Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris

作者：Siadat Seyed Omid Ranaei^*; Mol****lehi Hamidreza; Heydarzadeh Narjes

来源：Biotechnology Letters, 2016, 38(3): 483-488.

DOI：10.1007/s10529-015-1997-8

• Cellobiohydrolase
• Glycosylation
• Heterologous expression
• Pichia pastoris
• Sitedirected mutagenesis
• Trichoderma reesei

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 A degrees 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.

• 出版日期2016-3