Recombinant production of acidophilic L-arabinose isomerase from Lentilactobacillus parakefiri in Bacillus subtilis

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Abstract
Background: The monosaccharide D-tagatose is a promising alternative to sucrose because of its similar sweetness and lower glycemic index. A novel L-arabinose isomerase (L-AI) from Lentilactobacillus parakefiri DSM 10551 (L-AI-Lp) has been biochemically characterized and used to isomerize D-galactose to D-tagatose in skim milk ultrafiltration permeate at pH 4.5 and 6.5. However, like most L-AIs described in the literature, this enzyme has only been produced recombinantly in Escherichia coli . This study aimed to systematically investigate the intracellular recombinant production of L-AI-Lp in Bacillus subtilis , which has qualified for a presumption of safety (QPS) designation from the European Food Safety Authority. Results: The influence of four promoters on L-AI-Lp production in B. subtilis 007 was investigated in shake flask cultivations. Among these, the P AprE promoter yielded the highest volumetric L-AI activity of 69.2 ± 7.4 µkat Gal, 65 °C /L Culture . The production yield was further increased to 147.7 ± 1.0 µkat Gal, 65 °C /L Culture by using the nonsporulating, surfactin-deficient strain B. subtilis 007 ∆ sfp ∆ sigF , which was constructed by deleting sigF and sfp in B. subtilis 007. Furthermore, the influence of pH and dissolved oxygen (DO) on bioreactor cultivations of B. subtilis 007 ∆ sfp ∆ sigF was analyzed. In bioreactor cultivations, the highest L-AI activity of 88.6 ± 2.4 µkat Gal, 65 °C /L Culture was measured under unregulated pH and low oxygen conditions (DO ≤ 5%), representing a 3.2-fold increase compared with previous recombinant production in E . coli . The L-AI-Lp was subsequently partially purified by heat treatment and precipitation methods, resulting in a 7.8-fold increase in specific activity to 128.2 nkat Gal, 65 °C /mg and a yield of 84%. Conclusions: The L-AI-Lp was recombinantly produced for the first time in a microbial species with QPS status using the nonsporulating and surfactin-deficient strain B. subtilis 007 ∆ sfp ∆ sigF . The L-AI-Lp was subsequently partially purified via nonchromatographic methods, providing a basis for a low-cost downstream process. These results represent an important step toward potential industrial application of L-AI-Lp and highlight the potential of B. subtilis 007 ∆ sfp ∆ sigF as an expression host for the recombinant production of L-AIs compared with previously used hosts from the order Lactobacillales .
Funding Information
  • Universität Hohenheim

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