Reconstruction Of Thermostable Cellulases With Dual Catalytic Domains

Lignocellulose is a renewable resource that can be used to produce biofuels and other high value-added products.However,conversion of lignocellulosic biomass into fermentable sugars is a challenging task due to its complex plant cell wall structure.In lignocellulosic biomass,cellulose is wrapped between hemicellulose and lignin,which is difficult to be degraded by cellulase due to their covalent connection and the cross-linking of cellulose microfibers.Pretreatment of lignocellulose can destroy the hydrogen bond and enhance the accessibility of cellulase.However,conventional acid-base and ionic liquid pretreatment not only increase the production cost,but also lead to serious environmental pollution.CelA is a natural thermostable cellulase with dual catalytic domains from thermophilic bacterium Caldicellulosiruptor saccharolyticus.Crystalline cellulose?Avicel?can be directly hydrolyzed by CelA.Based on this discovery,the cellulase from thermophiles was reconstructed to obtain the biocatalytic thermostable cellulase for degradation of natural lignocellulose.In this study,the Exo5 and Endo5 genes of C.saccharolyticus were linked and inserted into the expression vector pET28a via the common restriction site.In order to improve the degradation of natural substrates,different carbohydrate binding modules?CBMs?were introduced between the two catalytic domains.Fused enzymes were successfully expressed in Escherichia coli,and a series of thermostable cellulases with bicatalytic domains were obtained.The optimal temperature,optimal pH,thermal stability,pH stability,influence of SDS,EDTA and metal ions on the activity of fusion enzyme and substrate specificity of the enzyme were tested.Fused enzymes possessed both?-glucosidase and exoglucanase activities.Filter paper?FP?,Avicel,steam-pretreated rice straw?SPRS?,rice straw?RS?and wheat straw?WS?were efficiently degraded by the action of these fused enzymes.Specific activities of the fusion enzymes on Avicel reached 34.4⁷6.4 U/?mol,which was 1.2².7 times as much as CBM3b-Exo5.The activity of Bgl1-3CBM-Exo5 on sodium carboxymethyl cellulose?CMC-Na?was 2.9 times as much as Bgl1-CBM-Exo5 and 1.5 times as much as Bgl1-2CBM-Exo5,respectively.The activities of these enzymes on CMC-Na were increased by128¹92%when 10 mM MnCl₂ added.The optimal temperature of Bgl1-3CBM-Exo5 was 70^°C,same as Bgl1.The optimal temperatures of the other two enzymes were 80^°C,same as Exo5.The optimal pH of fused enzymes was 4.5 same as that of Exo5.However,The optimal pH of fused enzymes was decreased by 1.0 compared to Bgl1.FP,Avicel,SPRS,RS,and WS were effectively degraded by these fused enzymes.Specific activities of the fusion enzymes on Avicel reached 0.167 U/mg,which was increased by 14.0 times as much as parental Endo5-CBM28.The acticity of Endo5-2CBM-Exo5 on CMC-Na was 4.2 times as much as Endo5-CBM3b-Exo5 and 1.9 times as much as Endo5-CBM28-Exo5,respectively.The activities of these enzymes on CMC-Na increased from 121 to 141%when10 mM MnCl₂ was added.The optimal temperature of Endo5-CBM3b-Exo5 was60^°C,which was reduced by 15^°C compared to Endo5.On the other hand,the optimal temperature of the other two enzymes was 80^°C,which was the same as Exo5.The optimal pH of Bgl1-2CBM-Exo5,Endo5-CBM3b-Exo5 and Endo5-CBM28-Exo5 were 4.0,4.5 and 4.5,respectively.However,The optimal pH of fused enzymes was decreased by 1.0¹.5 compared to Bgl1.The results indicated an improvement in the activity of bifunctional cellulases with CBMs on lignocellulose.CBMs contributed to further deconstruction of Avicel and other natural substrates.This research henceforth has laid the foundation for lignocellulosic conversion and application.