The Study On Substrate-Binding Mode Of A Thermophilic PET Hydrolase ICCG And Engineering The Enzyme To Enhance The Hydrolytic Efficacy

Polyethylene terephthalate(PET)is one of the most widely produced and used plastics,but the large amount ofPET waste that accumulates in the environment has become a serious threat to the ecosystem.The application ofPET hydrolases to depolymerisePET is an attractive measure to managePET pollution.AlthoughPET can be degraded by enzyme-mediated methods,to achieve the highest degradation effect,the enzyme needs to operate within or over this range glass-transition temperature range ofPET(65-70℃).In order to improve the efficiency of LCC,Tournier et al.introduced four amino acid mutations(F243I/D238C/S283C/Y127 G,named ICCG)with enhanced thermostability that can depolymerise 90% of amorphous or pretreatedPET material within 10 h at 72℃.The resulting TPA monomer can be used to resynthesisePET in line with commercial standards.In order to reveal the substrate binding mechanism of ICCG,the complex structure of the inactive mutant ICCG＿S165A and mono(2-hydroxyethyl)terephthalic acid(MHET)asPET analogue was obtained by soaking method.The complex structure shows that MHET is bound in a surface cleft composed of W190,V212,H242,I243,H164,A97,Y95 and M166.The carbonyl O of MHET forms a hydrogen bond with the backbone NH of Y95 and the carbonyl C is within the attack distance of the catalytic residue S165 of the wild-type enzyme.This indicating that MHET binds in a state that is prone to enzyme-catalyzed reactions.Two of the four mutations in ICCG(G127 and I243)are located near the MHET binding site,of which G127 is located on the surface of the protein,adjacent to the MHET binding cleft.It is speculated that G127 can allow the enzyme to contact the surface ofPET to function.The substitution of I243 expanding the substrate binding channel and increasing the capacity to bindPET.In this study,27 mutants were constructed with the goal of increasing the thermostability of ICCG by(1)increasing proline or hydrophilic interactions on the protein surface or(2)increasing hydrophobic interactions within the protein.All mutants were found to completely depolymerise the amorphous GfPET(GoodfellowPET film)at 70°C in 24 h.Most enzymes showed similar activity at 80°C,with only A59 H,A59R,A59 K,V63I,V75 R and N248 P showing higher activity than the original ICCG at 90°C.According to a recently discovered LCC homologous enzyme with high thermostability,which also carries a proline at position248.The seven triple mutants were constructed based on the retention of the N248 P in combination with other beneficial mutations and their activities were tested at the same temperature.All triple mutants showed higher activity than the parental ICCG at 90°C.The optimum reaction temperature was determined using GfPET,CcPET(commercialPET container)and ReinforcedPET(contains 30% glass fiber)as substrates.All enzymes showed the highest activity at 74°C when GfPET were used as substrates,but the mutants produced more hydrolysis products(～6 to 8 m M)than ICCG(～5 m M).The results were similar to the former toward CcPET.The optimum reaction temperature for the triple mutant is 80-85°C when using reinforcedPET as the substrate,compared to 80°C for ICCG.At the optimum reaction temperature,the amount of hydrolysis products produced by the mutant enzymes(～450 to 600 μM)was higher than that of ICCG(～360 μM).ThePET hydrolytic activity in a prolonged duration of KIP,KRP and RIP was examined by using ICCG as a control.The results showed that the concentrations of ICCG hydrolysis products were 600 μM at 80°C and 530 μM at 85°C in 7 days,respectively.All triple mutants exhibited higher hydrolytic activity than ICCG,releasing products up to 1070 μM.These results indicate that mutants have higher thermostability.Differential scanning calorimetry analysis showed that the melting temperatures of RIP,KIP and KRP were 3℃ higher than those of ICCG(98.0,98.9 and 98.6℃ vs 95.2℃,respectively)In this study,the structural details of KIP,RIP and KRP mutants were obtained by X-ray crystallography to explore the molecular mechanism of ICCG mutants with improved thermostability.In the KIP structure,The introduced P248 stacks with the adjacent indole ring of W104 may also provide additional interactions between the β8-α6 loop where it is located and α1.The methyl side chain of I63 strengthens hydrophobic interactions within the protein together with F71 and L117.K59 is located on the outermost β-strand,stabilizing the local structure through packing forces with Y78.Similar to K59 in KIP,R59 of RIP forms a hydrogen bond with OH of Y78.In the KRP structure,P248 and K59 have the same effects as in KIP.R75 forms two additional hydrogen bonds with Y77 and R107,resulting in the side chain of R107 being pushed towards R75 by a distance of about 1 ?.In summary,the introduction of hydrophobic proline,hydrophilic interactions on the protein surface and hydrophobic interactions in the protein interior have been recognized as important factors to improve the thermostability of the protein.This study suggest that the higher thermostability ofPET hydrolytic enzymes should be beneficial toPET biodegradation.It is of great important to understand the mechanism of action of the thermostablePET hydrolytic enzyme.The study provides pivotal guidance for designingPET biodegradation machineries and shall be beneficial to the development ofPET biodegradation platforms.