Molecular Modification Of Clostridium Thermocellum Endoglucanase And Cellobiase And Changes In Fusion Enzyme Activity

Endoglucanase CeID and cellobiase Ce1S that were known as the major active endo-cellulose and exo-cellulase in Clostridium thermocellum have received much attention in recent years.CelD randomly hydrolyzing ?-1,4-glycosidic bonds of the internal non-crystalline region of cellulose,and truncates the long-chain cellulose molecules to long-chain dextrin fibers.Crystalline cellulose can be hydrolyzed into cellobiose with the interaction of CelD with CipA.But now there are many problems of the application of CelD and CelS in cellulose degradation industrial.(1)CelD and CelS were found in C.thermocellum,a thermophilic anaerobic bacterium,thus it's difficult to study them in this circumstance;(2)Although there are studies of CelD and CelS cloned in E.coli,they are in the form of inclusion bodies,which therefore can not meet the demand of industrialization;(3)The activities of instable recombined CelD and CelS that expressed in E.coli were little in degrading crystalline cellulose.The gene celD from C.thermocellum encoding endoglucanase was cloned into high-level expression vector pHsh to produce recombinant enzyme CelD.Molecular modification was applied to constructed fusion enzyme NcelD by replacing the dockerin coding sequence in the C terminus of celD to CBD(Cellulose Binding Domain)coding sequence.Both CelD and NcelD were over-produced in E.coli in soluble form.CelD and NcelD were purified by heat treatment and an anion exchange chromatography.Enzyme activity analysis showed that the activity of NcelD to CMC and Avicel cellulose increased 86.32%and 115.53%respectively compared with that of CelD.The purified CelD and NcelD both show the highest activities at pH 4.5.The pH impacted the activity of CelD and NcelD significantly.The relatively activities were higher at the pH4.5 between 5.5 than others.The activities of CelD and NcelD decreased sharply when pH value was less than 4.5 or greater than 5.5.The most interested result was that the activity(Moore catalytic activity used in the work)of NcelD was significantly higher than the original CelD's under the same pH reaction conditions.In aspect of the enzyme stability of pH,CelD and NcelD were extremely unstable on the condition of pH4.0.The residual enzyme activity was less than 20%after 60 minutes incubation,and it was higher in the pH4.5?8.0,it is showed no loss on the condition of pH5.0?7 after 60 minutes in cubation.The results showed that the pH stability ofNcelD and the original CelD was similar,but the pH stability of NcelD in pH4.0 and pH8.0 was better than that of original CelD.The optimum reaction temperature of CelD and NcelD was 60? and 55?respectively.The relative activity of CelD were above 50%between 40??75?,and the relative activity was more than 80%between 40??70?.The molar catalytic activity of NcelD was significantly higher than CeD under the same reaction conditions of temperature especially at 55? on the substrate CMC.The residual activity of the CelD and NcelD was 92.5%and 93.51%respectively after incubation at 45? for 1 hours.The half-life of the CelD and NcelD was about 1 hours at 70?.The half-life trend on temperature stability of CelD and NcelD was similar.The temperature stability of NcelD was slightly better than the CelD.Compared with CelD,the NcelD stability in 45-70? temperature range was better,which was more conducive to the industrial application of endoglucanase.The gene celS from C.thermocellum ATCC 27405 encoding cellobiase was cloned into expression vectors pHsh.Molecular modification was applied to the gene celS,SDS-PAGE analysis shows the recombinant CelS did not expressed in E.coli in soluble form.Than the celS gene was cloned into extracellular expression vector pHsh-ex.The expression systempHsh-ex could secrete heterologous protein into the periplasmic space,reduce the concentration of intracellular proteins and facilitate correct folding of the heterologous protein.The vector pHsh-ex-Ce1S was constructed for secreting expression in E.coli.Enzyme activity assay and SDS-PAGE analysis showed that in periplasmic space protein,the soluble expression of CelS was at low level and weekly enzyme activity was increased,Due to the large molecular weight of CelS(82 kDa),or the signal peptide of vector pHsh-ex was not suitable in expressing cellobiose gene.