Expression, Purification And Characterization Of Recombinant SipoEXLX2 Synergistic Protein

As the most abundant biomass energy in the world,lignocellulose can be degraded into glucose and converted into fuel such as bioethanol.However,the enzymatic hydrolysis of lignocellulose is hindered by the highly complex crystal structure of lignocellulose.As a non-hydrolyzable protein,cellulose-enhancing protein has been considered as an option to increase the efficiency of cellulase hydrolysis and reduce the enzyme load.A recombinant strain containing a synergistic protein gene derived from Streptomyces ipomoeae was constructed in this laboratory,SipoEXLX2 is the expression product of the synergistic protein gene.The single-stranded SipoEXLX2 protein was not purified in the previous purification.Therefore,the Expression and purification of SipoEXLX2 synergistic protein,mechanism of action and degradation of pretreatment fiber substrate were carried out in this study.The experimental results are as follows:By reconstituting the plasmid vector,the His tag was added at the C-terminus to purify the SipoEXLX2 synergistic protein with a purity greater than 90%and synergistically greater than 1 fold.After optimization of the induction conditions,the expression of SipoEXLX2 protein in the target protein supernatant reached about 35 mg/L under the condition of IPTG of 1 M and induction at 16 ? for 12h.The influencing factors and mechanism of SipoEXLX2 synergistic protein synergistic with cellulase in degradation of filter paper were studied.The optimal synergistic conditions for SipoEXLX2 synergistic protein synergy with cellulase are pH 4.8,temperature 50 ?,250.g SipoEXLX2 synergistic protein and 0.06 FPU/g cellulose cellulase loading.SEM,FTIR,XRD,and universal testing machines were used to detect and analyze the filter paper before and after SipoEXLX2 synergistic protein pretreatment.After the filter paper was pretreated with SipoEXLX2 synergistic protein,SEM showed that the surface structure of the filter paper was loose and the crystallinity decreased;FTIR showed that the hydrogen bond strength and crystallinity of the filter paper decreased by 25.6%and 2.7%,respectively;due to the problem of filter paper treatment,XRD shows a 1.2%increase in crystallinity of filter paper due to filter paper handling issues;the universal testing machine showed a 6.4%drop in tensile strength of the filter paper.In addition,SipoEXLX2 synergistic protein enhances the thermal stability of cellulase.Investigation on the factors that affecting rice straw by SipoEXLX2 synergistic protein and enzymatic hydrolysis.The effect of SipoEXLX2 synergistic protein on the synergistic degradation of rice straw was the best under the condiation that the loading of SipoEXLX2 synergistic protein was 300 ug,the solid content of substrate was 2%and the enzyme loading was 0.90 U/g substrate respectively,the yield of reducing sugar was increased by 21.3%.The experimental results show that SipoEXLX2 synergistic protein can improve the degradation of pretreated fibrous substrate,which has potential application value in fiber raw material hydrolysis.