Researchers at the Lawrence Berkeley National Laboratory in the United States have recently discovered that it is very advantageous to produce biofuels from lignocellulose at 65 ° C-70 ° C. They used a promising technique to improve the ability of cellulase to decompose cellulose into fermented sugar in this temperature range. Using this technique, the researchers successfully improved a high-temperature enzyme mutant with better activity and stability in the required temperature range.
The researchers used the "factor B guided mutation" method to enhance the thermal stability of the endoglucanase EGI (TrEGI) of Trichoderma reesei. Trichoderma reesei is an important cellulase producing strain. The use of cellulase to hydrolyze lignocellulose at high temperatures has potential advantages, including reduced viscosity under high temperature pretreatment, reduced risk of microbial contamination, and enhanced compatibility, which results in a higher solid load and accelerated mass transfer And hydrolysis rate. However, Trichoderma reesei cellulase is not very stable above 50 ℃, the researchers used factor B method to improve the thermal stability of this cellulase.
Like all proteins, cellulase is formed by linking together a single chain of amino acids. Each amino acid has a "factor B" value in a given enzyme, which corresponds to the flexibility of this amino acid. The higher the flexibility of amino acids with higher factor B values, the better. The most active amino acids in enzymes are most easily separated under protein thermal stress. Therefore, it is necessary to fix these parts of the enzyme by mutating amino acids and reduce their B factor value to support the overall structure, which enhances the thermal stability value of the protein.
At the recent National Conference of the American Chemical Society, the researchers showed that they screened 11,000 TREGI mutants, which were then pretreated at 50 ° C, and finally confirmed about 500 mutant strain candidates. Using factor B to induce mutagenesis, the researchers modified the cellulase TrEGI to have twice the activity of the original cellulase on the insoluble lignocellulose matrix in the temperature range of 50 ° C to 60 ° C. The modified cellulase TrEGI in the model strain Neurospora crassa can hydrolyze lignocellulosic biomass at 60 ° C, and its conversion efficiency is the same as that of the original cellulase TrEGI at 50 ° C. After comparison, the researchers found that TrEGI mutants had lower activity at higher temperatures in E. coli extracts and model Saccharomyces cerevisiae.
Studies have shown that recombinant cellulase has a profound effect on the activity and stability of expressed enzymes. This will be of guiding significance for engineering optimization of enzyme performance to produce biofuels.
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