Purification And Characterization Of Active Enzyme Aggregates Induced By Self-assembly Peptides

With the development of genomics and proteomics,more and more recombinant proteins were produced.Once expressed,however,these products must be purified for further study or application.In the large-scale manufacture of recombinant proteins for industrial and therapeutic use,downstream purification is very costly and can account for up to 80%of the total production cost.Thus,the rapid and economical purification of recombinant proteins represents a persistent challenge in the filed of biotechnology.Traditional recombinant protein purification involves several steps such as ion exchanges,gel filtration,affinity chromatography and even revrese-phase chromatography when it comes to pharmaceutical application.Each step can be costly and time-consuming but decreases the final yield of the product.Recently,a class of self-assembly peptides such as 18A,R18A,ELK16,and L6KD has been studied and been applied to column-free recombinant protein purification.These short peptides can induce the fusion protein to form active aggregates,thus can be purified with economical procedures.In addition to economic considerations,thermostability also considered one of the crucial properties for enzymes in industrial processes.Many approaches through protein engineering,such as site directed,random,and saturated mutagenesis have been conducted to enhance the thermal tolerance of interest enzymes.In this study,four self-assembly peptides mentioned above were attached to the C termial of?-xylosidase?from Thermoanaerobacterium aotearoense SCUT27?and Acetylxylan esterase?from Bacillus pumilus PS213?,respectively.Then fusion proteins were functionally expressed in E.coli BL21?DE3?.For AXEs,SDS-PAGE analysis indicated the percentage of Insoluble expression of AXE-18A,AXE-R18A,AXE-ELK16,and AXE-L6KD accounted for79.3%,99.49%,93.54%,91.06%,and the recoveries were 69.33%,87.69%,87.70,and 99.17%,respectively.As for ThXylCs,only ELK16 induced the formation of active aggregates with98.6%of total activity,92.57%recovery and 95%purity reached.The enzyme kinetics parameters were also characterized.It is found that the Km of all enzyme aggregations were increased more or less,indicating that aggregation might affect the exposure of the active site.It should be noted that catalytic efficiency(K_cat/K_m)was increased from 21.31 mM^-1 s^-1 for ThXylC to 32.19 mM^-1 s^-1 for ThXylC-ELK16.Other than that,the optimum reaction temperature is increased by 5 degrees for ThXylC-ELK16,and half-life at65°C resulted in 6.2-fold increase.On the other hand,for the catalyze of 7-ACA,AXE-L6KD and AXE-His were purified and immobilized using sodium alginate followed by cross-linking with polyethyleneimine and glutaraldehyde,respectively.Immobilized particles AXE-L6KD-CLEAs were found to retain about 95%of the initial activity after 42 consecutive batche compare to 85%retaintion of AXE-His-CLEAs.