Expression, purification and characterization of a recombinant levan fructotransferase

Area of Science:

• Enzymology
• Molecular Biology
• Microbial Biochemistry

Background:

• Levan fructotransferase (LFTase) enzymes are crucial for modifying levan, a complex polysaccharide.
• Understanding the enzymatic properties of LFTase is important for biotechnological applications.

Purpose of the Study:

• To clone, express, and purify the lftM gene encoding LFTase from Microbacterium sp. AL-210.
• To characterize the enzymatic activity and kinetic properties of the recombinant LFTase.
• To investigate the catalytic mechanism and optimal conditions for DFA IV production.

Main Methods:

• Subcloning the lftM gene into a high-expression vector (pET-29b).
• Overexpression of recombinant LFTase in Escherichia coli.
• Purification using affinity and gel-filtration chromatography.
• Enzyme activity assays, kinetic analysis (K(m), V(max)), and pH-dependence studies.

Main Results:

• Recombinant LFTase was successfully overexpressed and purified 132-fold.
• The purified enzyme (approx. 54 kDa) efficiently converted levan to difructose anhydride IV (DFA IV).
• Optimal activity was observed between pH 5.0-8.0 at 40°C, with specific kinetic parameters determined.
• N-terminal sequencing indicated post-translational cleavage of 42 amino acids.

Conclusions:

• The characterized LFTase from Microbacterium sp. AL-210 is a potent biocatalyst for DFA IV production.
• The enzyme's broad pH optimum and specific kinetic properties are valuable for industrial applications.
• Acidic residues at the active site likely play a key role in the catalytic mechanism.