l-Threonine transaldolase could catalyze the transaldolation of l-threonine and aldehyde to generateβ-hydroxy-α-amino acids with high diastereoselectivity.A novel l-threonine transaldolase(PmLTTA)was identified from...l-Threonine transaldolase could catalyze the transaldolation of l-threonine and aldehyde to generateβ-hydroxy-α-amino acids with high diastereoselectivity.A novel l-threonine transaldolase(PmLTTA)was identified from Pseudomonas sp.through genome mining.PmLTTA exhibited high activity in the synthesis of l-threo-phenylserine from l-threonine and benzaldehyde,with specific activity of 5.48 U mg-1.However,the application of PmLTTA was impeded by the low conversion ratio and variable diastereoselectivity,which were caused by the toxicity of aldehydes and kinetic/thermodynamic controls in the transaldolation reaction.To solve these issues,alcohol dehydrogenase was used to remove the by-product acetaldehyde,and then carboxylic acid reductase was introduced to alleviate the inhibition of benzaldehyde and toxicity of DMSO.Finally,a multi-enzyme cascade reaction,comprising of PmLTTA,carboxylic acid reductase,alcohol dehydrogenase and glucose dehydrogenase,was constructed to prepare l-threo-phenylserine from cheap benzoic acid,in which alleviated inhibition of aldehydes and desirable diastereoselectivity were achieved.Under the optimized conditions,the conversion ratio of 57.1%and de value of 95.3%were reached.This study provides an efficient and green approach for the synthesis of chiral l-threo-phenylserine from industrial byproduct,and provides guidance for the development of cascade reactions influenced by the toxic intermediates and complicated kinetic/thermodynamic controls.展开更多
目的:探讨转醛醇酶(t ransaldolase,TAL)在氪激光诱导的BN大鼠脉络膜新生血管(choroida l neovascularization,CNV)中的表达变化及其可能的作用机制。方法:给予2组20只BN大鼠行氪激光光凝视网膜,诱导BN大鼠建立CNV模型。在光凝后21 d行...目的:探讨转醛醇酶(t ransaldolase,TAL)在氪激光诱导的BN大鼠脉络膜新生血管(choroida l neovascularization,CNV)中的表达变化及其可能的作用机制。方法:给予2组20只BN大鼠行氪激光光凝视网膜,诱导BN大鼠建立CNV模型。在光凝后21 d行荧光素眼底血管造影(fluorescein fundus angiography,FFA),确定模型建立成功后摘除眼球。采用蛋白质印迹法检测TAL、还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)和还原型谷胱甘肽(glutathione,GSH)蛋白质表达,并测定TAL活性。采用小干扰RNA(small interfering RNA,siRNA)抑制下TAL在人视网膜色素上皮(retinal pigment epithelium,RPE)ARPE-19细胞中的表达,蛋白质印迹法检测抑制前后TAL,NADPH和GSH表达水平的变化。结果:光凝后21 d见明显新生血管生成,表明模型成功建立。CNV增殖区TAL活性明显高于正常大鼠中TAL活性(67.16±3.38 vs 182.57±1.83,P<0.001);TAL蛋白质表达明显高于正常大鼠中TAL表达(0.87±0.33 vs 2.09±0.21,P=0.005);NADPH和GSH表达明显低于其在正常大鼠中的表达(0.92±0.20 vs 0.14±0.05,P<0.01;0.84±0.31 v s 0.29±0.11,P<0.05)。通过对人视网膜色素上皮ARPE-19细胞不做干预、加入LipofectamineTM 2000稀释液、加入TAL-siRNA/LipofectamineTM 2000复合物,将其分为正常对照组、阴性对照组及TAL-siRNA组,干预48 h后,TAL-siRNA组中TAL蛋白质表达明显低于正常对照组和阴性对照组(0.26±0.13 vs 1.39±0.42和1.15±0.19,P<0.01);NADPH和GSH表达明显高于正常对照组(P<0.01)和阴性对照组(P<0.05)。结论:CNV形成的过程中,TAL的活性增强、表达上调,证实TAL与CNV发生发展密切相关,其可能通过调控NADPH和GSH的表达参与CNV的发生发展。展开更多
基金We are grateful to the National Key Research and Development Program(2021YFC2102700)the National Natural Science Foundation of China(22077054,22078127)+1 种基金the National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-07)Program of Introducing Talents of Discipline to Universities(111-2-06)for the financial support of this research.
文摘l-Threonine transaldolase could catalyze the transaldolation of l-threonine and aldehyde to generateβ-hydroxy-α-amino acids with high diastereoselectivity.A novel l-threonine transaldolase(PmLTTA)was identified from Pseudomonas sp.through genome mining.PmLTTA exhibited high activity in the synthesis of l-threo-phenylserine from l-threonine and benzaldehyde,with specific activity of 5.48 U mg-1.However,the application of PmLTTA was impeded by the low conversion ratio and variable diastereoselectivity,which were caused by the toxicity of aldehydes and kinetic/thermodynamic controls in the transaldolation reaction.To solve these issues,alcohol dehydrogenase was used to remove the by-product acetaldehyde,and then carboxylic acid reductase was introduced to alleviate the inhibition of benzaldehyde and toxicity of DMSO.Finally,a multi-enzyme cascade reaction,comprising of PmLTTA,carboxylic acid reductase,alcohol dehydrogenase and glucose dehydrogenase,was constructed to prepare l-threo-phenylserine from cheap benzoic acid,in which alleviated inhibition of aldehydes and desirable diastereoselectivity were achieved.Under the optimized conditions,the conversion ratio of 57.1%and de value of 95.3%were reached.This study provides an efficient and green approach for the synthesis of chiral l-threo-phenylserine from industrial byproduct,and provides guidance for the development of cascade reactions influenced by the toxic intermediates and complicated kinetic/thermodynamic controls.
文摘目的:探讨转醛醇酶(t ransaldolase,TAL)在氪激光诱导的BN大鼠脉络膜新生血管(choroida l neovascularization,CNV)中的表达变化及其可能的作用机制。方法:给予2组20只BN大鼠行氪激光光凝视网膜,诱导BN大鼠建立CNV模型。在光凝后21 d行荧光素眼底血管造影(fluorescein fundus angiography,FFA),确定模型建立成功后摘除眼球。采用蛋白质印迹法检测TAL、还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)和还原型谷胱甘肽(glutathione,GSH)蛋白质表达,并测定TAL活性。采用小干扰RNA(small interfering RNA,siRNA)抑制下TAL在人视网膜色素上皮(retinal pigment epithelium,RPE)ARPE-19细胞中的表达,蛋白质印迹法检测抑制前后TAL,NADPH和GSH表达水平的变化。结果:光凝后21 d见明显新生血管生成,表明模型成功建立。CNV增殖区TAL活性明显高于正常大鼠中TAL活性(67.16±3.38 vs 182.57±1.83,P<0.001);TAL蛋白质表达明显高于正常大鼠中TAL表达(0.87±0.33 vs 2.09±0.21,P=0.005);NADPH和GSH表达明显低于其在正常大鼠中的表达(0.92±0.20 vs 0.14±0.05,P<0.01;0.84±0.31 v s 0.29±0.11,P<0.05)。通过对人视网膜色素上皮ARPE-19细胞不做干预、加入LipofectamineTM 2000稀释液、加入TAL-siRNA/LipofectamineTM 2000复合物,将其分为正常对照组、阴性对照组及TAL-siRNA组,干预48 h后,TAL-siRNA组中TAL蛋白质表达明显低于正常对照组和阴性对照组(0.26±0.13 vs 1.39±0.42和1.15±0.19,P<0.01);NADPH和GSH表达明显高于正常对照组(P<0.01)和阴性对照组(P<0.05)。结论:CNV形成的过程中,TAL的活性增强、表达上调,证实TAL与CNV发生发展密切相关,其可能通过调控NADPH和GSH的表达参与CNV的发生发展。
