Engineering highly thermostable xylanase variants using an enhanced combinatorial library method.

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• The GH11 xylanase 2 (BD-11) from Hypocrea jecorina has undergone extensive engineering over two decades.
• Improving enzyme thermostability is crucial for industrial and therapeutic applications.

Purpose of the Study:

• To enhance the thermostability of the wild-type GH11 xylanase 2 (BD-11) using novel directed evolution methods.
• To identify and combine beneficial mutations for increased enzyme stability.

Main Methods:

• Employed Look-Through Mutagenesis (LTM™) for rapid screening of protein sequence positions.
• Utilized Combinatorial Beneficial Mutagenesis (CBM™) to identify optimal mutation ensembles.
• Combined identified mutations into a single construct (Hjx-81).

Main Results:

• Discovered a diverse set of novel mutations, including N71D, Y73G, T95G, and Y96Q.
• The engineered enzyme construct (Hjx-81) demonstrated activity after incubation at 100°C for 20 minutes.
• Achieved significant thermostability enhancement in a time-effective manner.

Conclusions:

• The developed directed evolution method is efficient for improving enzyme properties.
• This approach is broadly applicable for enhancing the stability of industrial and therapeutic enzymes.
• The engineered GH11 xylanase exhibits exceptional thermostability for potential applications.