Expression And Modification Of Polyhydroxyalkanoate Depolymerase From Thermomonospora Umbrina And Its Application In The Degradation Of Stickies

The problem of stickies deposition is prone to occur during the production of recycled paper using waste paper as raw material,which can reduce the utilization rate of paper machine equipment and paper quality.The main component of domestic waste paper stickies is polyacrylate compounds,while the degradation efficiency of existing enzymes on polyacrylate is low,and there is no natural dedicated degradation enzyme for this type of polyester.This study explores and modifies the production of polyhydroxyalkanoate depolymerase（PHAD）with heat resistance,and investigates its degradation performance on typical representatives of PHA,polyhydroxybutyrate（PHB）,two stickies mode substrates,poly（ethylacrylate）（PEA）,poly（methyl methacrylate）（PMMA）,and actual stickies.The main research results are as follows:（1）PHAD screening and characterization of the enzymatic properties of Thermomonospora umbrina derived PHAD（TumPHAD）.Through database screening,structural simulation,and docking analysis,three PHA depolymerases were selected as target sequences and expressed in Escherichia coli BL21（DE3）.Among them,Shy PHAD and TumPHAD were well expressed,and their degradation ability to PHB and PEA was verified at50℃.When TumPHAD was treated with PHB powder for 4 hours,the degradation rate tended to stabilize at 4.2%.After re adding TumPHAD,the degradation rate increased to 5.8%,indicating poor thermal stability of the enzyme.TumPHAD can also degrade PEA.After 4hours of reaction,the turbidity of the reaction system decreased by 53.8%,the particle size decreased by 29.9%,and the absolute potential value increased by 41.5%.At the same time,the study also found that TumPHAD had better degradation ability for PHB and PEA than Shy PHAD.Therefore,TumPHAD was selected for purification and enzymatic properties determination.The results indicate that the specific activity of TumPHAD is 15.2 U·mg^-1,with an optimal temperature of 40℃and p H of 8.5.The half-life at 50℃is 20 minutes,indicating that TumPHAD has poor thermal stability and cannot meet industrial application conditions.（2）Thermal stability modification of TumPHAD.Through molecular dynamics simulation,the structure of TumPHAD was analyzed,and root mean square fluctuation data showed that amino acid residues within some ranges were relatively flexible.Based on the prediction results of Discolfide by Design 2,six mutant combinations incorporating disulfide bonds were designed and constructed.Among them,the half life of mutant A190C/V240C at 50℃was 7hours,21 times that of TumPHAD,with a specific activity of 15.4 U·mg^-1.The optimal temperature was increased from 40℃to 60℃,and the optimal p H was increased from 8.5 to9.0.Further analysis was conducted on the RMSF of mutant A190C/V240C,and the results showed that the flexibility of amino acid residues in the range of 180-200 and 230-240 positions of mutant A190C/V240C was lower than that of TumPHAD.（3）Research on the Application of Mutant A190C/V240C.Firstly,the degradation ability of mutant A190C/V240C to PHB was investigated,and it was found that its 4-hour degradation rate of PHB at 50℃was 12.0%,which is 2.9 times higher than the wild-type TumPHAD.Further research was conducted on the degradation of adhesive mode substrates by mutant A190C/V240C under 50℃conditions.Compared with the Humicola insolens derived keratinase（Hi C,better application performance than commercial enzyme Optimyze（?）525）obtained in our laboratory earlier,the turbidity and particle size of the PEA degradation system by mutant A190C/V240C were lower,and the absolute potential value was higher;At the same time,it also has excellent degradation ability for PMMA,with a degradation rate of 20%at 48hours.A large number of pores were observed on the surface of PMMA using scanning electron microscopy.Fourier transform infrared spectroscopy detection showed that the functional group change of PMMA film was more significant than that of Hi C treatment.By comparing the degradation ability of mutants A190C/V240C and Hi C on adhesive through turbidity changes,the turbidity of the mutant A190C/V240C group increased by 0.440 and the Hi C group increased by 0.361.The results showed that the degradation ability of the mutant A190C/V240C was stronger.Finally,the change in viscosity was measured using the inclined rolling ball method.After degradation by mutant A190C/V240C at 50℃,the viscosity of the adhesive decreased by 50%.