Fundamental Research On Bio-enzymatic Degradation Mechanism Of Anionic Polyacrylamide

Coal has always been occupied a dominant position in China’s energy structure,and the coal flotation is an effective way to realize its clean utilization.With the mechanization of coal mining,the grade of raw coal is decreased,and a large amount of coal slime water is urgent to be clarified treatment.Anionic polyacrylamide（HPAM）,as coal preparation flocculant,is often used for flocculation settlement of coal slime water.With the widespread use of HPAM,the difficulty and cost of treatment is increased by a large amount of polymer-containing wastewater accompanied.If sewage containing-HPAM enters the ecosystem,it will have a certain impact on the environment.HPAM itself is non-toxic,but it is easily broken down by physicochemical factors into the carcinogenic acrylamide（AM）monomer with cumulative neurotoxicity.Biological method has many advantages,such as,low cost,green,environmentally friendly and no secondary pollution etc.Hence,this method is used to degrade HPAM.In order to better degrade HPAM,the project carried out the interaction of enzymes with substrate,the dynamic transformation of complexes and the enzymatic degradation mechanism from the perspective of biological enzymes.These laid a foundation for revealing the enzymatic degradation process of HPAM and provided site supports for future mutation tests to modify the enzymes.Firstly,taking the representative polymer structure model with structural units of2～5 and the biological enzymes with known structure as the research entry point,the binding of enzyme to substrate and the characteristics and rules of enzymatic degradation were studied at the atomic or molecular level through molecular simulation.Among which Rhodococcus sp.amidase（Rh Amidase）,Bacillus subtilis laccase（Lac）and manganese peroxidase（Mn P）were selected.Then,the supposed mechanism of HPAM co-degraded by Rh Amidase and Lac was proposed combined with the degradation tests of HPAM by mixed bacteria.Finally,the HPAM degradation mechanism hypothesis by Mn P was proposed combined with the degradation experiments of HPAM by fungi.The primary conclusions are as follows:Rh Amidase-HPAM-2 enjoyed the highest affinity,and HPAM-2 was the most suitable substrate for Rh Amidase,while the binding of Rh Amidase with PAA-2 was the best.Rh Amidase tended to degrade the short-chain polymers.Strong hydrogen bond was the main reason for stabilizing the Rh Amidase-substrate complexes.SER171 and GLY193 had been involved in forming hydrogen bonds,and PHE146 had been participated in producing hydrophobic interactions.PHE146,ILE450,LYS96 and GLY193 were key residues in Rh Amidase degrading HPAM-2.GLY193 in Rh Amidase maximally affected its positive affinity with HPAM-2 mainly through forming the hydrogen bonds.ASP191ALA could improve the affinity of Rh Amidase with HPAM-2.Rh Amidase-HPAM-2 had the lowest total interaction energy between enzyme and substrate at 303 K.The skeletal vibration of enzyme at 303 K was smaller,and it reached equilibrium earlier than that at 298 K.The maximum fluctuation residues at 303 K was located in 0～50 in which had the smallest fluctuation compared with 298 K and 308 K.Thus,Rh Amidase-HPAM-2 had the optimal binding stability at303 K.The larger skeleton vibration of the tail residue region in Rh Amidase at 308 K resulted in the maximum RMSD values.Hence,Rh Amidase-HPAM-2 had the worst binding stability at 308 K.Lac-HPAM-3 acquired the highest affinity,and their binding was the optimal.HPAM-3 was the most suitable substrate for Lac.HPAM was more easily degraded by Lac than PAA.Lac could accommodate HPAM and PAA with a certain carbon chain length,but it was difficult to degrade high molecular weight polymers.Lac-HPAM-3had the highest affinity mainly due to the salt bridge.TYR133 was involved in forming hydrogen bonds to stabilize Lac-HPAM-2,Lac-HPAM-3,and Lac-HPAM-4.ARG487was involved in forming hydrophobic interactions to stabilize all Lac-substrate complexes.TYR118,TYR133,ARG487 and LYS135 in Lac were key residues for degrading HPAM-3.LYS135 had the greatest effect on the affinity of Lac with HPAM-3 by salt bridges,followed by the hydrophobic interaction by ARG487.Lac-HPAM-3 had the lowest total interaction energy between enzyme and substrate at298 K.The skeletal vibration of enzyme at 298 K was smallest.Moreover,all residues in Lac had the minimum RMSF values.Thus,Lac-HPAM-3 had the optimal binding stability at 298 K.The larger skeleton vibration in Lac at 308 K resulted in the maximum RMSD values.At this time,HPAM-3 deviated from the initial docking position.Hence,Lac-HPAM-3 had the worst binding stability at 308 K.The optimal degrdation time by mixed bacteria was 7 d.Meanwhile,the optimum parameters were determined by orthogonal experiment as temperature 35℃,p H=7 and inoculated dosage 2 m L.Under these conditions,the degradation rate was 39.24%.The effect of factors on the degradation rate followed a decreasing order,namely,temperature>p H>inoculated dosage.Accordingly,the HPAM co-degradation mechanism was put forward by amidase and laccase:1)The amide side chain of HPAM was degraded into-COOH under Rh Amidase to produce carbon skeleton PAA and NH₄⁺.The NH₄⁺released could be used as nitrogen source for bacterial growth;2)The residual PAA was oxidized and broken down under Lac to provide carbon source for bacterial growth.Mn P could accommodate a certain length of HPAM carbon chain.Mn P-HPAM-5had the highest affinity,and their binding was optimal.HPAM-5 was the most suitable substrate for Mn P.Strong hydrogen bond was the main reason for why the affinity of Mn P-HPAM-5 was the highest.ASP23,PHE93 and LYS96 were key residues in Mn P degrading HPAM-2.ASP23,PHE93,LYS96 and PHE353 were key residues in Mn P degrading HPAM-3.THR27,LYS96 and PHE353 were key residues in Mn P degrading HPAM-4.THR27,PRO92,PHE93,LYS96 and PHE353 were key residues in Mn P degrading HPAM-5.LYS96 was a key residue in all complexes,and Mn P-HPAM-5 had the largest number of key residues.THR27ILE could enhance the affinity of Mn P to HPAM-4.The vibration of enzyme skeleton was the minimum,and its residue region of 350～360 contained the minimum RMSF when Mn P binding to HPAM-4.Therefore,Mn P had the optimal binding stability with HPAM-4.All HPAM model compounds only vibrated near the binding site of Mn P.According to the results of simulation and degradation test,the mechanism of Mn P degrading HPAM was proposed:1)Mn P oxidized Mn²⁺into Mn³⁺by H₂O₂ under acidic conditions.2)Mn³⁺absorbed an electron from the secondary and tertiary carbon of HPAM and initiated a free radical chain reaction,which produced two cationic radicals without breaking the chemical bond.Moreover,Mn³⁺was reduced to Mn²⁺for recycling.Figure[60];Table[18];Reference[235]...