Improving The Catalytic Activity And Substrate Tolerance Of Deoxyribo Aldolases By Molecular Modification

2-Deoxy-D-Ribose Aldolase (EC 4.1.2.4, DERA) could catalyze aldol reaction, generate two chiral centers. In particular, it could accepet three molecules of acetaldehyde as substrate to produce chiral side chain of statins after two steps of aldol condensation reaction. Statins can reduce cholesterol levels in the human body, which is an important lipid-lowering drugs. Nevertheless, there are still some problems in the application of DERA:narrow spectrum of substrates, preference of phosphorylated substrate, low catalytic activity to non-phosphorylated substrate, and poor tolerance to high concentration of acetaldehyde. Therefore, based on the improved high-throughput screening method, homology modeling and molecular dynamics calculation, this issue used directed evolution and rational design comprehensively to improve the catalytic activity of non-phosphorylated substrates and the tolerance to high concentration of acetaldehyde, the reconstructed DERA would be used in the synthesis of chiral drug precursors.(1) The establishment of high-throughput fluorescent screening methods:In order to effectively screen new aldolase and assay the activity of reconstructed DERA, the fluorescent mechanism of coumarin derivatives was studied. The relationship between fluorescent intensity and the position of substituent group on parent ring was determined. Redesigned the fluorescent substrate by introducing benzimidazoly at 3rd postion and methoxy at 6th position, which made the substrate emit strong green fluorescence in day light. Comparing with the existing methods, the detection sensitivity increased by 58.2-fold.(2) The directed evolution of DERA:The DERAGth and DERASep were selected as the research object from the recombinant expression of 8 kinds of DERA in lab. They were both founded in E.coli BL21 (DE3) to achieve overexpression. The pure enzyme specific activity were 22.5U/mg and 16.5U/mg respectively. After directed evolution of DERAGth and DERASep, the mutant strain DERAGth(F159I, S209G) was obtained by high throughput screening method, and its specific activity was 105.8U/mg, which was 3.7 times higher than that of the original enzyme.(3) Rational design to improve DERA catalytical activity to N-APM:Invesgated the DERAGth and DERAsep catalytic activity to N-APM and acetaldehyde condensation reaction, DERAGth and DERAGth(F159I S209G) showed 0.52 U/mg and 0.71 U/mg of specific activity respectively, and DERAsep showed 6.2U/mg, DERAsep was chosen as the target enzyme to be reconstructed. DERATma (PDB1oOy) was used as template to construct homology modeling of DERAsep, and the enzyme substrate binding site was found to have similar structural characteristics by sequence alignment and active site. After molecular docking, Thr10, Ser205, Ala206 was found to hinder the side chain of N-APM.These three sites were chosen to be simulated according to the docking energy and binding free energy of N-AMP and mutates. The binding free energy of DERAsep (A206G) and DERASep (S205E) were -12.39kcal/mol and -7.11kcal/mol (DERASep -3.17 kcal/mol), which indicated that these two site mutation may cause activity change. After site directed mutagenesis, the changes of the catalytic activity of the mutants were investigated. The results showed that the specific activity of DERAsep (A206G) was 22.8U/mg,2.7-fold more than that of DERASep.(4) Rational design to improve acetaldehyde tolerance of DERA:DERAsep and DERAsep (A206G) were selected as the research object. According to the research report, there is a positive correlation between the thermal stability of enzyme and its t organic solvents. Therefore, it is proposed to enhance the stability of DERAsep to enhance its tolerance to acetaldehyde. The structural model of DERAsep was virtually mutation scanned on the Discovery Studio. The single amino acid mutation energy was obtained. Based on the operation rules of software, low mutation energy means the structure more stable, D15F, S16I, T41C, T120I, G174Y, G174, G213C were selected,double and multiple vitual mutation was done base on these seven sites.The Variant10 DERASep (T120C, G174I, G213C) and Variant" DERAsep(T120C, G174I,, A206G,, G213C) were obtained, the acetaldehyde tolerance was assayed, the results showed that Variant10 and Variant11 remained 70.5% and 66.7% of their initial activity after incubation for 2h, under 300mM acetaldehyde while the extensively studied DERAEco almost lost all its activity under the same conditions. In the condensation reaction of N-AMP (46.2mM) and acetaldehyde (166.7mM) catalyze by whole cell. Variant11 consumed 43.6% of substrate after reation for 24h, which was almost 1.32 times and 3.1 times than that of DERAsep and DERAEco,1-55 times higher than those reported DERA in the literature.(5) The immobilization of DERAEco:DERAEco was conenectd to Fe3O4 nanoparticles by covalently coupling. The optimum crosslinking conditions were:the ratio of the enzyme amount to the carrier content was 1:10, the best crosslinking pH was 6, and the cross-linking time was 5h. After the preparation of the cross-linked, 76.8% of the enzyme was linked to the carrier, the highest recovery rate of enzyme activity was 65.04%,which significantly improved the enzyme thermal stability and tolerance to acetaldehyde.DERAEco remained 61.4% of its cleavage activity after incubation in 300mM acetaldehyde for 10h at 25℃.