Endoxylanase II from Trichoderma reesei has several isoforms with different isoelectric points

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Trichoderma reesei Rut C30 produces multiple xylanases, enzymes crucial for breaking down xylan.
• Previous isolation of XYL II (pI 9.0) from this fungus provides a reference point for enzyme characterization.

Purpose of the Study:

• To purify and characterize two minor xylanases from T. reesei Rut C30 cultivation broth.
• To investigate the relationship between these minor xylanases and the previously identified XYL II.

Main Methods:

• Enzyme purification using ion-exchange, hydrophobic-interaction, and gel chromatography.
• Characterization of enzyme properties including pI, molecular mass, specific activity, and kinetic parameters.
• Amino acid sequencing of tryptic peptides and in vitro modification studies.

Main Results:

• A mixture of two active xylanases (pI 7.1 and 8.1, 20 kDa) was purified.
• These enzymes exhibited properties similar to XYL II but with significantly lower specific activity.
• Amino acid sequence analysis confirmed identity with XYL II, and in vitro modifications demonstrated conversion of XYL II to pI 8.1 and pI 7.1 forms.
• The primary sequence of XYL II contains numerous glutamine and asparagine residues, supporting deamination as a mechanism for pI modification.

Conclusions:

• The purified minor xylanases are likely deamidated forms of the major xylanase XYL II.
• Deamidation of glutamine and asparagine residues in XYL II can explain the observed lower pI values and altered properties.
• This study elucidates post-translational modifications affecting xylanase function in T. reesei.