Study On A Cellulase Activity Enhancing Protein From Bacterium

Low energy consumption,mild reaction conditions,and environment friendly were the advantages of degradating cellulose with cellulase,while the application of the process was limited by the low efficiency of fiber hydrolysis with cellulase.So far,proteins that could promote the enzymatic hydrolysis efficiency of cellulose have been reported,such as expansins from plant and expansin-like proteins from fungi.However,the cellulase activity enhancing proteins from bacteria was rarely reported,and its mechanism was unclear also.Therefore,the screening of cellulase-activity-enhancing-protein producing bacteria,prodution,isolation and purification of the protein,the synergistic mechanism between cellulase and the protein were researched in this paper.Specific works and the results were as follows:(1)A method to screen the cellulase-activity-enhancing-protein producing strain was established.A strain named POEPI,having the ability of producing cellulase-activity-enhancing-protein and no cellulase activity,was screened from the 21 strains isolated from soil and then identified as Pseudomonas according to the 16S rDNA gene sequencing analysis(99%).(2)The enhancing-protein was separated and purified from the culture medium through a seriese of procedures including ultrafiltration,ammonium sulphate precipitation,Sephadex G-75 and DEAE Sehadex A-50 column chromatography,the molecular weight of the protein was estimated to be 60 kDa by SDS-PAGE.Mass spectrometry analysis indicated that this purified protein was an unknown protein and this new protein was named POEPI.RL.(3)The synergistic conditions between the POEP1-RL and cellulase were as follows:pH 4.8,50 mg of ilter paper,5 FPA of cellulase,37 ?g of the enhancing protein,3mL of reaction volume,12 hours of reaction time.Under these conditions,a maximum synergistic efficiency was observed to be 245%.(4)XRD,SEM and UTM analisisshowed that POEp1-RL would promote the loose of cellulose crystalline region,destructe the surface structure of the tubular fiber and reduce the filter paper tensile strength.(5)Single factor,Placket-Burman design,Box-Behnken experimental design were used to optimize fermentation condictions for increasing the protein productivity of POEP1 strain,and the synergistic activity was improved 1.74 times higher than that before optimization.