L-比率> 100）尽管存在这些不同的立体特异性，LATA在一级结构中显示出与LSLTA的显著同源性。通过易错PCR将随机点突变引入LATA基因中，以改变其对Thr异构体的立体特异性。在筛选的800个突变体中，菌株F48不仅对L-allo-Thr而且对L-Thr显示TA活性（L-allo-/L-比率[约等于] 10）。在对F48的DNA序列分析中，发现了两个突变（765和876位的T分别被C和A取代），前一个突变在氨基酸序列上是沉默的，后一个突变将292位的Ser变为Arg。从突变株F48的无细胞提取物中分离得到两种不同分子量的TA。这两种酶均由4个或8个相同的亚基组成，分子量均为38,000，N端氨基酸序列相同。四聚体结构为天然状态，四聚体结合可能形成八聚体。四聚体酶比八聚体酶具有更高的活性。

项目成果

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