Abstract
BackgroundL-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed.ResultsThe coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn2+ at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (kcat/Km) for L-arabinose and D-galactose were 8.7 mM-1 min-1 and 1.0 mM-1 min-1 respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L-1 and 250 g L-1 D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h).ConclusionsIn this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn2+. Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0286-5) contains supplementary material, which is available to authorized users.
Highlights
L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose
Over-expression and purification of Bacillus coagulans NL01 AI (BCAI) The araA gene contained an open reading frame (ORF) of 1422 base pairs encoding a protein of 473 amino acids
Protein sequence alignments showed that, among the AIs with high activities toward D-galactose, BCAI was mostly similar to Lactobacillus sakei AI and Pediococcus pentosaceus AI with identities of 68.8 and 67.5 % respectively
Summary
L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. D-tagatose is a natural rare ketohexose that possesses 92 % of the sweetness, but only 38 % of the calories of sucrose [1] Since it attained GRAS (Generally Recognized As Safe) status under U.S Food and Drug Administration (FDA) regulations, D-tagatose has become a promising functional sweetener on the food market. One mature method for D-tagatose production is the direct isomerization of D-galactose into D-tagatose with metal hydroxides as the chemical catalysts under basic conditions [2] This process was applied into commercial food grade D-tagatose production by Arla Food Company between 2002 and 2006 [3]. It has been gradually dismissed because of the drastic reaction conditions and high cost of the subsequent purification steps. The method has some significant advantages over the chemical process, such as a lower alkali dosage and less unexpected by-products [3]
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