A novel thermotolerant l-rhamnose isomerase variant for biocatalytic conversion of d-allulose to d-allose

Abstract

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 (~ 80%) in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges. Optimal L-RIM 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-RIM catalysis conducted at slightly acidic pH of 6.0 and 70 °C achieved biosynthesis of about 30 g L−1 from 100 g L−1d-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-RIM)• L-RIMexhibits substantial (80% or more) activity in a broad spectrum of pH (6.0 to 9.0) and temperature (70 to 80 °C) ranges• L-RIMis excessively heat stable, displaying a half-life of about 12 days and 5 days at 65 °C and 70 °C, respectively