Study On The Mechanism Of Polycaprolactone Depolymerase Combined With Polyester Substrates

Degradation of insoluble polymer polyester materials by the enzyme must first be adsorbed on the surface of the substrate.Current research shows that this type of adsorption is mostly achieved by specialized substrate binding domains.In the lab,we have screened a polycaprolactone depolymerase(PCLase)which has degradation activity on various polyester substrates.Structural analysis revealed that the enzyme is a single domain enzyme with only a catalytic domain.The depolymerase can degrade the solid-phase polyester substrate efficiently,but it is not yet known how the depolymerase combine with the solid-phase polyester substrate,without the substratebinding domainIn this experiment,the adsorption and binding of PCLase and polyester substrate were mainly studied,and we speculate that the surface amino acids that may participate in the substrate binding process though the structural analysis of the enzyme.Based on the hydrophobic properties of amino acids,we construct 23 mutants by a one-step plasmid site-directed mutagenesis PCR method.Then we tested the capacity of wild-type PCLase and mutants on adsorption and degradation of insoluble PCL membranes and the capacity of wild-type PCLase and mutants on the binding and catalytic to soluble substrates p-NPC12 and PCL emulsions,clarifing the mechanism of single domain PCLase binding to substrates.In addition,we add the substrate-binding domain to PCLase though protein-engineering method.We explore the effect of the modification of the substrate-binding domain on the single-domain enzyme by detecting the change of the binding activity of the recombinant hybrid enzyme to the substrate.The results are as follows:(1)PCLase binds to PCL substrate through hydrophobic amino acids on the surface.During this process,the four amino acids outside the active pocket play an important role.The G79 in Hy R1 region,I174,G175,L176,and G177 in Hy R2 region,G197,I200,I202,and L205 in Hy R3 region,and F229 in Hy R4 region have a significant effect when the enzyme adsorpt on polyester substrate surface.In addition,the experimental results also show that the G79 in the Hy R1 region has an important effect on the binding of the enzyme to the molecular chain,and I174,G175,L176,G177,G197,I200,I202,and L205 in the Hy R2 and Hy R3 regions may also be involved in binding process.The T82 in the Hy R1 region and P231 in the Hy R4 region may have space resistance effects during the enzyme-substrate binding process.(2)By adding the different substrate binding domain SBD of homologous PHBase to the single domain PCLase,the hybrid enzymes PCLase S1,PCLase S2 and PCLase S12 containing different substrate binding domains were constructed.Then we detected that hybrid enzymes’ adsorption capacity of PCL polyester surface and degradation capacity of PCL polyester and other substrates.The results showed that the capacity of the hybrid enzyme on the adsorption of PCL polyester surface has been enhanced,PCLase S1 and PCLase S2 on the solid substrate adsorption rate was1.1 times and 1.03 times compared with wild type,The adsorption capacity of PCLase S12 on the solid substrate was the same as that of the wild type.These hybrid enzymes has no significant change in the degradation activity of the PCL emulsion substrate,but the degradation efficiency of the PCL film was significantly improved.Compared with wild type,the degradation rate of the PCLase S1,PCLase S2,and PCLase S12 were 1.29 times,1.13 times and 1.05 times,respectively.It indicates that the degradation of PCL may promote the change of the surface of the thin film,and the exposure of the crystalline region is more conducive to the adsorption of the substrate-binding domain.In addition,we tested the enzymatic properties of the hybrid enzyme,and the results showed that the temperature stability of the hybrid enzyme was increased when the substrate binding domain was added.The most optimal reaction temperature of PCLase with SBD1 and SBD12 was increased from 55°C to 60°C.The p H stability of PCLase with SBD2 and SBD12 was improved under acidic conditions.This part of the experimental results shows that the substrate binding domain has a modification effect on PCLase,not only can increase its adsorption and degradation on the solid substrate,but also can improve other properties of the enzyme.Molecular modification of the polyester degrading enzyme by domain recombination may be an effective means to improve the enzymatic degradation activity and properties.