Optimized Expression Of The Expansin-like Proteins TrSwol And BsEXLX1in Pichia Pastoris And Study Of Their Functional Properties

Energy problem is one of the most critical problems that draw attention all overthe world. It has become the focus of research in the world to know how to uselignocellulose resources efficiently to produce biofuels as the nonrenewable resourcesrun down. However, the high crystallinity of lignocellulose and the high cost resultedfrom the inefficiency of cellulose degradation have been the bottleneck in the efficientconversion and exploitation of biomass.Expanin-like protein can enhance cellulase activity by disrupting hydrogenbonds in cellulose, and have a structure and function similar to that of plant expanin.Therefore, the discovery of the protein will be of important significance to degradecellulose efficiently. TrSwol from Trichoderma reesei and BsEXLX1from Bacillussubtilis also can enhance cellulase activity by disrupting cellulose. However, theexpression levels of the proteins are too low to satisfy the demand of detailedfunctional study, and some important biochemical properties of the proteins have notbeen uncovered yet.The expanin-like genes from Trichoderma reesei (TrSwol) and Bacillus subtilis(BsEXLX1) were successfully cloned and expressed in Pichia pastoris. The effects ofinitial pH and several protease inhibitors on the expression of recombinant proteinswere investigated. And the results showed that the protease inhibitor has a crucial rolein increasing the protein production. The yields of two recombinant proteins weresignificantly improved by PMSF and a commercial protease inhibitor cocktail,whereas EDTA had no significant effect on the production of recombinant proteins.After optimizing expression conditions, the highest TrSwol expression level reachedwas approximate120mg l-1, which was almost2.4fold compared to the highestexpression level in other host cells. And the highest BsEXLX1expression level wasapproximate860mg l-1, which was almost86fold compared to the previouslyreported expression level. Recombinant proteins were purified by Ni-NTA His-bindResin in order to carry on a comparative study on the synergistic activity of BsEXLX1and TrSwol and investigate unknown biochemical properties. BsEXLX1/TrSwoldisplayed synergism in cellulose hydrolysis in combination with endoglucanase, andthe maximum amount of reducing sugars released was almost2.0/2.5fold comparedto that in reaction mixture without the expansin-like proteins. Under several enzymatic hydrolysis conditions, the synergistic activity of TrSwol was higher thanthat of BsEXLX1, indicating that TrSwol exhibits stronger disrupting activity oncellulose than BsEXLX1. The effect of the concentration of expansin-like proteins onsynergistic activity was explored, and the synergistic effect reached the maximumlevel when1mg of target protein per g of filter paper was loaded. Both proteinsexhibited relatively high thermal stability at temperature50,70and90°C, andretained more than45%residual activities after1h of pre-incubation at100°C,suggesting remarkable heat tolerance. They also showed resistance to denaturation byurea and SDS. The study optimized BsEXLX1/TrSwol production, and uncoveredtheir biochemical properties such as thermal stability, which provides valuable cluesfor industrial application of expanin-like proteins.