Bifidobacterium bifidum has the characteristics of adhering to the intestine and alleviating inflammatory reactions,but the mechanism by which its surface structure functions is not yet clear.In this study,the recombi...Bifidobacterium bifidum has the characteristics of adhering to the intestine and alleviating inflammatory reactions,but the mechanism by which its surface structure functions is not yet clear.In this study,the recombinant expression of the transaldolase(TAL)of Bifidobacterium bifidum E3(B.bifidum E3),and the TAL protein had adhesion and anti-inflammatory activity.The binding mode of TAL protein to mucins(MUC1 and MUC2)was simulated by molecular docking.Then,the anti-inflammatory effect of TAL protein on IEC-6 cells was investigated in vitro.The TAL protein of B.bifidum E3 was successfully expressed in Escherichia coli M15.Simulation results showed that TAL protein mainly was bound to MUC1 and MUC2 proteins through hydrogen bonding forces.Studies on the anti-inflammatory activity of TAL protein indicated that it was non-toxic to IEC-6 cells.The protein had an alleviating effect on TNF-α-induced inflammation in IEC-6 cells by promoting cell proliferation,reducing apoptosis,regulating cytokine balance and signaling pathways related to intestinal inflammation.Further,TAL protein reduced the nuclear translocation of the p65 subunit in the NF-κB signaling pathway and downregulated the expression of the NF-κB signaling pathway related genes p65 and p50,and upregulated the expression of the PI3K/AKT signaling pathway related genes PI3K and AKT,at both gene and protein levels.Therefore,based on the above results,it was indicated that the transaldolase of B.bifidum E3 bound to mucin receptors and had anti-inflammatory properties,providing a theoretical basis for the development of Bifidobacterium surface proteins as bioactive substances.展开更多
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
基金supported by China Postdoctoral Science Foundation,Top Project,Study on the Role of Bifidobacterium dentis N8 in Improving Intestinal Inflammation and Related Mechanisms(2022M721071)National Key Laboratory of Food Science and Resource Mining,Open Project,Study on the Role of Bifidobacterium dentis N8 in Improving Intestinal Inflammation and Related Mechanisms(SKLF-KF-202310).
文摘Bifidobacterium bifidum has the characteristics of adhering to the intestine and alleviating inflammatory reactions,but the mechanism by which its surface structure functions is not yet clear.In this study,the recombinant expression of the transaldolase(TAL)of Bifidobacterium bifidum E3(B.bifidum E3),and the TAL protein had adhesion and anti-inflammatory activity.The binding mode of TAL protein to mucins(MUC1 and MUC2)was simulated by molecular docking.Then,the anti-inflammatory effect of TAL protein on IEC-6 cells was investigated in vitro.The TAL protein of B.bifidum E3 was successfully expressed in Escherichia coli M15.Simulation results showed that TAL protein mainly was bound to MUC1 and MUC2 proteins through hydrogen bonding forces.Studies on the anti-inflammatory activity of TAL protein indicated that it was non-toxic to IEC-6 cells.The protein had an alleviating effect on TNF-α-induced inflammation in IEC-6 cells by promoting cell proliferation,reducing apoptosis,regulating cytokine balance and signaling pathways related to intestinal inflammation.Further,TAL protein reduced the nuclear translocation of the p65 subunit in the NF-κB signaling pathway and downregulated the expression of the NF-κB signaling pathway related genes p65 and p50,and upregulated the expression of the PI3K/AKT signaling pathway related genes PI3K and AKT,at both gene and protein levels.Therefore,based on the above results,it was indicated that the transaldolase of B.bifidum E3 bound to mucin receptors and had anti-inflammatory properties,providing a theoretical basis for the development of Bifidobacterium surface proteins as bioactive substances.
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.
