Biotechnology and Bioprocess Engineering 2022; 27(3): 432-442  
Characterization of a Recombinant L-rhamnose Isomerase from Paenibacillus baekrokdamisoli to Produce D-allose from D-allulose
Sang Jin Kim, Min Su Choi, and Chang-Su Park
Sang Jin Kim, Min Su Choi, Chang-Su Park*
Department of Food Science and Technology, Daegu Catholic University, Gyeongsan 38430, Korea
Tel: +82-53-850-3216; Fax: +82-53-359-6585
E-mail: parkcs@cu.ac.kr
Received: November 2, 2021; Revised: December 23, 2021; Accepted: December 26, 2021; Published online: June 30, 2022.
© 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
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 Gel-filtration 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. baekrokdamisoli L-RI could be applied as a D-allose producer.
Keywords: D-allose, D-allulose, enzymatic production, L-rhamnose isomerase, Paenibacillus baekrokdamisoli


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