A novel thermotolerant L-rhamnose isomerase variant for biocatalytic conversion of D-allulose to D-allose.

A novel L-rhamnose isomerase was identified and cloned from an extreme-temperature aquatic habitat metagenome. The deduced amino acid sequence homology suggested the possible source of this metagenomic sequence to be Chloroflexus islandicus. The gene expression was performed in a heterologous host, Escherichia coli, and the recombinant protein L-rhamnose isomerase (L-RI M ) was extracted and purified. The catalytic function of L-RI M was characterized for D-allulose to D-allose bioconversion. D-Allose is a sweet, rare sugar molecule with anti-tumour, anti-hypertensive, cryoprotective, and antioxidative properties. The characterization experiments showed L-RI M to be a Co ++ - or Mn ++ -dependent metalloenzyme. L-RI M was remarkably active (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RI M activity with D-allulose as the substrate occurred at pH 7.0 and 75 °C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively. L-RI M catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L -1 from 100 g L -1 D-allulose in 3 h. KEY POINTS: • The present study explored an extreme temperature metagenome to identify a novel gene that encodes a thermostable l-rhamnose isomerase (L-RI M ) • L-RI M exhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges • L-RI M is excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively.