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-RIM) was extracted and purified. The catalytic function of L-RIM 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-RIM to be a Co++- or Mn++-dependent metalloenzyme. L-RIM was remarkably active (similar to 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 degrees C) ranges. Optimal L-RIM activity with d-allulose as the substrate occurred at pH 7.0 and 75 degrees C. The enzyme was found to be excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 degrees C and 70 degrees C, respectively. L-RIM catalysis conducted at slightly acidic pH of 6.0 and 70 degrees C achieved biosynthesis of about 30 g L-1 from 100 g L(-1)d-allulose in 3 h.