Study On The Biodegradation Of Polycaprolactone And Polybutylene Succinate By CutE

Cutinase is a multifunctional enzyme that can degrade and surface-modify a variety of soluble esters,insoluble triglycerides,natural polyesters,synthetic polyesters,etc.It has huge application potential in the fields of food,chemistry,and environmental protection.In recent years,it has been discovered that some cutinases have a degrading effect on synthetic polymer polyesters,but the specific degradation mechanism is still unclear.There are still few reports on how single-domain cutinases can be combined with solid-phase polyester materials.In this paper,the model cutinase CutE derived from F.solani pisi was selected and expressed heterologously in Pichia pastoris GS115,and the pure enzyme components were studied for the degradation characteristics and degradation process of two artificial high-molecular polyesters of polycaprolactone(PCL)and polybutylene succinate(PBS);at the same time,the molecules of cutinase CutE and solid-phase polyester substrate for adsorption and molecular chain binding were studied using site-directed mutagenesis technology basis.The specific research results are as follows:(1)Study on the expression,purification and properties of cutinase CutEThe cutinase gene of F.sonlai pisi was obtained by gene synthesis technology,and it was heterologously expressed in Pichia pastoris GS115,and purified by affinity chromatography to obtain a pure enzyme component with a molecular weight of 25 k Da.It has been tested that the recombinase has degrading activity on PCL and PBS polyester.Using two polyesters as substrates,the enzymatic properties of CutE were measured.The results showed that the optimal enzymatic reaction temperature is 50?,and it has good thermal stability in the range of 4??40?;the optimal p H value of the enzyme reaction is 9.0,and the corresponding buffer system is Tris-HCl buffer,which is very stable in the range of p H 3.0 to 11.0.The metal ions Mn²⁺and Ca²⁺at low concentrations promote the enzyme binding to the substrate,K⁺and Na⁺have no obvious effect in the process of enzyme binding to the substrate,while Zn²⁺and Co²⁺have a significant inhibitory effect on enzyme degradation of PBS,but the effect on its degradation of PCL is not significant;the organic reagents Tween-80 and Triton X-100 have a significant inhibitory effect on the enzyme,and methanol and ethanol only have a certain inhibitory effect at high concentrations.The products of cutinase CutE degradation of polyester were analyzed by mass spectrometry.The results showed that the main enzymatic hydrolysis products of PCL were hydroxycaproic acid monomers and dimers,and the main enzymatic hydrolysis products of PBS were succinic acid-butanediol monomers and some oligomers,including dimers,trimers,tetramers,etc.These products are all oligomers with alcohol end groups.(2)Study on the degradation process of cutinase CutE on solid-phase materialsThe cutinase CutE has a significant difference in the degradation rate of PBS and PCL solid-phase substrates.The results show that under the same enzyme concentration,the cutinase CutE degrades the PCL film at a much higher rate than the PBS film.At the same time,scanning electron microscope(SEM),differential scanning calorimeter(DSC)and thermogravimetry(TGA)were used to study the degradation process of the film.The results show that as the degradation rate increases,the film is gradually enzymatically degraded,the surface structure is gradually destroyed,and the stability gradually decreases.The crystallinity of the polyester increases first and then decreases,indicating that the enzyme preferentially degrades the amorphous area and exposes the crystalline area.The increase of crystallinity restricts the movement of polymer chains,thereby degrading the crystalline area,and reducing the overall crystallinity.At the same time,it was also found that the degradation of PBS and PCL films by cutinase CutE followed different degradation modes.The degradation of PBS was mainly surface erosion type,while for PCL,it followed a layer-by-layer degradation mode.(3)Research on the structural basis of cutinase CutE adsorption and binding to polyester substratesBy analyzing the structure of cutinase CutE,the Loop I region that may be related to its substrate binding was selected for research.Based on amino acid hydrophobicity and spatial structure,8 mutants were constructed using site-directed mutagenesis to detect its adsorption on solid-phase films,and simultaneously determine its binding and catalytic ability to soluble short-chain substrates and suspended long-chain substrates.The adsorption results showed that the adsorption rate of mutants L176A,V177A,L182A,I183A,V184A and L189A to PBS and PCL films decreased,indicating that the enzyme was adsorbed to the surface of the solid-phase polyester material through the hydrophobic effect of amino acids,among them,the 177 and 189 amino acids have the most significant influence on the adsorption.At the same time,these two amino acids are adjacent to the active center.It is speculated that they use their own hydrophobicity to anchor the active center of the enzyme at the position closest to the substrate surface,which is conducive to the occurrence of enzyme catalysis on the substrate.The results of the combination of cutinase and soluble short-chain substrates showed that the Km values of mutants N172A,T173A,L176A,V177A,L182A,I183A and V184A all decreased compared with the wild type,and only the Km value of L189A is higher than that of wild type,and its affinity for p NP substrate is significantly lower.Comparing the changes of Km value,it is indicated that positions 177,182,183 and 189 play a role in the process of enzyme binding to soluble short-chain substrates,and position 189 plays an important role in the process of enzyme binding to p NP substrate due to its proximity to the active center.The results of the binding of cutinase to the suspended long-chain substrate showed that the affinity of mutants L176A,V177A,L182A,I183A and V184A to PBS and PCL substrates all increased to varying degrees.Among them,177 and 184 have significantly increased affinity for PCL substrates;mutants L189A and N172A had increased affinity for PBS substrates,but significantly decreased affinity for PCL substrates.It indicates that the 172,177,184 and 189 amino acids may be the key positions that affect the molecular chain to enter the active pocket,which may be related to the position,structure and substrate structure of the amino acid.At the same time,it was also found that the role of amino acids at the same position in the process of enzyme interaction with substrates of different structures is also different.