Screening And Purification Of Cellulase Synergistic Protein From Actinomycetes

Lignocellulose is the most abundant biomass,which can be degraded into fermentable monosaccharide for energy or platform chemicals process.Though the cellulose enzymatic degradation of lignocellulose takes the advantages of low energy consumption,mild reaction conditions and eco-friendliness,the cellulose enzyme is high-cost,easy deactivation and low hydrolysis efficiency become one of the bottlenecks in industrialization.In recent years,application of synergistic proteins in enzymatic reaction mixtures of cellulose or lignocellulose increase hydrolysis yield,which may lead to cellulase loading reduction in enzymatic hydrolysis.Synergistic protein from actinomycetes has been less reported.Therefore,the screening of actinomycete producing synergistic protein,the separation and purification of synergistic protein and study on its synergistic mechanism were carried out in this study.The detailed contents and results were as follows:A screening method of actinomycetes producing synergistic protein of cellulose enzyme was established and 8 different morphology strains were isolated from soil samples.Three isolated strains M1?M2?M3 had the synergistic activity and no cellulose enzyme activity,and the synergistic activity were 112%,148%and 192%respectively;the bacteria intracellular proteins treated with salting out,synergistic activity were 80%,136%and 118%respectively.M2 was chosen as the cellulose enzyme production efficiency strain for further studies.The sequencing of M2 16S rRNA and phylogenetic tree revealed M2 was closely related with actinomycetes(99%).The sequencing of M3 16S rRNA and phylogenetic tree revealed M3 was closely related with Actinobacterium R13(99%).The intracellular proteins in bacteria M2 was isolated with the separation methods of salting out,flat ultrafiltration,first by DEAE sepharose FF anion exchange chromatography separation of bacteria M2 intracellular proteins,the peak 1 and the corresponding concentration of 0.3M elution peak 3 has a synergistic activity,the synergistic activity were 73%,132%,peak 3 protein was separated by Sephadex G-75 gel chromatography,to obtain a total of two erude protein isolate(named peak and peak 3-1?3-2)have a synergistic activity,its synergistic activity were 80%,127%.Preliminary studies have shown that the isolated protein from M2 with Sephadex G-75 gel chromatography(peaks 3-2,named synergistic protein)has three functions:destructive filter structure,when filter paper was pretreatmented by the synergistic protein,which was 2.6-fold greater than the control;stabilization(to prevent heat inactivated enzyme),when the reaction temperature was 60?,70? time(above optimum temperature 50°C),added synergistic protein make reservations cellulase activity increased 1.5-fold and 3.0-fold;cooperative hydrolysis natural cellulose substrates of bagasse,its coordination mechanism was different from the "bovine serum albumin invalid adsorption of lignin by improving the free enzyme content" synergy mechanism,the conversion ratio were increased by more than 10%when different pretreatment of bagasse(lignin content different)were incubated with cellulase and synergistic protein,but the specific mechanism remains to be further explored.