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

苏氨酸缩醛酶(TA)催化β-羟基-α-氨基酸可逆降解为甘氨酸和相应的醛。我们发现并鉴定了几种具有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，建立了生产光学纯β-羟基-α-氨基酸的酶促拆分工艺。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。低特异性假单胞菌L-Tas(LSLTA)。NCIMB 10558、酿酒酵母和大肠埃希氏菌均能切割L异构体和L异构体，而简达气单胞菌的L异构体(LATA)几乎特异性地作用于L异构体(L异构体/L异构体&GT；100)。为了改变LATA基因对苏氨酸异构体的立体特异性，用易错聚合酶链式反应将随机点突变引入LATA基因。在筛选的800个突变株中，菌株F48不仅对L-allo-Thr具有TA活性，而且对L-Thr(L-allo-/L-比率[约等于]10)也具有TA活性。在对变异体F48的DNA序列分析中，发现了两个替换(765和876位的T分别替换为C和A)。前者的氨基酸序列没有变化，而后者将292位的Ser改变为Arg。从突变菌株F48的无细胞提取液中分离到两个不同分子质量的TA。这两种酶由4个或8个相同的亚基组成，分子质量为38,000，显示了相同的N-末端氨基酸序列。四聚体结构是天然状态，八聚体可能由四聚体结合而成。四聚体酶比八聚体具有更高的活性。

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.