title: Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Protein Research Center (PRC), Shahid Beheshti University, GC, Evin, Tehran, 1983963113, Iran. o_ranaei@sbu.ac.ir.
authors.forename: Seyed Omid
authors.identifier: http://orcid.org/0000-0002-6466-0222
authors.initials: SO
authors.lastname: Ranaei Siadat
authors.affiliation: Protein Research Center (PRC), Shahid Beheshti University, GC, Evin, Tehran, 1983963113, Iran.
authors.forename: Hamidreza
authors.initials: H
authors.lastname: Mollasalehi
authors.affiliation: Protein Research Center (PRC), Shahid Beheshti University, GC, Evin, Tehran, 1983963113, Iran.
authors.forename: Narjes
authors.initials: N
authors.lastname: Heydarzadeh
mesh_terms: D019943:Amino Acid Substitution; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D050505:Mutant Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase; D002266:Carboxymethylcellulose Sodium
keywords: Cellobiohydrolase; Glycosylation; Heterologous expression; Pichia pastoris; Site-directed mutagenesis; Trichoderma reesei