The typically used chemical method, also known as non-enzymatic isomerization [
21], uses metal hydroxides as catalysts. This method consists of three steps: firstly, an insoluble
d-tagatose complex is generated when
d-galactose interacts with metal hydroxides, which are stable under alkaline conditions; in the second stage, the insoluble
d-tagatose complex is neutralized with an acid to produce an insoluble salt; finally, filtering is used to separate
d-tagatose from the insoluble salt. During the isomerization, the metal hydroxides perform two functions: isomerizing
d-galactose to
d-tagatose and degrading
d-galactose into dicarbonyl compounds and acidic substances. This method has a high yield (>70%) but includes the following disadvantages: severe
d-galactose degradation leads to a reduction in the yield of
d-tagatose, while the decrease in the quality of the syrup makes the production of crystalline
d-tagatose difficult; the removal of the degradation products requires complex extraction steps; and a large number of metal hydroxides and acids are consumed in the reaction, which ultimately has many negative impacts on the cost and the environment. There are also other methods such as supercritical fluid (<24%) [
22], triethylamine (<34%), arginine (<16.8%) [
23], hydrotalcite (<27%) [
24], Sn-β zeolite (<26%), etc. However, all of these non-enzymatic pathways have low
d-tagatose yields [
25]. Moreover, these methods require reactions under extreme conditions, which can lead to increased energy consumption and damage to substrates and products.
Tagatose 4-epimerase can epimerize
d-fructose into
d-tagatose. However, there are few reports of enzymes with
d-fructose epimeric activity at the C-4 position in nature. Therefore, obtaining such enzymes by screening new ones or modifying old ones is the main work in this production pathway. Shin et al. [
27] developed a new tagatose 4-epimerase through rational design and directed the evolution of the tagaturonate 3-epimerase from
Thermotoga petrophila. The modified enzyme exhibited 184-fold-higher epimeric activity towards
d-fructose compared to the original enzyme. Under optimal conditions of 80 °C, pH 8.5, and 1.5 mM Ni
2+, the enzyme was able to produce 213 g/L of
d-tagatose from 700 g/L of
d-fructose within 2 h, with a conversion rate of 30%. Jeon et al. [
28] expressed tagatose 4-epimerase from
Thermotoga neapolitana in
Corynebacterium glutamicum and improved the enzyme expression level by optimizing plasmid copy numbers. Under conditions of 60 °C and a specific amount of metal ions, the conversion rate reached 21.7%.
2. The Production of d-Tagatose by L-AIs
2.1. Molecular Structure and Catalytic Mechanism of L-AIs
The crystal structures of L-AIs from
E. coli (PDB code: 2AJT) [
38],
Lactobacillus fermentum CGMCC2921 (PDB code: 4LQL) [
39],
Geobacillus kaustophilus (PDB code: 4R1O), and
Thermotoga maritima MSB8 (PDB code: 7CWV) have been determined. Taking the L-AI from
E. coli (ECAI) as an example [
40], it is a hexamer with a total molecular weight of 336 kDa. Three asymmetric units of L-AI subunits form a trimer, as shown in
Figure 4a. Two such trimers stack together to form the complete hexamer. Each subunit contains three structural domains: the N-terminal domain, the central domain, and the C-terminal domain. Within the complete hexamer of ECAI, there are six active sites located at the monomer–monomer interfaces (
Figure 4a) and situated in the conjugate regions of adjacent subunits.
Figure 4. (a) The substrate and active site are located near the interface of two identical protein subunits; (b) ECAI catalytic residues Glu306 and Glu333 and amino acid residues in the region surrounding the active site; (c) Catalytic mechanism of L-AI—enediol intermediate.
The catalysis of
d-galactose to
d-tagatose by L-AIs follows the mechanism of enediol intermediate formation [
41]. Taking ECAI as an example (
Figure 4b), the residues E306 and E333 act as essential catalytic residues, while H350 and H450 stabilize the active site together with manganese ions. During isomerization (
Figure 4), the O
ε2 of Glu306 nucleophilically attacks the hydrogen atom of C-2 of
d-galactose to deprotonate it and form a carbon–carbon double bond. The carbon–oxygen double bond is broken to form an oxygen anion, which combines with a proton to form an enediol intermediate; the O
ε2 of Glu333 nucleophilically attacks the hydroxyl hydrogen of C-2 of
d-galactose to generate a carbon–oxygen double bond at the C-2 position. This results in the cleavage of the carbon–carbon double bond between C-1 and C-2, producing a carbon anion that combines with a proton, ultimately yielding
d-tagatose. This mechanism involves two proton transfers in
d-galactose, one from O-2 to O-1 and the other from C-2 to C-1.
2.2. Properties of L-AIs
In order to efficiently produce
d-tagatose, it is crucial to delve into the properties of L-AIs from various sources. The enzyme comes from a wide range of microbial sources, including
Lactobacillus plantarum NC8 [
42],
Anoxybacillus flavithermus [
43],
Bacillus coagulans NL01 [
44],
Pediococcus pentosaceus PC-5 [
45],
Clostridium hylemonae [
46],
Lactobacillus sakei 23K [
47],
Lactobacillus fermentum CGMCC2921 [
48],
Bifidobacterium adolescentis [
49],
Thermo toga maritima [
50],
Thermotoga neapolitana [
51],
Lactococcus lactis [
52],
Bacillus thermoglucosidasius [
53],
Arthrobacter species 22c [
54],
Shewanella species ANA-3 [
55],
Bacillus licheniformis [
56],
Bacillus subtilis 168 [
57], etc.
L-AIs from different microbial sources have different optimal temperature ranges. L-AIs sourced from mesophilic microorganisms exhibit optimal temperatures between 30 and 50 °C; those from thermophilic microorganisms have optimal temperatures ranging from 50 to 80 °C; and L-AIs from hyperthermophilic microorganisms have optimal temperatures exceeding 80 °C. Although L-AIs from hyperthermophiles have higher optimal temperatures and excellent thermal stability, their need for Co
2+ to stabilize their structure at ultrahigh temperatures limits their application in the food industry [
59,
60]. Therefore, in actual industrial applications, L-AIs from thermophilic microorganisms are usually preferred.
L-AIs from different microbial sources exhibit variations in their optimal pH. Most reported L-AIs display their maximum activity under neutral or alkaline conditions. However, L-AIs with an optimal pH in the weakly acidic range offer advantages in industrial applications, including faster reaction rates and reduced by-product formation.
Metal ions play a crucial role in the activity and stability of L-AIs. Although not all L-AIs necessarily rely on the divalent metal ions to maintain their activity [
61], for most L-AIs, divalent metal ions, especially Mn
2+ and Co
2+, are essential for exerting their activity and maintaining thermal stability. Since Co
2+ is not permitted in the food industry, the addition of Mn
2+ is more appropriate. Choi et al. [
62] explored the structure of the L-AI from
Geobacillus kaustophilus and found that the addition of Mn
2+ transforms the L-AI structure from low oligomers to complete hexamers, indicating the significant role of metal ions in the oligomerization process. The authors also determined the melting temperatures of the hexamers by differential scanning calorimetry (DSC) and showed that the complete hexameric form is thermodynamically more stable.
Although most of the reported L-AIs were specific for
l-arabinose and
d-galactose, the specificity for
l-arabinose was significantly higher than that for
d-galactose. However, there are some exceptions. For example, the L-AIs from
Bacillus subtilis 168 and
Pseudoalteromonas haloplanktis [
63] showed unique substrate specificity exclusively for
l-arabinose. In addition, there are some enzymes classified as
d-galactose isomerase because their optimal substrate is
d-galactose instead of
l-arabinose, such as the L-AI from
Bifidobacterium adolescentis.
In addition to L-AIs, various sugar phosphate isomerases are also general aldose-ketose isomerases that can catalyze the biotransformation of non-phosphorylated monosaccharides. Patel et al. [
64] characterized the phosphoglucose isomerase from
Pseudomonas aeruginosa PAO1. This enzyme can also isomerize
d-galactose to
d-tagatose. The
d-tagatose yield was 56% and the optimal activity was observed at 60 °C and pH 7.
A high temperature and slightly acidic conditions are considered ideal conditions for the efficient isomerization of
d-galactose into
d-tagatose. This is because the
d-galactose isomerization process requires a high Gibbs free energy (4.96 kJ/mol) [
65]. Therefore, the proper thermal conditions are necessary to overcome this energy barrier, which usually requires high temperature conditions [
37]. High temperature conditions provide several advantages in this process. Firstly, within a specific range, higher temperatures lead to faster reaction rates, favoring the production of
d-tagatose and improving the conversion; increasing the substrate solubility; reducing the viscosity of the reaction mixture, which improves the mass transfer efficiency and makes the reaction more efficient; and causing the risk of microbial contamination to be relatively low. However, it should be noted that extremely high temperature conditions above 80 °C may cause browning of the product and the generation of unnecessary by-products [
66]. Therefore, the temperature range between 60 °C and 80 °C is usually chosen for industrial production.
d-tagatose exhibits stability under acidic conditions and can remain relatively stable within the pH range of 3–7 [
67], while side reactions will increase under alkaline conditions [
68]. In addition, a high temperature and high
d-galactose concentration may result in a decrease in the pH due to the Maillard reaction [
69].
In conclusion, the L-AI from
Lactobacillus sakei 23K showed optimal conditions at a low temperature and under acidic conditions, making it particularly suitable for the conversion of
d-galactose to
d-tagatose during the storage of milk and yogurt [
70]. Xu et al. screened the L-AI from
Lactobacillus fermentum CGMCC2921 and found its optimum temperature of 65 °C and the optimal pH of 6.5, which was within the range of suitability for industrial applications.
2.3. Production of d-Tagatose Using Lactose as Raw Material
d-galactose can usually be easily obtained from raw materials containing lactose. Therefore, it is necessary to combine lactose hydrolysis with the enzymatic isomerization of
d-galactose to
d-tagatose. This process can be accomplished under optimal conditions for hydrolysis and isomerization or through a one-pot method where hydrolysis and isomerization are carried out simultaneously [
71]. The stepwise method allows hydrolysis and isomerization to be optimized separately under their respective optimal conditions, but high concentrations of
d-glucose and
d-galactose can lead to feedback inhibition of lactose hydrolysis, ultimately resulting in low conversion rates and yields. In such cases, the one-pot method is more advantageous as it simplifies the operations and minimizes the accumulation of
d-galactose, thus improving the final yield of
d-tagatose.
Furthermore, fully utilizing the residual
d-glucose and
d-galactose in the process is also an important consideration. These residues are typically wasted, and the similar physicochemical properties of
d-galactose and
d-tagatose introduce complex purification steps in downstream processes. These residual substances can serve multiple purposes. They can be used as energy sources to sustain cell viability or further converted into other products such as
d-fructose and ethanol. Rhimi et al. [
72] successfully co-expressed the L-AI from
Bacillus stearothermophilus US100 and
d-glucose isomerase from
Streptomyces SK in
E. coli to isomerize the remaining D-glucose into
d-fructose. Zheng et al. [
73] expressed the L-AI from
Bacillus coagulans NL01 in
E. coli and combined it with self-expressed β-galactosidase for the crude enzyme conversion of lactose at 50 °C, and then the residual
d-glucose and
d-galactose were further fermented to bioethanol using
Saccharomyces cerevisiae NL22. This approach enhanced the ethanol production competitiveness and simplified the purification process of
d-tagatose. Zhang et al. [
74] utilized cheese whey for
d-tagatose-production through whole-cell biotransformation in
E. coli. Then, they fermented the residual
d-glucose and
d-galactose into
d-arabitol and
d-galactitol using
Metschnikowia pulcherrima E1 yeast to maximize the conversion of lactose in the cheese whey into three high-value rare sugars (
Figure 5).
Figure 5. Utilization of lactose raw materials to produce d-tagatose, d-fructose, and bioethanol.