Study On Enhancement Of Catalytic Activity And Thermostability Of ?-glucosidase And PET Hydrolase Through Rational Design

Enzymes are a key link in the development of biofuel technology,the high catalytic activity and high thermal stability of enzymes are desirable qualities in industrial production.so,it is of great significance to improve the catalytic activity and thermostability of enzymes for their industrial application.Currently,there are mainly three strategies for enzyme engineering including rational protein design,irrational protein design and semi-rational protein design.Although rational protein design could be greatly helpful to reduce the experimental workload,its efficiency is relatively low.Recently,with the development of bioinformatics and the indepth understanding of protein structure,the efficiency of rational protein design has been greatly improved.In this study,the catalytic activity and thermostability of?-glucosidase TrCellb from Trichoderma reesei and PET hydrolytic enzyme IsPETase from Isdeonella sakaiensis were improved through protein engineering based on bioinformatics analysis.TrCellb catalyzes the synthesis of oligosaccharides using glucose as substrate,however the low reverse hydrolytic activity limits its scaled-up industrial application.Therefore,we proposed a strategy of HIFEA based on hydrophobic index to improve the catalytic activity,and at the same time proposed a C-terminal-mediated multimerization strategy to enhance the thermostability of TrCellb.Furthermore,two strategies were combined to improve the catalytic activity of TrCel1b.Finally,the strategy of C-terminal-mediated multimerization was applied in the rational design of IsPETase.The HIFEA strategy based on the hydrophobic index and the C-terminal-mediated multimerization strategy provide a new reference for the rational design of other enzymes.The main results were listed as following:1 Rational evolution of TrCel1b to improve its reverse hydrolysis activityThe gene sequence of ?-glucosidase TrCellb was obtained by reverse transcription technology,which was 1455 bp in length and encoded a total of 484 amino acids and the efficient heterologous expression of ?-glucosidase TrCel1b in Escherichia coli is achieved.The three-dimensional structure of TrCel1b was obtained by SWISS-MODEL and docked with cellobiose as the model of disaccharides.Based on the analysis of the docking results,we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis activity and the decrease of the hydropathy index of the key amino acid in catalytic site may be beneficial to the synthesis of disaccharides.To verify our deduction,the Hydropathy Index For Enzyme Activity(HIFEA)strategy was devised.Three hydrophobic amino acid residues in the catalytic site were mutated into hydrophilic residues,which generated the maximal change in the hydropathy index.Five variants TrCellbW173H,TrCel1bI174s,TrCeI1bI177S,TrCel1bI177S/I174S,and TrCel1bI177S/I174S/W173H were obtained.Additionally,a negative variant TrCel1bN24o1 was obtained to improve the hydrophobicity in the catalytic site.The production of synthesized disaccharides by the six variants were investigated.The maximal productions of total disaccharides of TrCel1b,TrCel1bW173H,TrCel1b1774S,TrCel1bI177s,TrCel1bI177S/1174S,and TrCel1bI177S/I174S/W173H were 44.0,32.9,80.2,148.8,152.9,195.8 mg/ml/mg enzyme.The production of synthesized total disaccharides of TrCell bI177S/I174S/W173H was increased by 3.5 times,compared to that of the wild type.However,the negative variant TrCel1b,N2401 lose disaccharide synthesis activity.This HIFEA strategy based on hydropathy index provides a new perspective for the modification of enzyme catalytic activity.2 Rational design of ?-glucosidase TrCel1b to improve its thermostabilityBioinformatic analyzing structures of reported ?-glucosidases belonging to GH1 family from thermophilic bacteria revealed that most of them forms multimers.Furthermore,the amino acid sequences of TrCellb and hyperthermophilic?-glucosidases PfCelB were aligned and found that the C-terminal sequence of the hyperthermophilic ?-glucosidase PfCelB was longer than the C-terminal sequence of TrCel1b protein.Previous studies showed that the C-terminal sequence of the enzyme was related to its thermal stability.We speculated that the multimerization of?-glucosidase may help it resist the stress of heat denaturation caused by high temperature.Therefore,a hypothesized was proposed that protein multimerization is related to its thermostability,which can be realized by C-terminal sequence substitution.Based on this hypothesis,A strategy to enhance protein thermostability based on artificial multimerization were proposed.The C-terminus of TrCellb was modified with the C-terminus of PfCelB and then six amino acid residues(RVKVAA)at C-terminal of TrCellb were replaced with different amino acid residues(IPEEL,IPEELAHLA,IPEELAHLADLKF,IPEELAHLADLKFVTRK)from C-terminal of PfCelB.Thus,the variants-TrCel1b-H5,TrCel1b-H9,TrCel1b-H13,TrCellb-H17 were designed.Enzymatic properties showed that the optimal pH of TrCellb,TrCel1b-H5,TrCel1b-H9,TrCel1b-H13,TrCel1b-H17 was 6,6,7,7 or 7 respectively and their optimal temperature was 20,20,10,75 or 30 0C.the half-life period against thermal inactivation of TrCel1b,TrCel1b-H5,TrCel1b-H9,TrCel1b-H13,TrCel1b-H17 at 65? was 0.22,0.22,0.22,118.20 or 0.22 h and their specific activity at 65? was 0.02,0.48,0.10,64.46 or 0.12 U/mg.Compared with the wild-type TrCel1b,the optimal catalytic temperature and Tm value of TrCel1b-H13 was increased by 55? and 20?respectively,the half-life of thermal stability and hydrolysis activity was increased by 537 times and 3223 times respectively at 65?.To study the molecular mechanism of enhanced thermal stability of TrCel1b-H13,we crystallized TrCel1b-H13 and the result showed that TrCellb-H13 was a centrosymmetric trimeric structure.Site-directed mutagenesis indicated that five amino acid residues-E28,H60,E99,K106 and K471,located in trimeric interface of the adjacent subunits,were related to the thermal stability of TrCel1b-H13.They maintained the trimeric structure of TrCel1b-H13 through hydrogen bond and hydrophobic interaction.This strategy of the C-terminal mediated multimerization might be a starting point for the development of a low-cost alternative for increasing the thermal stability of protein in the future.3 Rational design of TrCellb-H13 to improve its catalytic activityTrCel1bI177S/I174S/W173H-H13 was obtained by combining the hydrophobic index based HIFFA strategy and the C-terminal mediated multimerization strategy,and it synthesized laminaribiose,sophorose and cellobiose with glucose as substrate.The maximal reverse hydrolysis activity of TrCel1bI177S/I174S/W173H-H13 was 47.9 mg/ml/mg enzyme and the hydrolytic activity was 0.003 U/mg at 30?.Compared with TrCel1bI177S/I174S/W173H,the reverse hydrolysis activity of TrCel1bI177S/I174S/W173H-H13 was enhanced by 100%at 45?,indicating that the reverse hydrolysis activity of TrCellbI177s/I174s/W173H-H13 was affected by C-terminal amino acid sequences.We speculated that the substitution of C-terminal amino acid sequences of TrCel1bI177S/I174S/W173H-HI3 may improve its thermostability,resulting in the increase of the reverse hydrolysis activity of TrCel1bI177S/I174S/W173H-H13 at 45?.4 Rational design of IsPETaseIsPETase consists of 290 amino acids encoded by 873 nucleotides,which has a core structure of eight ?/? sheets.The predicted molecular weight of IsPETase is 27.6 kDa and its isoelectric point is 9.41.Three strategies including loop substitution,B-factor and C-terminal mediated multimerization were proposed based on the structure of IsPETase.Results showed that the C-terminal mediated multimerization strategy was better than loop substitution and B-factor strategy.The optimal catalytic temperature of IsPETase and IsPETase-H 17 was 35 ?.The overall output of PET degradation products produced by IsPETase,IsPETase-H9 and IsPETase-H17 was 0.005 and 0.028 mg/ml at 45? for 10 d.Compared with wild-type IsPETase,the Tm value and catalytic activity of IsPETase-H9 were increased by 20.6? and 4.1 times.The thermostability of IsPETase-H9 may enhance through the formation of dimers.The dimeric structure of IsPETase-H9 showed that the hydrogen bonds and hydrophobic interactions between the ?-helix structure formed by the substituted C-terminal sequence and the adjacent subunits played an important role in maintaining the thermal stability of IsPETase-H9.