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

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Abstract

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.

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Acknowledgments

The authors appreciate the support of Research Council of Shahid Beheshti University.

Conflict of interest

The authors declare that there is no conflict of interest regarding the manuscript content.

Supporting Information

Supplementary Table 1 Primers used in this study for colony PCR and site directed mutagenesis.

Supplementary Fig. 1 CBH I gene sequences from Trichoderma reesei synthesized according to the codon usage of Pichia pastoris.

Author information

Authors and Affiliations

  1. Protein Research Center (PRC), Shahid Beheshti University, GC, Evin, Tehran, 1983963113, Iran

    Seyed Omid Ranaei Siadat, Hamidreza Mollasalehi & Narjes Heydarzadeh

Authors
  1. Seyed Omid Ranaei Siadat
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  2. Hamidreza Mollasalehi
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  3. Narjes Heydarzadeh
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Corresponding author

Correspondence to Seyed Omid Ranaei Siadat.

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Ranaei Siadat, S.O., Mollasalehi, H. & Heydarzadeh, N. Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris . Biotechnol Lett 38, 483–488 (2016). https://doi.org/10.1007/s10529-015-1997-8

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  • DOI: https://doi.org/10.1007/s10529-015-1997-8

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