Heterologous Expression Of Lignocellulase Genes From Termites And Their Symbiotic Bacteria

Lignocellulose is the most abundant renewable resource on earth.Natural lignocellulose materials contain cellulose,hemicellulose and lignin.Cellulose is the most simple component of lignocellulose.Hemicellulose and lignin are wrapped around the cellulose to form a natural barrier.Except protecting the plants,this natural barriers makes the biodegration conversion artificially inefficient and cannot meet the requirements of large-scale industrialization.There are some natural and efficient lignocellulose degradation systems in nature.Lignocellulase from termites and their symbiotic bacteria has high enzyme activity and good alkali resistance.In this paper,the synergistic effect of lignocellulase was explored with termites.At the same time,an anaerobic bacteria,Dysgonomonas macrotermitis?was isolated from the hindgut previously.Dysgonomonas is the second dominant group of intestinal tract bacteria.The strain D.macrotermitis has lignocellulose degradation ability.In order to obtain lignocellulase with better enzymatic properties and accelerate its industrial development.The main results of this study are summaried as follows:Cloning and expression of xylanase genes from D.macrotermitis in the hindgut of M.barneyi.Five xylanase genes were identified by genome sequencing and blast comparison.These gene fragments were cloned and expressed in E.coli JM109.Only DysxynB(orf-00078)and DysxynE(orf-03642)had xylanase activity,and their molecular weights were 40 kDa and 33 kDa,belonging to GH10 and GH11 families respectively.The optimum temperature and pH of the two xylanases were 45? and 6.5-7.0 respectively.The two xylanases had good alkali resistance.The specific activities were 259.5 U/mg and 151.2 U/mg respectively.The results of TLC showed that the two xylanases had no activity of beta-xylanase,and they were both beta-1,4-xylanase.Cloning and expression of beta-1,4-endoglucanase genes from D.macrotermitis of M.barneyis hindgut.beta-1,4-endoglucanase was found by genome analysis and blast comparison of D.macrotermitis.We successfully cloned it in E.coli JM109.Only DysengE(orf-01678)belonging to GH5 family had EG activity and its molecular weight was 20 kDa.The optimum temperature and pH of the EG enzyme were 40 ? and 8.0.The specific activity of the EG enzyme was 0.58 U/mg.Bioinformatics analysis showed that DysengE enzyme was the only GH5 family source of termite intestinal symbiotic bacteria so far.Four lignocellulase genes encoding ?-glucosidase,endo-1,4-?-glucanase,xylanase and CotA from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli.After SDS-PAGE analysis,the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities.We used phosphoric acid swollen cellulose(PASC)as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme.The co-expressed enzymes increased the degradation efficiency of PASC by 44%compared with the single enzyme mixture;while the degradation rate increased by 34%and 20%,respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates.The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.Secretory expression of xylanase from Paenibacillus sp.termites in Pichia pastoris.Xylanase has good prospects for industrial applications.Secretory expression can greatly reduce production costs.At the same time,yeast cells have advanced foreign protein folding system,strong secretory capacity and post-processing ability.So P.pastoris GS115 was selected to express Xy1Mb1.The xylanase XylMbl was secreted and expressed by pPICZa-A plasmid.The molecular weight of XylMbl expressed in GS115 was 35 kDa higher than the theoretical value(20 kDa).In conclusion,we cloned and expressed xylanases and EG enzymes derived D.macrotermitis,and analyzed their enzymatic properties.At the same time,we co-expressed lignocellulases derived from termites and their symbiotic,and identified their synergistic degradation of some natural substrates.In order to accelerate the application of lignocellulases in industry,we used P.pastoris system to expression the xylanase Xy1Mb1.