酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用

目的:建立谷氨酸依赖型氨基转移酶-谷氨酸脱氢酶偶联反应的96孔板高通量筛选方法,并用于大肠杆菌氨基转移酶Wec E突变库的筛选。方法:通过优化偶联指示酶-谷氨酸脱氢酶、信号分子NADH浓度及双酶偶联反应时间,建立了光学法测定氨基转移酶活性的氨基转移酶-谷氨酸脱氢酶偶联反应方法;通过定点饱和突变技术构建了大肠杆菌氨基转移酶WecE的突变库;采用96孔板高通量初筛、摇瓶复筛获得了高活性的转氨酶突变体,并对纯化的突变体进行催化活力分析。结果:建立了谷氨酸依赖型氨基转移酶目标反应与0.5 U/ml L-谷氨酸脱氢酶和0.4 mmol/L NADH信号指示反应相偶联的筛选方法;构建了氨基转移酶WecE Tyr 321饱和突变库,通过96孔板高通量筛选,获得了催化活性比野生型提高3.4倍的突变体Y321F。结论:所建立高通量筛选方法背景干扰小,准确性高,为谷氨酸依赖型氨基转移酶分子进化提供了可行性方案。     

老年维持性血液透析患者认知功能障碍的影响因素及其对脑血流动力学、肠道菌群和预后的影响

摘要 目的：探讨老年维持性血液透析（MHD）患者认知功能障碍（CI）的影响因素及其对脑血流动力学、肠道菌群和预后的影响。方法：选取2017年2月～2021年1月长治医学院附属晋城医院收治的411例老年MHD患者,根据蒙特利尔认知评估量表（MoCA）评分结果,将其分为CI组（n=321）和非CI组（n=90）。收集所有老年MHD患者临床资料,检测、分析其脑血流动力学和肠道菌群水平。采用Logistic回归分析老年MHD患者CI的影响因素。随访18个月,采用Kaplan-Meier法绘制两组患者生存曲线,对比其生存预后情况。结果：411例老年MHD患者CI发生率为78.10％（321/411）。单因素分析显示,CI组年龄大于非CI组,体质指数（BMI）、单室尿素清除指数（spKt/V）、白蛋白水平低于非CI组,受教育年限＞12年比例少于非CI组,透析龄长于非CI组,高血压、糖尿病比例和全段甲状旁腺素高于非CI组（P＜0.05）。多因素Logistic回归分析显示,年龄及透析龄增加和高血压、糖尿病为老年MHD患者CI的独立危险因素,受教育年限＞12年和BMI、spKt/V、白蛋白水平升高为其独立保护因素（P＜0.05）。与非CI组比较,CI组大脑中动脉-平均血流速度（MCA-Vm）、大脑前动脉-平均血流速度（ACA-Vm）降低,大脑中动脉-搏动指数（MCA-PI）、大脑前动脉-搏动指数（ACA-PI）升高（P＜0.05）。与非CI组比较,CI组肠球菌和大肠埃希菌数量增加,乳杆菌和双歧杆菌数量减少（P＜0.05）。Kaplan-Meier生存曲线分析显示,CI组累积生存率低于非CI组（P＜0.05）。结论：年龄、BMI、受教育年限、透析龄、高血压、糖尿病、spKt/V、白蛋白是老年MHD患者CI的影响因素,CI与老年MHD患者脑血流动力学紊乱、肠道菌群失衡和预后不良有关。