Methylglyoxal synthase derived from Clostridium difficile strain 630 containing a Strep-tag and a method for producing methylglyoxal using the same

Technology for Mesophilic Methylglyoxal Production Using N-terminal Strep-tagged Clostridium-derived Enzyme

This technology features an enzyme created by attaching a Strep-tag to the N-terminus of a methylglyoxal synthase derived from the Clostridium difficile 630 strain. It includes the enzyme, the gene encoding it, recombinant vectors, transformed microorganisms, and a method for producing methylglyoxal through enzymatic reaction.

Petrochemical-based 1,2-propanediol production involves propylene oxide, which generates harmful byproducts. Research on enzymes that produce methylglyoxal—a key intermediate in bio-processes—has been limited to a few strains, resulting in a shortage of candidates with high activity at room or mesophilic temperatures.

This technology exhibits activity in the mesophilic range of 20–65°C and pH 5–7, with a specific activity approximately 1.37 times higher than that of the Bacillus subtilis-derived enzyme, which peaks at 30°C. The N-terminal Strep-tag simplifies enzyme purification. It can be applied to biochemical processes operating at standard microbial fermentation temperatures, allowing for the efficient production of methylglyoxal and 1,2-propanediol precursors without the need for high-temperature equipment.

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Key Features:

• Methylglyoxal synthase with an N-terminal Strep-tag derived from Clostridium difficile strain 630
• Methylglyoxal synthase with a specific activity of at least 0.139 U/mg at pH 5–7 and at least 0.063 U/mg at 20–65°C
• Polynucleotide, recombinant vector, and transformed microorganism with a Strep-tag coding sequence attached to the 5'-end of the enzyme-coding sequence
• Step of synthesizing methylglyoxal by adding dihydroxyacetone phosphate to the N-terminal Strep-tagged synthase at pH 5–7

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This invention was developed with support from the Ministry of Science and ICT's C1 Gas Conversion Technology common research project.

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