Stabilization of Enzymes by Using Thermophiles

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biocatalysis

Background:

• Steroid compounds are vital in pharmaceuticals, but chemical synthesis is complex.
• Enzyme-based bioconversion offers a more efficient alternative, with thermostability being a key desirable trait.
• Thermostable enzymes enhance reaction yields by increasing enzyme half-life and substrate/product solubility.

Purpose of the Study:

• To develop a protocol for selecting thermostable variants of steroid-processing enzymes.
• To overcome limitations of metagenomic screening for prokaryotic thermostable enzymes.
• To improve enzyme-based bioconversion processes for pharmaceutical applications.

Main Methods:

• Utilized error-prone PCR (epPCR) to introduce random mutations into enzyme genes.
• Created a library of enzyme variants for selection.
• Employed folding interference vectors in Thermus thermophilus for selection of thermostable variants.
• Linked enzyme folding to a selectable reporter (kanamycin resistance) for life-or-death selection.

Main Results:

• Successfully generated a library of enzyme variants with enhanced thermostability.
• The selection method identified variants with reinforced folding at high temperatures.
• The protocol facilitates the selection of thermostable enzymes independent of their specific catalytic activity.

Conclusions:

• The described protocol offers an effective strategy for engineering thermostable enzymes for industrial applications.
• This method provides a valuable tool for improving biocatalytic processes in the pharmaceutical industry.
• The folding interference vector system enables efficient selection of enzyme variants with improved stability.