Mannanase Man23 mutant library construction based on a novel cell-free protein expression system

Area of Science:

• Enzymology
• Protein Engineering
• Industrial Biotechnology

Background:

• Mannanases are crucial enzymes in agriculture and food processing.
• Improving mannanase performance is key for industrial applications.
• Rational design and mutant libraries are used for enzyme engineering.

Purpose of the Study:

• To engineer a high-performance mannanase mutant from Man23.
• To utilize the Brevibacillus brevis B16 cell-free system for efficient mutant library expression.
• To characterize the activity and stability of engineered mannanase variants.

Main Methods:

• Rational design of mutations on loops around the catalytic region of mannanase Man23.
• Expression of the mutant library using the Brevibacillus brevis B16 cell-free system.
• Activity measurement of mutants using a multi-volume spectrophotometer.

Main Results:

• A sextet mutant, Man1606, with six specific mutations (N146G, S147H, S156P, T157Y, Q206S, T249H) was identified.
• Man1606 demonstrated significantly higher activity and stability compared to the original Man23.
• Man1606 exhibited an increased optimal temperature (60 °C) and a broader optimal pH range.

Conclusions:

• The Brevibacillus brevis cell-free system is an efficient tool for rapid mannanase engineering.
• Engineered mannanase Man1606 shows enhanced properties suitable for industrial use.
• This approach accelerates enzyme development for biotechnological applications.