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KMID : 1094720220270030432
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Biotechnology and Bioprocess Engineering
2022 Volume.27 No. 3 p.432 ~ p.442
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Characterization of a Recombinant L-rhamnose Isomerase from Paenibacillus baekrokdamisoli to Produce D-allose from D-allulose
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Kim Sang-Jin
Choi Min-Su
Park Chang-Su
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Abstract
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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. baekrokdamisoli L-RI could be applied as a D-allose producer.
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KEYWORD
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D-allose, D-allulose, enzymatic production, L-rhamnose isomerase, Paenibacillus baekrokdamisoli
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