Effect Of Codon Optimized α-factor On Expression Of Endoglucanase Gene In Pichia Pastoris

Cellulase is a kind of multienzyme compound that can degenerate cellulose into monosaccharide. According to its potential usage and its conversion ability of renewable cellulosic biomass to simple sugars for fermentation to ethanol, it is believed that this enzyme could be applied to deal with various problems in agriculture and renewable sources of energy as well as environmental pollution. Hence, it has been now widely used not only in textile industry, food and feed industry but also in pharmacy. However, its practical use is far beyond its anticipated use because of low production and low enzyme activity. Therefore, it is significant to find a way to increase its production and activity.Based on the experiments done before, this research focuses on codon optimization. We optimized the a-factor gene and synthesized it according to Pichia Pastoris codon usage bias. This newly-synthesized gene called HS replaced the original one in plasmid pPIC9K-End/Ends. Then we successfully constructed recombinants containing plasmid pPIC9K-HS-End/Ends which are detected by PCR and sequencing. Further more, we extracted the plasmids, linearize it with SacI and then transformed it into Pichia Pastoris by electroporation. Finally, we successfully screened out two kinds of Pichia Pastoris using MD/MM plates and these two reconbinants are named9KHSEG and9KHSEGS.Further experiments were conducted to check the enzyme activity of their products. It showed that9KHSEG and9KHSEGS can obtain their highest enzyme avtivities726.8UmL and865.5U/mL, which are as good as the original ones, after84hours-induction under26℃. When the inducing temperature is changed to a higer or lower one, the enzyme activities will decrease. When the inducing temperature goes to30℃, they displayed79.57%and74.04%lower enzyme activities respectively compared to the ones whose a-factor genes are not optimized. Besides, the total proteins are decreaced by14.1%and20.10%respectively. However, their specific activities are comparatively steady and are around92.6%of their original ones. This reaserch reveals that the optimal inducing temperature is26℃and optimal inducing time is84h for these two yeast recombinants. Meantime, all these showed a negative correlation between the expression and the optimization. It also demonstrates that α-factor non-optimal codons can influence the folding of exported protein and it is significant in maintiaining the expression of endoglucanase.Ultimately, the SDS-PAGE analysis was carried for the expressed endoglucanase of9KHSEG and9KHSEGS. According to it, the molecular sizes are approximate79.82KD, which were the same as the expressed ones in Pichia Pastoris GS115-End and pPIC-Ends. What is more, after subxulturing for10generations, it comfirmed that these two newly constructed reconbinants are own genetic stability.