Characterization of a Recombinant l-rhamnose Isomerase from Paenibacillus baekrokdamisoli to Produce d-allose from d-allulose

A putative l-rhamnose isomerase (l-RI) gene from Paenibacillus baekrokdamisoli was expressed as a recombinant enzyme in Escherichia coli BL21(DE3) and characterized as a producer of d-allose from d-allulose. Recombinant l-RI from P. baekrokdamisoli was homogeneously purified on SDS-PAGE with a 46 kDa molecular mass and specific activity of 1.27 U/mg by His-Trap affinity chromatography. The enzyme was estimated to be a tetramer in enzyme active form because its molecular mass was determined to be approximately 190 kDa by Gelfiltration chromatography. In the isomerization reaction between d-allose and d-allulose, recombinant l-RI exhibited the highest activity at pH 8.0 and 60°C in the presence of 0.5 mM Mn2+. The half-lives of the enzyme at 50, 55, 60, 65, 70, and 75°C were 417, 57, 27, 20, 3.3, and 0.2 h, respectively. The Michaelis-Menten constants (Km), turnover numbers (kcat), and catalytic efficiencies (kcat/Km) of the enzyme for d-allose and d-allulose were 33 mM, 13.79 s−1, and 0.4 mM−1s−1 and 45.24 mM, 6.58 s−1, and 0.14 mM−1s−1, respectively. The enzyme showed isomerization activity for aldoses with right-handed configuration of hydroxyl group at the C-2 and C-3 positions, such as l-mannose, l-lyxose, d-gulose, d-allose, and d-ribose. During production of d-allose from d-allulose, the enzyme produced 125 g/L of d-allose from 500 g/L of d-allulose in 3 h with 41.6 g/L/h productivity with 104 U/mL enzyme. We first reported the l-RI from the Paenibacillus genus, and the results suggested that the P. baekrokdamisolil-RI could be applied as a d-allose producer.