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Updated: Mar 29, 2026
Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants
Published on: June 13, 2014
Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei
Seyed Omid Ranaei Siadat1, Hamidreza Mollasalehi2, Narjes Heydarzadeh2
1Protein Research Center (PRC), Shahid Beheshti University, GC, Evin, Tehran, 1983963113, Iran. o_ranaei@sbu.ac.ir.
Objectives:
To modify two main N-glycosylation residues of cellobiohydrolase I from Trichoderma reesei by site-directed mutagenesis for decreasing the extent of glycosylation and exploring possible effects on its properties.
Results:
Asparagine 45 and 64 residues were mutated to alanine to make single/double mutants and expressed in P. pastoris. Decreasing N-glycosylation of the recombinant CBH I resulted in an increased affinity of the enzyme for carboxymethylcellulose and also improved the Kcat/Km while the specific activity was decreased. Also, the enzymes were stable up to 80 °C. There was no significant change of the optimum pH and temperature by decrease of glycosylation in the mutated enzymes in comparison to the wild-type at constant incubation time of assay.
Conclusion:
Post-translational glycan-modification of CBH I in P. pastoris has different impacts on the properties of the secreted enzymes. Substrate affinity and catalytic efficiency were improved significantly while the activity and high temperature stability were negatively affected.
