Cut The N-glycosylation On The Expression In The Pichia Pastoris And Trichoderma Reesei Cellulase Enzyme Activities

Converting cellulose to ethanol has become an important task in the world. Hydrolysing cellulose to glucose is key step for this conversion. Until now, there are many methods to hydrolyse cellulose, and acid or high temperature is in common use, which may result in unwilling byproducts and polluting environments and even damaging glucose. More and more emphasis has been put on enzymatic hydrolysis. But low efficiency of cellulase astrict its extensive application at present. To solve the problem, protein engineering technology is practiced to modify CBHI or other cellulases from filamentous fungi. A high efficiency system is needed to express cellulase. However, T. reesei and E.coli were proved to be unfit expression systems. P. pastoris is evoluted from 80's, which can express single enzyme and secrete little self proteins, which could be used as a perfect cellulase expression system.Nowadays, it is seldom reported that cellulase was expressed in P. pastoris. Shubhada Godbole reported in 1999 that the V_max of cellobiohydrolase I expressed in P. pastoris was one percent than that of the natural CBHI. He found that the expressed CBHI in P. pastoris had been hyperglycosylated and the activity was significantly lower than that of T. reesei CBH I also. Shubhada Godbole believed that hyperglycosylation of rCBHI in P. pastoris is the reason for its low activity.To understand the effect hyperglycosylation on recombinant CBH I from T. reesei in P. pastoris, asparagines of the four N—glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of CBH I were replaced by serines using site-directed mutagenesis. These four mutants and the cbh1 gene from T. reesei were transformed into P. pastoris', then the enzymes expressed were purified and analyzed. Data showed that the activity of mutant enzyme N64S was higher than that of rCBHI expressed in P. pastoris. It indicated that the Asn64 glycosylation has an effect on the activity of the CBHI expressed in P. pastoris. Comparing the activities of CBHI from different mutants and native CBH I, experimental data showed that hyperglycosylation is only a limited factor for the low activity of recombinant CBHI in P. pastoris.