Isomerization Of D-galactose Research Articles

Improving Catalytic Efficiency of L-Arabinose Isomerase from Lactobacillus plantarum CY6 towards D-Galactose by Molecular Modification.

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

Exploring an l-arabinose isomerase from cryophile bacteria Arthrobacter psychrolactophilus B7 for d-tagatose production

A novel l-arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d-galactose into a rare sugar d-tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (kcat/Km) and Km for d-galactose were 0.32 mM−1 min−1 and 51.43 mM, respectively, while for l-arabinose, were 0.64 mM−1 min−1 and 23.41 mM, respectively. It had the highest activity at pH 7.0–7.5 and 60 °C in the presence of 0.250 mM Mn2+. Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l-arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d-tagatose mass-production due to its industrially competitive temperature.

Highly efficient production and simultaneous purification of d-tagatose through one-pot extraction-assisted isomerization of d-galactose

A one-pot extraction-assisted d-galactose-to-d-tagatose isomerization strategy was proposed based on the selective extraction of d-tagatose by phenylborate anions. 4-Vinylphenylboronic acid was selected with high extraction efficiency and selectivity towards d-tagatose. The extracted sugars could be desorbed through a two-staged stripping process with the purity of d-tagatose significantly increased. In-situ extraction-assisted d-galactose-to-d-tagatose isomerization was implemented for the first time ever reported, and the effect of boron-to-sugar ratio (boron: sugar) was investigated. The conversion yield of d-tagatose at 60 °C increased from ∼ 39 % (boron: sugar = 0.5) to ∼ 56 % (boron: sugar = 1) but then decreased to ∼ 44 % (boron: sugar = 1.5). With temperature increased to 70 °C, the conversion yield of d-tagatose was further improved to ∼ 61 % (boron: sugar = 1.5), with the minimized formation of byproducts. Moreover, high purity (∼83 %) and concentrated d-tagatose solution (∼40 g/L) was obtained after sequential desorption. The proposed extraction-assisted isomerization strategy achieved improving the yield and purity of d-tagatose, proving its feasibility in industrial applications.

Galactose to tagatose isomerization by the l-arabinose isomerase from Bacillus subtilis: A biorefinery approach for Gelidium sesquipedale valorisation

Tagatose is a rare sugar with increasing commercial interest as sweetener. Biotechnological production of d-tagatose by enzymatic isomerization of d-galactose provides an alternative to chemical processes. In the last years, l-arabinose isomerases (L-AIs) from different origins have been studied to increase the effectiveness of tagatose production. In this work, the L-AI from Bacillus subtilis, previously reported to have unique substrate specificity for l-arabinose, was expressed in Escherichia coli and studied for isomerization of d-galactose to d-tagatose. The recombinant enzyme demonstrated, for the first time, tagatose bioconversion capacity, reaching ~59% conversion. Furthermore, a sustainable tagatose production strategy was developed by using Gelidium sesquipedale red seaweed and its undervalued processing residues as source of galactose. L-AI successfully converted the galactose-rich hydrolysate, obtained from direct acid hydrolysis of seaweed, to tagatose (50.9% conversion). Additionally, the process combining autohydrolysis of G. sesquipedale and acid hydrolysis of the remaining residue allowed a full integral valorisation of polysaccharides: 13.33 g of agar, an important hydrocolloid, coupled with the production of 5.97 g of tagatose. These results confirmed that seaweed biomass and waste-derived are promising substrates for tagatose production by L-AI, contributing to the advancement of circular economy and to the actual needs of food industry.

Whole-cell biosynthesis of d-tagatose from maltodextrin by engineered Escherichia coli with multi-enzyme co-expression system

d-tagatose is a functional sweetener that occurs in small quantity in nature. It is mainly produced through the isomerization of d-galactose by l-arabinose isomerase (l-AI; EC 5.3.1.4). However, the cost of d-galactose is much higher than those commonly used for the production of functional sweeteners such as glucose, maltodextrin, or starch. Here, a multi-enzyme catalytic system consists of five enzymes that utilizes maltodextrin as substrate to synthesize d-tagatose were co-expressed in E. coli, resulting in recombinant cells harboring the plasmids pETDuet-αgp-pgm and pCDFDuet-pgi-gatz-pgp. The activity of this whole-cell catalyst was optimal at 60 °C and pH 7.5, and 1 mM Mg2+ and 50 mM phosphate were the optimal cofactors for activity. Under the optimal reaction conditions, 2.08 and 3.2 g L−1d-tagatose were produced by using 10 and 20 g L−1 maltodextrin as substrates with recombinant cells for 24 h. This co-expression system provides a one-pot synthesis approach for the production of d-tagatose using inexpensive substrate, avoiding enzymes purification steps and supplementation of expensive cofactors.

Stabilization of immobilized l-arabinose isomerase for the production of d-tagatose from d-galactose.

The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d-galactose to d-tagatose at high temperature. l-Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)6 -tagged protein, immobilized on a copper-chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d-galactose to d-tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi-subunit structure of an enzyme immobilized on a metal-chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate-epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.

Elegant and Efficient Biotransformation for Dual Production of d-Tagatose and Bioethanol from Cheese Whey Powder.

In this study, the dual production of valuable d-tagatose and bioethanol from lactose and cheese whey powder is presented. First, a one-pot biosynthesis involving lactose hydrolysis and d-galactose isomerization for d-tagatose production was established using crude enzymes of recombinant Escherichia coli with l-arabinose isomerase (L-AI) at 50 °C. Compared to the current enzymatic system, only L-AI was overexpressed, because of the unexpectedly thermotolerant β-galactosidase in E. coli BL21(DE3). Moreover, this high temperature rendered the d-glucose catabolism of E. coli inactive, while retaining all fermentable sugars for bioethanol fermentation. Thereafter, the mixed sugar syrup was fermented by Saccharomyces cerevisiae NL22. A total of 23.5 g/L d-tagatose and 26.9 g/L bioethanol was achieved from cheese whey powder containing 100 g/L lactose. This bioprocess not only provides an efficient method for the functionalization of byproduct whey, but also offsets the high production cost of d-tagatose and bioethanol.

Towards efficient enzymatic conversion of D-galactose to D-tagatose: purification and characterization of L-arabinose isomerase from Lactobacillus brevis.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75°C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65°C and 7.0, respectively. Besides, we found that Ca2+, Cu2+, and Ba2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg2+, Mn2+, or Co2+ ions. The optimum concentration of Mn2+ and Co2+ was determined to be 1mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the Km (129mM) and the Vmax (0.045mM min- 1) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.

D-Tagatose production by permeabilized and immobilized Lactobacillus plantarum using whey permeate

The aim of the work is to produce d-Tagatose by direct addition of alginate immobilized Lactobacillus plantarum cells to lactose hydrolysed whey permeate. The cells were untreated and immobilized (UIC), permeabilized and immobilized (PIC) and the relative activities were compared with purified l-arabinose isomerase (l-AI) for d-galactose isomerization. Successive lactose hydrolysis by β-galactosidase from Escherichia coli and d-galactose isomerization using l-AI from Lactobacillus plantarum was performed to investigate the in vivo production of d-tagatose in whey permeate. In whey permeate, maximum conversion of 38% and 33% (w/w) d-galactose isomerization by PIC and UIC has been obtained. 162mg/g and 141mg/g of d-tagatose production was recorded in a 48h reaction time at 50°C, pH 7.0 with 5mM Mn2+ ion concentration in the initial substrate mixture.

Bioproduction of D-Tagatose from D-Galactose Using Phosphoglucose Isomerase from Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 phosphoglucose isomerase was purified as an active soluble form by a single-step purification using Ni-NTA chromatography that showed homogeneity on SDS-PAGE with molecular mass ∼62kDa. The optimum temperature and pH for the maximum isomerization activity with D-galactose were 60°C and 7.0, respectively. Generally, sugar phosphate isomerases show metal-independent activity but PA-PGI exhibited metal-dependent isomerization activity with aldosugars and optimally catalyzed the D-galactose isomerization in the presence of 1.0mM MnCl2. The apparent Km and Vmax for D-galactose under standardized conditions were calculated to be 1029mM (±31.30 with S.E.) and 5.95 U/mg (±0.9 with S.E.), respectively. Equilibrium reached after 180min with production of 567.51μM D-tagatose from 1000mM of D-galactose. Though, the bioconversion ratio is low but it can be increased by immobilization and enzyme engineering. Although various L-arabinose isomerases have been characterized for bioproduction of D-tagatose, P. aeruginosa glucose phosphate isomerase is distinguished from the other L-arabinose isomerases by its optimal temperature (60°C) for D-tagatose production being mesophilic bacteria, making it an alternate choice for bulk production.

The Study of Food-Grade Induced Expression and Enzymatic Properties of L-Arabinose Isomerase from <i>Lactobacillus plantarum WU</i>14 with High D-Tagatose Yield

L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60&degC, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60&degC. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50&degC, pH 7.17 and D-galactose concentration was 0.6 mol/L.

The secreted l-arabinose isomerase displays anti-hyperglycemic effects in mice

BackgroundThe l-arabinose isomerase is an intracellular enzyme which converts l-arabinose into l-ribulose in living systems and d-galactose into d-tagatose in industrial processes and at industrial scales. d-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The d-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive l-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of d-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts.ResultsThe l-arabinose isomerase (l-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SPUsp45). The l-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakeil-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant l-AI with the SPUsp45. Th l-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the l-AI and galactose, tagatose was produced in vivo and reduced the glycemia index.ConclusionsWe report for the first time the secretion of the intracellular l-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted l-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the l-arabinose isomerase has the ability to produce d-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.

Engineering of Alicyclobacillus hesperidum l-Arabinose Isomerase for Improved Catalytic Activity and Reduced pH Optimum Using Random and Site-Directed Mutagenesis

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the L-arabinose isomerase (L-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for D-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60°C. D478A, D478N, and D478Q retained more than 80% of the maximum relative activity of the wild-type L-AI at 75°C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0-6.5). All of the variant L-AIs could be significantly activated by the addition of Co(2+) and Mn(2+). D478N and D478Q showed higher catalytic efficiencies (k cat/K m) toward D-galactose than that of wild-type L-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of D-galactose to D-tagatose at 6.0 than the wild-type L-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.

Production of rare sugars from common sugars in subcritical aqueous ethanol

A new isomerization reaction was developed to synthesize rare ketoses. d-Tagatose, d-xylulose, and d-ribulose were obtained in the maximum yields of 24%, 38%, and 40%, respectively, from the corresponding aldoses, d-galactose, d-xylose, and d-ribose, by treating the aldoses with 80% (v/v) subcritical aqueous ethanol at 180°C. The maximum productivity of d-tagatose was ca. 80g/(Lh). Increasing the concentration of ethanol significantly increased the isomerization of d-galactose. Variation in the reaction temperature did not significantly affect the production of d-tagatose from d-galactose. Subcritical aqueous ethanol converted both 2,3-threo and 2,3-erythro aldoses to the corresponding C-2 ketoses in high yields. Thus, the treatment of common aldoses in subcritical aqueous ethanol can be regarded as a new method to synthesize the corresponding rare sugars.

Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of d-tagatose production

d-Tagatose is an innovative natural low-calorie bulk sweetener with a broad potential for low-calorie and low-glycaemic foods and drinks. Production of this healthy sweetener is realized through enzymatic d-galactose isomerization. d-Galactose needs to be produced in situ due to its limited availability. Whey permeate contains a substantial amount of lactose, which is an interesting source for d-galactose production through enzymatic lactose hydrolysis. In this context, the cold-active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis was studied. Optimal parameters for efficient lactose hydrolysis in whey permeate have been deduced, viz. optimal incubation temperature, pH and lactose concentration. Hydrolysis efficiencies above 96.0% were realized within 24h at 23°C and pH 7.0 in whey permeate with a maximum dry matter content of 10.0% (w/w). In addition, the effect of the presence of d-glucose and d-galactose was investigated up to concentrations of 100gl−1. d-Glucose inhibited lactose hydrolysis more strongly compared to d-galactose. Also, the operational stability of the cold-active β-galactosidase was studied. Hydrolysis efficiencies above 90.0% were maintained during 7 subsequent hydrolysis cycles.

Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

L-Arabinose isomerase (L-AI) catalyzes the isomerization of L-arabinose to L-ribulose and D-galactose to D-tagatose. Most reported L-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial D-tagatose production. Lactobacillus fermentum L-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for D-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62h at pH6.0), which are more favorable for industrial applications. With the addition of borate, D-galactose was isomerized into D-tagatose by D268K/D269K/D299K at pH5.0, resulting in a high conversion rate of 62%. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other L-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of L-AIs that can be modified for desired optimum pH and better pH stability, which are useful in D-tagatose bioproduction.

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

BackgroundD-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.ResultsIn 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.ConclusionsThe 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.

EFFICIENT BIOTRANSFORMATION OF D-GALACTOSE TO D-TAGATOSE BY ACIDOTHERMUS CELLULOLYTICS ATCC 43068

ABSTRACT An efficient method for biotransformation of D-galactose to D-tagatose was achieved by a whole-cell L-arabinose isomerase reaction of Acidothermus cellulolytics ATCC 43068 for the first time. It was observed that the strain grew well on the most carbon sources, whereas cells grown on cellobiose demonstrated the highest conversion capability. In addition, effect of reaction parameters such as pH, temperature and substrate concentration on the conversion of the isomerization was investigated. The optimum conversion ratio of D-galactose to D-tagatose reached 50.9% at pH 7.5, 75C when the substrate concentration was 50 mM. D-tagatose was separated to the purity of 95% by Ca2+cation exchange resin with a recovery of 83% and the identity of D-tagatose was confirmed by high-performance liquid chromatography analysis and 13C-Nuclear Magnetic Resonance spectra. Acidothermus cellulolytics ATCC 43068 will be a good candidate of L-AI origin for D-tagatose production on a mass scale. PRACTICAL APPLICATIONS D-tagatose, as one of the rare sugars, has been found to be natural and safe low-calorie sweetener in food products and is classified as a generally recognized as safe substance in the United States. It has attracted a great deal of attention in recent years as a low calorie sugar-substituting sweetener, an intermediate for synthesis of optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulations. L-arabinose isomerase (L-AI, EC 5.3.1.4), catalyzing the isomerization of D-galactose to and D-tagatose, is regarded as the most feasible catalyst for D-tagatose biotransformation. Isomerization at high temperature (≥60C) increases the reaction rate and allows a shift in the equilibrium toward D-tagatose, which is desirable for industrial use. In this article, a thermophile bacterium capable of producing L-arabinose isomerase was screened for the biotransformation of D-tagatose from D-galactose, which will be a good candidate of L-AI origin for industrial applications.

An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization

The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 75 degrees C and pH 7.5. It required divalent metal ions, either Mn(2+) or Co(2+), for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent K (m), V (max), and catalytic efficiency (k (cat)/K (m)) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM(-1) min(-1), respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after 12 h under optimal conditions, suggesting its potential in D-tagatose production.