D-Tagatose 3-epimerase

Rare sugars were defined as monosaccharides and their derivatives that rarely exist in nature. It has been recognized as a functional sweetener with peculiar health promoting properties and potential values in pharmaceutical industries. D-tagatose 3-epimerase (DTE,EC5.3.1.-) is the key enzyme in rare sugars bioproduction. To probe a new DTE Family enzyme, the investigations were carried out by four steps, enzyme-producing-microorganism screening, fermentation process optimization, enzyme purification and characterization, and gene recombination and recombinant enzyme characterization. The main results were described as follows:1. A strain numbered as SK011 producing D-tagatose 3-epimerase was screened from fish pond sludge. The strain was identified as Rhodobacter sphaeroides (R. sphaeroides) based on the morphous, biological, biochemical characteristics, and 16S rDNA analysis. It was named R. sphaeroides SK011.2. Fermentation process was optimized for DTE production from R. sphaeroides SK011. The results showed that enzyme activity was the highest (13.4 U/L) in medium containing 1% of glucose, yeast extract 2%, tryptone 1.25%, NaCl 0.3%, NaH₂PO₄.2H₂O 0.3 %, MgSO4 0.05%, and D-tagatose 0.2% when R. sphaeroides SK011 was cultured at 30℃, 180 rpm for 36 h in dark, and the initial pH was adjusted to 7.0.3. The enzyme was purified to electrophoresis homogeneity by a combination of ammonium sulfate precipitation and a serial of chromatographic stages. The molecular mass estimated to be 64 kDa by size exclusion chromatography and 32 kDa by SDS-PAGE. It exhibited maximal activity at 40℃and pH 9.0 and was stable below 40℃in the pH range of 8-10 with Mn²⁺ as a cofactor. Cu²⁺ and Zn²⁺ caused its inhibition. Its substrate specificity was greatest for D-tagatose.4. The N-terminal amino acid sequence of the purified enzyme was MKNPVGIISM and showed no experimentally confirmed homologies, but we found computationally annotated sequences, Rsph17029₃406. To our knowledge, it is the first report on the characterization of D-tagatose 3-epimerase from R. sphaeroides and it was named R. sphaeroides DTE.5. Rsph17029₃406 was amplified and inserted into the pET-22b (+) plasmid and transformed into Escherichia coli BL21 (DE3). Then, the recombinant BL21 (DE3) (pET-dte) was constructed. After cultured with IPTG as inducer, the recombinant protein was produced and purified by nickel-equilibrated chelating Sepharose Fast Flow. It exhibited maximal activity at 40℃and pH 9.0 and was stable below 40℃in the pH range of 8-10 with Mn²⁺ as a cofactor. Cu²⁺ and Zn²⁺ caused its inhibition. The enzyme properties obtained were similar to that of DTE purified from SK011. It was suggested that Rsph17029₃406 was expressed successfully in BL21 (DE3) (pET-dte) and recombinant DTE was produced. Rsph17029₃406 was qualified as the gene encoded R. sphaeroides DTE and was assigned in GenBank with a gene accession number FJ851309 and a corresponding protein id, ACO59490. 6. On substrate specificity, the recombinant R. sphaeroides DTE was greatest for D-fructose and second for D-tagatose, that was different from the authentic R. sphaeroides DTE. At 40℃and pH 9.0, the equilibrium ratio value between D-psicose and D-fructose was 23:77.