Characteristics and Kinetic Properties of L-Rhamnose Isomerase from Bacillus Subtilis by Isothermal Titration Calorimetry for the Production of D-Allose

Abstract

L-Rhamnose isomerase, an aldose-ketose isomerase, is an important enzyme for rare sugar production. In this paper, L-rhamnose isomerase (L-RhI) from Bacillus subtilis 168 (B. subtilis L-RhI) was cloned and expressed in Bacillus subtilis WB600 for the production of D-allose. The obtained crude L-RhI was then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The characteristics of purified L-RhI were analyzed subsequently. The enzyme was activated in presence of Mn²⁺, and it had thermal advantages as its optimum temperature was 70°C, and 85% of its activity was retained after 4 hours' incubation at 60°C. In addition, isothermal titration calorimetry (ITC) was for the first time employed to determine the isomerization of rare sugars, and the kinetic data showed that B. subtilis L-RhI had relatively low K_m values on the substrates of L-rhamnose, L-mannose, D-ribose and D-allose. Using D-psicose as the substrate, no by-products but only D-allose was generated catalyzed by B. subtilis L-RhI, and the yield of 37.51% was obtained when the reaction reached its equilibrium at 60°C.