Tagatose Production Research Articles

Tagatose Production by Immobilized Recombinant Escherichia coli Cells Containing Geobacillus stearothermophilusl-Arabinose Isomerase Mutant in a Packed-Bed Bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.

Tagatose Production with pH Control in a Stirred Tank Reactor Containing Immobilized l-Arabinose Isomerase from Thermotoga neapolitana

Chitopearl beads were used as immobilization supports for D-tagatose production from D-galactose by L-arabinose isomerase from Thermotoga neapolitana because chitopearl beads were more stable than alginate beads at temperatures above 60 degrees C. The pH and temperature for the maximum isomerization of galactose were 7.5 and 90 degrees C, respectively. In thermostability experiments, the half-lives of the immobilized enzyme at 70, 75, 80, 85, and 90 degrees C were 388, 106, 54, 36, and 22 h, respectively. The reaction temperature was determined to be 70 degrees C because the enzyme is highly stable up to 70 degrees C during the reaction. When the reaction time, galactose concentration, and temperature were increased, the pH of a mixture containing enzyme and galactose decreased by the Maillard reaction, resulting in decreased tagatose production. With pH control at 7.5, tagatose production (138 g/L) at 70 degrees C in a stirred tank reactor containing immobilized enzyme and 300 g/L galactose increased two times higher, comparing that without pH control.

Current studies on biological tagatose production using L-arabinose isomerase: a review and future perspective.

D-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of D-galactose and is rarely found in nature. Recently, there has been industrial interest in D-tagatose as a low-calorie sugar-substituting sweetener. This article describes the properties and metabolism of tagatose as well as its commercial importance. The comparison between the biological tagatose production and the chemical production was reviewed based on the example of the glucose isomerization into fructose. The industrial problems facing its commercial application is described and evolving potential solutions are suggested.

Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501.

A gene (thaI) corresponding to L-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the L-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60 degrees C at pH 8 for 30 min of reaction. Zn2+ and Ni2+ inactivated the catalytic activity of ThaI, 5 mM Mn2+ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from D-galactose to D-tagatose was obtained by recombinant ThaI at 60 degrees C over 3 d.

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe2+ and Mn2+ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu2+, Zn2+, Hg2+, and Fe3+ ions inhibited the reaction completely. In the presence of 1 mM Fe2+ ions, the K m for galactose was found to be 300 mM.

Biotransformation of d-galactitol to tagatose by acetic acid bacteria

Tagatose, a ketohexose C-4 fructose epimer present in nature in low concentration, is a potential low calorie bulking sweetener that can be obtained by the microbial oxidation of the corresponding polyalcohol galactitol. The screening of strains belonging to the acetic acid bacteria resulted in 100–160 mg tagatose/l, produced at 24 h in non growing conditions, while 260–340 mg tagatose/l was obtained at 48 h with growing cells of Gluconobacter strains, with a specific activity rate of tagatose production, 1.4×10 −3 l/h. After galactitol adaptation the Gluconobacter oxydans DSM 2343 strain gave a notable increase in tagatose yield, reaching 3160 mg/l with a corresponding 6.6×10 −3 l/h specific activity rate at 24 h of reaction. Preliminary enzyme characterisation experiments indicated that the dehydrogenase activity may be attributable to a sorbitol dehydrogenase (SDH).

Development of an immobilization method of L-arabinose isomerase for industrial production of tagatose

Of the immobilization methods tested, alginate beads treated with glutaraldehyde gave the most stable and economic method for immobilizing l-arabinose isomerase for the industrial production of tagatose. l-Arabinose isomerase immobilized in a packed-bed reactor produced an average of 30 g tagatose l−1 day−1 from 100 g galactose l−1 for 8 days.

Preparation of L-arabinose isomerase originated from Escherichia coli as a biocatalyst for D-tagatose production

l-Arabinose isomerase for tagatose production from recombinant Escherichia coli was partially purified 15-fold with a specific activity of 70 U mg−1 protein. The purified enzyme had a major band when it was subjected to SDS/PAGE. With the purified l-arabinose isomerase, 17.7 g tagatose l−1 was produced from 50 g galactose l−1 in 168 h which corresponds to a 34% equilibrium.