Biotechnology and Bioprocess Engineering 2021; 26(6): 976-984  
D-Allulose Production from D-fructose by Putative Dolichol Phosphate Mannose Synthase from Bacillus sp. with Potential D-allulose 3-epimrase Activity
Min-Ju Seo, Eun Ryeong Kwon, Sang Jin Kim, Min Su Choi, Yeong-Su Kim, and Chang-Su Park
Min-Ju Seo
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minneapolis, MN 55108, USA
Eun Ryeong Kwon, Sang Jin Kim, Min Su Choi, Chang-Su Park*
Department of Food Science and Technology, Catholic University of Daegu, Gyeongsan 38430, Korea
Tel: +82-53-850-3216; Fax:+82-53-359-6585
E-mail: parkcs@cu.ac.kr
Yeong-Su Kim
Division of Wild Plat Industrialization Research, Baekdudaegan National Arboretum, Bonghwa 26209, Korea
Min-Ju Seo and Eun Ryeong Kwon have contributed equally to this work.
Received: January 6, 2021; Revised: February 16, 2021; Accepted: February 21, 2021; Published online: December 31, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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 Dallulose from D-fructose. The epimerization activity of Basp_DAEase toward D-fructose was maximum at pH 7.5 and 40°C with 1 mM Co2+. The half-lives of Basp_DAEase at 35°C, 40°C, 45°C, 50°C, and 55°C were 45.8, 4.18, 1.24, 0.46, and 0.17 h, respectively. At pH 7.5, 40°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 Dallulose production from D-fructose.
Keywords: Bacillus sp., dolichol phosphate mannose synthase, D-allulose, D-psicose, D-fructose, enzymatic production


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