类弹性蛋白修饰R型ω转氨酶用于催化R苯乙胺-化学工程与技术专业毕业论文.docxVIP

的酶系统，在反应介质中，ELP．RTA自组装形成微米大小的酶集群，底 的酶系统，在反应介质中，ELP．RTA自组装形成微米大小的酶集群，底 物R-苯乙胺也形成了微米大小的液滴。酶集群和底物液滴的亲密接 触提供了高底物浓度的微环境来接近酶，促进底物分子的扩散到活性 位点。对于双酶系统，ELP．RTA和ELP融合DAAO组装形成双酶复 合物，形成集群的幅度比单一酶大得多，大大地提高了转化率。 最后，利用DAAO催化D．Ala的反应产生丙酮酸和H202，而CAT 可以又将H202分解成H20和02，应用构建的内含肽介导的 RTA．DAAO和CAT-DAAO双酶剪接体，探究了在30mM R-苯乙胺， 2mM丙酮酸钠条件下RTA．DAAO和CAT-DAAO两个体内剪接体构 成的三种多酶催化体系的转化效果。从实验结果可以看出， 利用体 内剪接体RTA—DAAO并引入了CAT的体系转化速率很快。 关键词：R型∞转氨酶，类弹性蛋白，Intein，多酶催化 万方数据 AbstractElastin Abstract Elastin Proteins Modified R-∞．transaminase Used for Catalytic R-phenylethylamine Abstract R-0)一transaminase is a kind of enzyme that refers to selectively catalytic R-amine generate transamination reaction which the substrate or product must have no a-amino acids or a-keto acids．5-pyridoxal phosphate(PLP)assisted to complete the whole amino exchange catalytic process．R-to—transaminase is widely researched and applied in the resolution of raceme and synthesis of chiral alllJlle．Started with R-o)一 transaminase genes from Arthrobacter sp．KNKl68，we constructed several expression vectors to express and purify,then explored the multiple enzyme system in the application of catalytic R-phenylethylamine． First of all，we constructed expression vector of pET28a-RTA through genetic method．In order to use more simple and low—cost method for purification，ELP was introduced as a kind of elastic protein purification tag．So we constructed expression vector of pET28a-1 0ELP- llI 万方数据 万方数据 万方数据 北京化工大学硕士学位论文RTA．Compared 北京化工大学硕士学位论文 RTA．Compared with enzyme activity,we found the single enzyme RTA enzyme activity is very similar with that of 1 0ELP—RTA．May safely draw the conclusion that ELP did not affect enzyme activity of RTA．Besides， the two proteins have similar secondary structures in the results of circular dichroism spectrum． Then，in order to move forward the balance of reaction under the condition of lack of pyruvic acid，using DAAO of converting the by-product D—Ala to pyruvic acid，SO as to realize pyruvic acid cycle， DAAO is in