A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting β-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp. 22c

Abstract

Background: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a β-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose.

Results: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting β-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting β-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose.

Conclusions: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting β-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.

Figure 1

Multiple sequence alignment ofArthrobactersp. 22c L-arabinose isomerase with other L-arabinose isomerases. 22cAI – Arthrobacter sp. 22c L-AI, ACAI – Acidothermus cellulolytics ATCC 43068 L-AI, TSAI – Thermus sp. IM6501 L-AI, BStAI – Bacillus stearothermophilus IAM 11001 L-AI, BSAI – Bacillus stearothermophilus US100 L-AI, GSAI – Geobacillus stearothermophilus L-AI, AFAI - Anoxybacillus flavithermus L-AI, AAAI – Alicyclobacillus acidocaldarius L-AI, GTAI – Geobacillus thermodenitrificans L-AI, BHAI – Bacillus halodurans L-AI, TNAI – Thermotoga neapolitana L-AI, TMAI – Thermotoga maritima L-AI, ECAI – Escherichia coli str. K-12 substr. W3110 L-AI, LPAI – Lactobacillus plantarum NC8 L-AI, LFAI – Lactobacillus fermentum CGMCC2921 L-AI, LSAI – Lactobacillus sakei 23 K L-AI, TaMAI – Thermoanaerobacter mathranii L-AI. Three levels of conserved residues are indicated by blue (100%), green (80%) and yellow (60%) backgrounds. Catalytic residues are shaded red. The alignment was performed using Clustalx 2.0.11 program.

Figure 2

SDS-PAGE analysis of the fractions obtained by expression and purification ofArthrobactersp. 22c L-arabinose isomerase. Lane 1 – Unstained Protein Molecular Weight Marker (Fermentas): 116, 66.2, 45, 35, 25, 18.4 and 14.4 kDa, lane 2 – cell extract of E. coli BL21(DE3)pLysS + pET30araA22c after araA gene expression, lane 3 – purified L-arabinose isomerase after ion exchange chromatography on Fractogel EMD DEAE column, lane 4 - purified L-arabinose isomerase after ion exchange chromatography on Fractogel EMD TMAE column.

Figure 3

Effect of temperature on activity of recombinantArthrobactersp. 22c L-arabinose isomerase. Reaction mixtures containing 0.2 mg mL^-1Arthrobacter sp. 22c L-arabinose isomerase and 4% (w/v) D-galactose in 20 mM potassium phosphate buffer pH 7.0 were incubated at temperatures from 25 to 65°C for 12 h.

Figure 4

Effect of pH on activity of recombinantArthrobactersp. 22c L-arabinose isomerase. Reaction mixtures containing 0.2 mg mL^-1Arthrobacter sp. 22c L-arabinose isomerase and 4% (w/v) D-galactose in 20 mM citrate buffer pH 5–6 (♦), 20 mM potassium phosphate buffer pH 6–8 (■) and 20 mM Tris–HCl buffer pH 8–9 (▴) were incubated at 50°C for 12 h.

Figure 5

Time course of D-tagatose production duringArthrobactersp. 22c L-AI-catalyzed isomerization of D-galactose. Reaction mixtures containing 0.2 mg mL^-1Arthrobacter sp. 22c L-arabinose isomerase and 4% (w/v) D-galactose in 20 mM potassium phosphate buffer pH 7.0 were incubated at 50°C.

Figure 6

Time course of D-galactose production from whey permeate usingP. pastorisstrain secreting β-D-galactosidaseA. chlorophenolicus: (□) lactose, (♦) D-galactose. Panel A: whey permeate containing 60 g L^-1 lactose before inoculation with recombinant P. pastoris strain. Panel B: whey permeate containing 140 g L^-1 lactose before inoculation with recombinant P. pastoris strain. Panel C: whey permeate containing 200 g L^-1 lactose before inoculation with recombinant P. pastoris strain. Panel D: whey permeate containing 300 g L^-1 lactose before inoculation with recombinant P. pastoris strain.

Figure 7

Time course of D-tagatose production from whey permeate usingP. pastorisstrain secreting β-D-galactosidaseA. chlorophenolicusand recombinantArthrobactersp. 22c L-arabinose isomerase: (□) lactose, (♦) D-galactose, (▴) D-tagatose.