Abstract：
l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L-1) or 67.02 mM (15.16 g L-1) l-Car was produced at the substrate concentrations of 6 M β-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.
摘要：
l-肌肽(l-Car),内源性二肽存在于各种脊椎动物的肌肉和脑组织中，具有广泛的应用价值。1-Car的酶法制备是一种有前途的合成方法，因为它避免了保护和脱保护步骤。在本研究中,克隆并表征了来自产气荚膜梭菌的具有高l-Car合成活性的二肽酶基因(CpPeD)。为了提高这种酶的性能，我们使用CpPepD作为模板进行了位点饱和诱变。通过邻苯二甲醛(OPA)衍生的高通量筛选方法,获得了具有2.2倍增强的合成活性的突变体A171S。研究了CpPepD和突变体A171S的酶学性质。在优化条件下,63.94mM（14.46gL-1）或67.02mM（15.16gL-1）l-Car在6Mβ-Ala和0.2Ml-His的底物浓度下使用野生型或突变型A171S酶产生，分别。虽然突变增强了酶的活性，反应平衡几乎没有受到影响。
