【作者】 张晓霞；

【导师】 陈涛；

【作者基本信息】 天津大学 ， 生物化工， 2019， 硕士

【摘要】 乳酰辅酶A是一些草食动物肠道微生物乳酸代谢过程中的重要中间物,其主要来源是乳酸还原的非随机化途径。以乳酰辅酶A为直接前体物的乳酸基聚合物,因在可降解塑料的生产、生物医学方面具有重要的应用,对解决当前日益严重的环境问题、医疗问题具有重大研究意义。而乙酰辅酶A:乳酸辅酶A转移酶（ALCT）催化的转化乳酸生产乳酰辅酶A的反应是其生物法生产的关键步骤。然而,目前可选择的ALCT较少。为了探索新的、有效的ALCT,使用来自丙酸梭菌（Clostridium propionicum）和埃氏巨型球菌（Megasphaera elsdenii）的丙酰辅酶A转移酶的氨基酸序列做模板在NCBI数据库中进行BLAST筛选,筛选得到三个候选的辅酶A转移酶,分别来源于Megasphaera sp.DISK 18,Clostridium lactatifermentans An7和Firmicutes bacteria CAG:466。对其进行序列分析与亲缘树分析,发现其符合Ⅰ类辅酶A转移酶的序列特点,且进化树分支符合预期。将三个候选酶和两个模板的基因分别连接到p ET28a载体上进行表达纯化,鉴定了五个酶都具有ALCT活性。首次系统地研究了五个酶的最适反应条件,以及它们对两个主要底物的动力学参数和稳定性。之后表征了它们在菌体内表达的催化效果。最终,在五个酶中来源于埃式巨型球菌的丙酰辅酶A转移酶（ME-PCT）无论对乙酰辅酶A(K_cat/K_m=264.22 s^-1m M^-1)还是乳酸(K_cat/K_m=84.18 s^-1 m M^-1)都具有最高的催化效率,并且显示出良好的热适应性和稳定性。此外,在大肠杆菌内表达ME-PCT,也显示出最佳的乳酰辅酶A催化效果。本研究是率先开展的ALCT的系统研究,为乳酰辅酶A的生成提供了新的、有效的候选酶。其中酶学性能最好的ME-PCT必将对以乳酰辅酶A为重要中间体的产品合成提供巨大的驱动力,也可以为乳酸衍生产品的新途径的开发提供更有力的新工具。更多还原

【Abstract】 Lactoyl-CoA is an important intermediate in the metabolism of intestinal microorganism in some herbivores,which is mainly from the nonrandomizing pathway of lactate reduction.The lactate-based copolymers are widely used in the production of biodegradable plastics and biomedicine,which is important for environment protection and biomedicine production.The conversion of lactate to lactoyl-CoA catalyzed by acetyl-CoA:lactate CoA-transferase（ALCT）is the key step in the lactate-based copolymers production with biosysthesis.However,there are few alternatives for ALCTs.To exploit novel and efficient lactoyl-CoA transferases,the amino acid sequence similarity was analysised by BLAST with the well-known CoA-transferases from Clostridium propionicum and Megasphaera elsdenii as templates.Three candidate CoA-transferases were screened,which are derived from Megasphaera sp.DISK 18,Clostridium lactatifermentans An7 and Firmicutes bacteria CAG:466,respectively.Sequence analysis and phylogenetic tree analysis were performed.They were found to be consistent with the sequence characteristics of class I CoA-transferases,and the phylogenetic tree branches were distributed as expected.The genes of three candidates and two known ALCTs were integrated into the p ET28a.Then,the enzyme activity for converting lactate and acetyl-CoA into lactoyl-CoA and acetate were indentified.Then,the optimal reaction conditions,stability and kinetic properties of the five ALCTs for both D-lactate and acetyl-CoA were systematically investigated.The catalytic efficiency of ALCTs were then characterized in vivo.Among the five ALCTs,the propionyl-CoA from Megasphaera elsdenii（ME-PCT）exhibited the highest catalytic efficiency for both acetyl-CoA(264.22 s^-1 m M^-1)and D-lactate(84.18 s^-1 m M^-1)and showed a broad thermal adaptability and good stability.Moreover,it also showed the highest catalytic efficiency in vivo.We initially studied on ALCT,which can provide novel and efficient enzyme candidates for lactoyl-CoA generation.The ME-PCT with the best enzymatic properties will surely provide a huge driving force for the synthesis of products with lactoyl-CoA as an important intermediate.It can also offer valuable new tools for the engineering of pathways that rely on these enzymes.更多还原