Site Directed Mutant Design And Activity Study Based On 3D Model Of PHA Polymerase PhaC

Polyhydroxyalkanoates（PHAs）are high molecular particles deposited in cells by a variety of microorganisms under the condition of excessive carbon source and lack of nitrogen,sulfur and phosphorus.Because of its thermoplastic,hydrophobic,optical activity,biocompatibility and biodegradability,it has attracted much attention as a substitute for petroleum based plastics.PHAs synthetic strains of Pseudomonas have the ability to synthesize a variety of material properties from brittle to elastic.PhaC,the key synthetase of PHA synthesis from Pseudomonas sp.SG4502,has extensive substrate specificity,and the amino acid mutation near the activity center of PhaC1_Ps can change the substrate specificity and activity of the enzyme and create new PHAs.In this study,the 3D structure of PHA class II synthetase PhaC1_Ps derived from Pseudomonas sp.SG4502 and the known class I synthetase PhaC_Re of Cupriavidus necator H16 will be homologous modeled,and eight mutants will be constructed by designing mutation sites for the changes of synthetase spatial conformation caused by specific amino acid mutations that may affect the active center.Then eight mutant synthetase were expressed and purified by prokaryotic expression system,and the activities of eight mutants were studied by using eight substrates.Firstly,using SWISS-MODEL software,taking the PhaC_Re 3D structure of Cupriavidus necator H16 as the model,the 3D structure model was established by using the amino acid sequence of PHA class II synthetase PhaC1_Ps from Pseudomonas sp.SG4502.According to the spatial position of the catalytic triad（Cys117,Asp272,His300）,on the basis of considering the size,polarity and hydrophilicity of amino acids,single-point and double-point mutations（F52L/I/M/H,F52L/I/M/HA116L）were designed for amino acids at F52 and A116.The minimum binding free energy between the mutated synthetase and the substrates 2-hydroxybenzoic acid（2HBZ）,3-hydroxybenzoic acid（3HBZ）,4-hydroxybenzoic acid（4HBZ）and（R）-3-hydroxybutyric acid[（R）-3HB]was calculated by Auto Dock software,which proved the possibility of substrate polymerization of PhaC1_Ps mutant in theory.Secondly,the recombinant plasmid p QE-80L-pha C1 containing PhaC1_Ps of Pseudomonas sp.SG4502 was constructed and used as the template.The recombinant plasmids of eight mutants containing single point and double point mutations were successfully constructed by using KOD-Plus-Mutagenesis Kit.Nine engineering strains were screened by transferring the recombinant plasmid into BL21（DE3）:wild-type BL21-p QE-80L-pha C1,four single point mutations BL21-p QE-80L-pha C1_F52L/I/M/H and four double point mutations BL21-p QE-80L-pha C1_{F52L/I/M/HA116L}.Finally,wild-type and eight mutant engineering strains were cultured to express the target protein in vitro and purified with His-tag,and nine purified proteins were obtained.The concentrations of wild-type PhaC1_Ps were 3.2 mg/m L,the concentrations of single point mutants were 3.8 mg/m L,3.0 mg/m L,4.9 mg/m L and 4.0 mg/m L,and the concentrations of double point mutants were 2.7 mg/m L,2.6 mg/m L,3.1 mg/m L and 2.5mg/m L,respectively.The protein concentration increased partially after single point mutation and decreased after double point mutation.Using（R）-3HB,（R）-2-hydroxybutyric acid[（R）-2HB],3-hydroxypropionic acid（3HP）,D-lactic acid（D-LA）,（R/S）-3-hydroxycaproic acid[（R/S）-3HHx],（R/S）-2-hydroxyvaleric acid[（R/S）-2HV],（R）-mandelic acid[（R）-MA]and 4HBZ as substrates,the activities of eight PhaC1_Ps mutants were analyzed by 5,5′-disulfide bis（2-nitrobenzoic acid）（DTNB）.The results showed that when（R）-3HB,（R）-2HB and 3HP were used as substrates,the specific activities of PhaC1_Ps were 0.038 U/mg,0.107 U/mg and 0.046 U/mg respectively,and the specific activities of mutant PhaC1_F52L were significantly increased,which were0.052 U/mg,0.143 U/mg and 0.071 U/mg respectively.On this basis,the specific activities of mutant PhaC1_F52LA116L were roughly the same as those of PhaC1_F52L.When D-LA,（R/S）-3HHx,（R/S）-2HV,（R）-MA and 4HBZ were used as substrates,the specific activity of the mutant synthetase did not change significantly.These results showed that when F52 near the synthetase activity center was mutated to L,the activity of the synthetase to（R）-3HB,（R）-2HB and 3HP substrates could be significantly changed.