Enzymatic Synthesis of Cystathionine by Microbial Enzymes

l -半胱硫氨酸是生物体内重要的转硫中间体。由于分子中有两个不对称碳，用化学方法合成l -半胱硫氨酸并不容易。此外，l -半胱硫氨酸是目前非常昂贵的氨基酸。l -半胱氨酸在未治疗的同型半胱氨酸患者和胎儿大脑中明显缺乏。因此，建立一种简单、经济的合成l -半胱硫氨酸的方法对今后的医学研究具有重要意义。因此，我们研究了利用微生物半胱硫氨酸< γ > -裂解酶和半胱硫氨酸< γ > -合酶的酶法合成l -半胱硫氨酸。在嗜铬链霉菌中存在的半胱氨酸< γ > -裂解酶可以催化l -同型丝氨酸在l -半胱氨酸存在下的< γ > -替代反应，生成l -半胱氨酸。考察了酶法合成l -半胱硫氨酸的最佳反应条件。由于l -半胱氨酸浓度大于10 mM会抑制<g…More amma - > -取代反应，因此l -半胱氨酸浓度保持在10 mM左右是l -半胱氨酸高产率的必要条件。在最佳反应条件下，即产率最高的条件下，以1L的反应混合物为原料，酶促合成半胱硫氨酸（3g）。o -琥珀酰- l-高丝氨酸和dl -乙烯基甘氨酸是比l-高丝氨酸更好的底物。在最佳条件下，由o -琥珀酰-L-高丝氨酸合成L-半胱氨酸（11 g/L），底物转化率为100%。其次，利用胡萝卜Erwinia carotovora和球形芽孢杆菌中丰富的半胱硫氨酸< γ > -合成酶，优化酶促生产l -半胱硫氨酸的反应条件。在最优条件下，Erwinia和Bacillus酶合成L-半胱硫氨酸（41g/L），转化率约为90%。综上所述，半胱硫氨酸< γ - > -合酶比半胱硫氨酸< γ - > -裂解酶更适合于酶法生产l -半胱硫氨酸，原因如下：(1)半胱硫氨酸< γ > -合成酶不催化l -半胱硫氨酸形成降解反应。(2)半胱硫氨酸< γ > -合成酶对变构抑制剂l -半胱氨酸不敏感。(3)半胱硫氨酸< γ > -合成酶比活性远高于半胱硫氨酸< γ > -裂解酶。此外，利用半胱硫氨酸< γ > -裂解酶和半胱硫氨酸< γ > -合酶还可以合成各种l -同型半胱氨酸相关氨基酸。少

L-Cystathionine is an important intermediate in transsulfuration in a living thinsg. As it has two asymmetric carbons in the molecule, it is not easy to synthesize L-cystathionine chemically. In addition, L-cystathionine is very expensive amino acid at present. L-Cystathionine is markedly deficient in the brains of untreated homocystinuric patients and fetal brains. Thus, it is meaningful to establish the simple and economical ways of synthesizing L-cystathionine for use in future medical research. We, therefore, investigated enzymatic synthesis of L-cystathionine using microbial cystathionine <gamma> -lyase and cystathionine <gamma> -synthase.Cystathionine <gamma> -lyase occurring in Streptomyces phaeochromogenes can catalyze the <gamma> -replacement reaction of L-homoserine in the presence of L-cysteine to form L-cystathionine. Optimal reaction conditions for the enzymatic synthesis of L-cystathionine were examined. Since concentrations of greater than 10 mM L-cysteine inhibit the <g … More amma> -replacement reaction, it is necessary to keep the concentration of L-cysteine around 10 mM for a high yield of L-cystathionine. Under optimal reaction conditions, defined as the conditions producing the highest yield, cystathionine (3 g) was synthesized enzymatically in 1L of reaction mixture. O-Succinyl-L-homoserine and DL-vinylglycine were better substrates than L-homoserine. Under the optimal conditions, L-cystathionine (11 g/L) was synthesized from O-succinyl-L- homoserine with a substrate conversion ratio of 100%. Next, the reaction conditions for the enzymatic production of L-cystathionine were optimized, using cystathionine <gamma> -synthase, which is abundant in the cells of Erwinia carotovora and Bacillus sphaericus. Under optimal conditions, L-cystathionine (41g/L) was synthesized with conversion ratio of about 90% with the Erwinia and Bacillus enzymes.It can be concluded that cystathionine <gamma> -synthase is more promising and suitable for the enzymatic production of L-cystathionine than cystathionine <gamma> -lyase based on the following reasons;(1) Cystathionine <gamma> -synthase does not catalyze the degradation reaction of L-cystathionine formed.(2) Cystathionine <gamma> -synthase is not sensitive to the allosteric inhibitor , L-cysteine.(3) Specific activity of cystathionine <gamma> -synthase is much higher than cystathionine <gamma> -lyase.In addition, it was also possible to synthesize various kinds of L-homocysteine related amino acids using the cystathionine <gamma> -lyase and cystathionine <gamma> -synthase. Less