d-Allulose Production from d-fructose by Putative Dolichol Phosphate Mannose Synthase from Bacillus sp. with Potential d-allulose 3-epimrase Activity

We found a putative dolichol phosphate mannose synthases (DPMS) from Bacillus sp., which exhibited the highest specific activity toward d-allulose, one of a functional rare sugars and also known as d-psicose, and identified it as d-allulose 3-epimerase (Basp_DAEase). The gene of Basp_DAEase from Bacillus sp. was cloned to the expression vector (pET-28a(+)) and then expressed as a recombinant enzyme in the Escherichia coli BL21(DE3). The recombinant enzyme was purified homogeneously on the SDS-PAGE with 34 kDa by 6XHis-tagging affinity chromatography and it was found to exist in dimer form as its activity form by Sephacryl S 200 HR 16/60 gelfiltration chromatography. The reaction conditions for a recombinant Basp_DAEase were optimized to produce d-allulose from d-fructose. The epimerization activity of Basp_DAEase toward d-fructose was maximum at pH 7.5 and 40 degrees C with 1 mM Co2+. The half-lives of Basp_DAEase at 35 degrees C, 40 degrees C, 45 degrees C, 50 degrees C, and 55 degrees C were 45.8, 4.18, 1.24, 0.46, and 0.17 h, respectively. At pH 7.5, 40 degrees C, and 1mM Co2+, 215 g/L of d-allulose was produced from 700 g/L of d-fructose by 20 U/mL of Basp_DAEase after 50 min of enzyme reaction with a conversion yield of 31% and productivity of 258 g/L/h. This is the highest productivity of d-allulose from d-fructose reported to date. From this result, it is strongly suggested that Basp_DAEase has high potential application value in the industrial d-allulose production from d-fructose.