Heterologous Expression And Functional Studies Of Endogenous Endoglucanase Of Reticulitermes Flavipes

The lower termite gut is a natural bioreactor that can destroy the recalcitrance of lignocellulosic biomass for further degrading and utilizing the polysaccharide components.Pretreatment of the lignocellulosic biomass to remove its structural resistance barrier mainly processed in the foregut and midgut of the lower termite,which is benefit for the symbiotic microbial community of the termite hindgut to degrade the exposed polysaccharide components by secreting various enzymes.Endoglucanase(EG)is regarded as one of the main enzymes for cellulose hydrolysis.Some studies have found that EG is mainly distributed in the termite hindgut,and secreted by termite symbiotic microorganisms.However,our previous studies showed that a large amount of EG was endogenously expressed by the termite host in the foregut and midgut,where the lignin structure barrier is removed.In order to further explore whether the endogenous EG of termites played a role in the removal of the resistance barrier of lignocellulose,an endogenous RfEG from Reticulitermes flavipes was studied.The Rhodococcus opacus PD630 as a new heterologous expression system on secretory expression of EG was explored.And the enzymatic properties of recombinant EG protein obtained by heterologous expression of the system was compared with that of the recombinant protein obtained by the traditional protein expression system Escherichia coli and Pichia pastoris.Moreover,the relationship between the reaction of RfEG and Fenton reaction in the process of removing the resistance barrier of lignocellulose structure was further explored.The main research results are as follows:1.R.flavipes intestinal transcriptome data was obtained from a public database,which showed it had strong ability of lignocellulose degradation according to functional annotation analysis.The endogenous RfEG gene sequence was obtained from R.flavipes,and the protein structure of RfEG was analyzed by bioinformatics.RfEG belongs to the glycoside hydrolase family 9 and has the typical slit-type catalytic active center of cellulase.Compared with the classical endoglucanase structure,there are 2 additional?-sheets and 8 short?-helices on RfEG.2.The heterologous secretory expression system of RfEG with R.opacus PD630 as the host was constructed.The signal peptide OPAG?Pro,which can efficiently enhance the secretion of RfEG,was obtained by screening the biologically active of extracellular recombinant RfEG?Ro protein.The nitrogen source concentration for the protein expression was optimized.In addition,the recombinant protein RfEG?Ec and RfEG?Pp were obtained by heterologous expression of RfEG using the traditional protein expression system E.coli BL21(DE3)and P.pastoris X-33.RfEG?Ec was completely inactive.The enzymatic properties of RfEG expressed by PD630 and P.pastoris X-33 revealed that the proteins secreted by the two different expression systems have significant differences in specific activity,thermal stability and the optimum temperature and p H.The specific activities of RfEG?Ro and RfEG?Pp with CMC-Na as substrates were 138.9 U/g and 63.8U/g,respectively.The thermal stability of RfEG?Ro was significantly higher than that of RfEG?Pp,and the residual activity of 48.97%for RfEG?Ro was obtained after incubation at 50?for 2 hours.The p H optima of RfEG?Ro is around 5.0,while the optimal p H of RfEG?Pp is around 4.0.The specific activity of RfEG?Ro is 93.58 U/g at p H 6.0(similar to the p H condition of termite foregut),which is higher than that of 53.38 U/g RfEG?Pp.These results indicate that R.opacus PD630 is more suitable for heterologous secretory expression of RfEG.3.RfEG and Fenton reaction with low H₂O₂ concentration could have a synergistic effect on the removal of the resistance barrier of lignocellulose structure,thereby improving the pretreatment efficiency of lignocellulose.The results showed that after pre-treated by RfEG and Fenton reaction with 50?M H₂O₂ concentration,the enzymatic saccharification of the milled poplar significantly increased by 16.54%.Environmental scanning electron microscopy showed that the surface of poplar powder was destroyed seriously after RfEG-Fenton treatment.Py-GC/MS analysis further showed that synergistic treatment of RfEG and Fenton reaction can significantly reduce the lignin content in poplar wood.In summary,the non-typeable protein expression host R.opacus PD630 was used for heterologous secretory expression of endogenous RfEG from termite,and it was proved that the obtained protein had higher superiority in enzymatic properties than that obtained by traditional yeast expression system.This result provides a new idea for the selection of heterologous expression systems of animal endogenous proteins.The joint treatment of milled poplar with RfEG and Fenton reaction further reveals that RfEG has played an important role in removing the lignin structural barrier,in addition to the traditional cellulose hydrolysis function.These works establish a now theoretical basis on the research of lignocellulosic biomass degradation mechanism and also provide a new idea for the design of the efficient biorefinery process of lignocellulose?...