Overview

Lactose is composed of β-D-glucose and β-D-galactose. For lactose to be absorbed by the human body, it must be hydrolyzed into monosaccharides with the help of lactase and β-T-galactosidase. These enzymes are abundant in newborns; however, their activity gradually decreases with age, a phenomenon known as lactase non-persistence (LNP). Lactose intolerance refers to a condition where individuals experience digestive symptoms—such as bloating, diarrhea, and flatulence—after consuming lactose-containing foods or beverages. According to data from the National Institutes of Health (NIH), approximately 68% of the global population suffers from lactose malabsorption (NIDDK, 2018) ^[1]. To address this issue, this project developed a dairy product tailored for lactose-intolerant individuals. Specifically, β-T-Galactosidase (β-T-Gal) and glucose oxidase (GOD) were used to decompose dissolved lactose in milk into prebiotics, including galactooligosaccharide (GOS) and glucuronic acid.

1.  The Construction of Plasmid

1.1 PCR amplification of β-T-Gal and GOD

PCR amplification was performed to obtain the target genes encoding β-T-Galactosidase (β-T-Gal) and glucose oxidase (GOD), with lengths of 2209 bp and 1294 bp, respectively. As shown in Fig. 1, distinct and sharp bands were observed, which matched the expected sizes of the two target genes. This result confirms the successful amplification of β-T-Gal and GOD.

Fig 1. Gel Electrophoresis Validation of PCR-Amplified β-T-Gal and GOD

1.2 Linearization of pPICZαA

After double digestion, linearized pPICZαA vector fragments were obtained. Gel electrophoresis (Fig. 2) showed clear bands at the expected size, verifying both the successful linearization of the vector and the correctness of the fragment length.

Fig 2. Gel Electrophoresis Validation of Double-Digested pPICZαA

1.3 Homologous Recombination of pPICZαA-β-T-Gal and pPICZαA-GOD

Recombinant plasmids (after homologous recombination) were transformed into competent E. coli DH5α cells using a standard heat-shock transformation protocol. As shown in Fig. 3, zeocin-resistant colonies were clearly observed, indicating that the recombinant plasmids had been successfully introduced into E. coli DH5α. Since the LB solid medium was supplemented with zeocin, and the original E. coli DH5α strain is inherently sensitive to zeocin, only E. coli cells harboring the recombinant plasmids (which confer zeocin resistance) could survive and form colonies on this selective medium.

Fig 3. Heat-Shock Transformation of E. coli DH5α

Plasmids were extracted from cultured E. coli DH5α cells. Gel electrophoresis (Fig. 4A) showed clear bands for the recombinant plasmids pPICZαA-β-T-Gal and pPICZαA-GOD, with lengths significantly longer than that of the empty pPICZαA vector—confirming the successful homologous recombination of the target genes into the vector. To rule out the presence of random mutations that might impair protein function, the purified recombinant plasmids were subjected to full-length sequencing by Beijing Tsingke Biotech Co., Ltd. The sequencing results (Fig. 4B for pPICZαA-GOD and Fig. 4C for pPICZαA-β-T-Gal) confirmed that both expression vectors were correctly constructed without mutations.

 Fig 4. Colony PCR and Sequencing results of E. coli DH5α

 2. Protein Expression

2.1 Linearization of Recombinant Plasmids

Gel electrophoresis of the treated plasmids showed distinct and sharp bands (Fig. 5A for pPICZαA-GOD and Fig. 5B for pPICZαA-β-T-Gal). The lengths of these bands were consistent with the predicted sizes in the plasmid maps (Fig. 5C for pPICZαA-β-T-Gal and Fig. 5D for pPICZαA-GOD), providing clear evidence of successful plasmid linearization.

Fig 5. Linearization of pPICZαA-GOD and pPICZαA-β-T-Gal

 2.2 Plasmid Transformation

YPD petri dishes were supplemented with zeocin, which specifically inhibits the growth of wild-type Pichia pastoris. Thus, only Pichia pastoris cells that successfully took up the recombinant plasmids (pPICZαA-GOD or pPICZαA-β-T-Gal, which carry zeocin resistance genes) could survive. As shown in Fig. 6 (Fig. 6A for pPICZαA-β-T-Gal and Fig. 6B for pPICZαA-GOD), dense colonies were observed on the YPD plates, confirming the successful transformation of recombinant plasmids into Pichia pastoris host cells.

Fig 6. Pichia pastoris Cultured on YPD Solid Medium

 2.3 Detection of Recombinant Plasmids in Pichia pastoris

Plasmids were extracted from Pichia pastoris colonies and subjected to PCR amplification. Gel electrophoresis (Fig. 7) was used to verify the integrity of the recombinant plasmids (pPICZαA-GOD and pPICZαA-β-T-Gal) in the host cells. The results showed that the lengths of the amplified fragments were consistent with the expected sizes of the recombinant plasmids, indicating that the plasmid structure remained unchanged after transformation into Pichia pastoris.

Fig 7. Yeast Colony PCR Results

 2.4 SDS-PAGE Analysis of pPICZαA-β-T-Gal and pPICZαA-GOD

β-T-Gal and GOD have molecular weights of ~80 kDa and ~48 kDa, respectively. SDS-PAGE analysis was performed to verify the expression of these two proteins. As shown in Fig. 8A (for GOD) and Fig. 8B (for β-T-Gal), clear bands were observed at the expected molecular weights, confirming the successful expression of both recombinant proteins.

Fig 8. SDS-PAGE Analysis of GOD and β-T-Gal 

3. Function Test

3.1 β-T-Gal Hydrolysis Activity Test

To evaluate the catalytic activity of the purified β-T-galactosidase (β-T-Gal), this study utilized its specific hydrolysis of o-nitrophenyl-β-D-galactoside (ONPG) to produce the yellow product o-nitrophenol (ONP). Enzyme activity was quantified by measuring the characteristic absorbance of ONP at 420 nm. The experimental method is briefly described as follows: Enzyme solutions of β-T-Gal at different concentrations were reacted with ONPG substrate at 40°C and pH 7.0 for a specific duration. The reaction was terminated by adding sodium carbonate solution, followed by immediate absorbance measurement using a microplate reader. As shown in Table 1, three parallel experiments were conducted at each concentration to ensure data reliability.

Table 1. Absorbance measurement at 420 nm

mg/ml	A420
0	0.032	0.034	0.031
1	0.071	0.078	0.077
2	0.084	0.089	0.091
5	0.126	0.133	0.132
10	0.232	0.239	0.239
15	0.305	0.289	0.295
20	0.296	0.299	0.298

 Results demonstrated that the purified β-T-Gal exhibited significant catalytic activity. As shown in Fig. 9, the absorbance value of the reaction system increased significantly with rising enzyme concentration, exhibiting a good linear relationship between the two. This linear trend clearly confirmed that ONP production is proportional to enzyme concentration, indicating the successful purification of β-T-Gal with highly efficient and concentration-dependent catalytic activity.

Fig 9. Results of β-T-Gal Hydrolysis Activity Test 

3.2 GOD Activity Test

The activity of purified glucose oxidase (GOD) was determined using a glucose oxidase (GOD)-peroxidase (POD) coupled reaction system. The principle is as follows: GOD catalyzes the oxidation of glucose to produce H₂O₂. Subsequently, POD catalyzes the decomposition of H₂O₂ in the presence of oxygen. The resulting reactive oxygen species oxidize colorless o-anisidine to form a brownish-yellow product. The absorbance of this product at a specific wavelength is positively correlated with GOD activity. A series of GOD dilutions (Table 2) was measured for reaction absorbance using a Multiskan™ FC microplate reader. 

Table 2. Absorbance measurement at 500 nm

mg/ml	A500
0	0.076	0.074	0.075
0.5	0.178	0.179	0.178
1	0.184	0.183	0.183
2.5	0.469	0.467	0.465
5	0.725	0.723	0.721
10	1.251	1.249	1.247
20	1.508	1.503	1.5

 Results demonstrated (Fig. 10) that although the absorbance curve was not strictly linear, its overall trend of significant increase with rising enzyme concentration clearly confirmed that the purified GOD retained potent catalytic activity.

Fig 10. Results of the GOD Hydrolysis Activity Test 

3.3 TLC Test for β-T-Gal Activity

Thin-layer chromatography (TLC) was used to assess β-T-Gal activity under two experimental conditions: (1) fixed β-T-Gal concentration with gradient lactose concentrations; (2) fixed lactose concentration with gradient time. Fig 11 shows the results of the lactose-gradient experiment: the migration distance of samples increased with increasing lactose concentration. This indicates that when the substrate is insufficient and β-T-Gal is not saturated, the lactose concentration acts as the limiting factor for the hydrolysis reaction, and the amount of product increases with the increase in substrate. As the reaction time extends from 0 min to 60 min, there is little difference in the intensity of the "lactose substrate spot" on the TLC plate, and no obvious distinction in the resolution of spots between adjacent time points. This may be because β-T-Gal has completed the hydrolysis of most lactose within 15 min; the change in the amount of substrate is minimal in the subsequent time, resulting in insignificant differences between spots at adjacent time points and making it impossible to clearly distinguish the degree of hydrolysis. The time gradient needs to be adjusted in subsequent experiments to enable such a distinction.

Fig 11. TLC Analysis of β-T-Gal Activity 

At a fixed enzyme concentration, increasing lactose concentration not only significantly elevated the yield of the primary hydrolysis product glucose. Notably, in the high-concentration lactose system (5%), galacto-oligosaccharides (GOS) were also observed to form alongside glucose. Moreover, GOS production increased with rising lactose concentration, indicating that high substrate concentration promotes β-T-Gal-catalyzed glycosylation reactions, thereby synthesizing GOS. 

3.4 TLC Analysis of Dual-Enzyme Synergy

To investigate the efficiency of lactose hydrolysis under the synergistic action of β-T-Gal and GOD, we comparatively analyzed the hydrolysis effects in both pure lactose solution and fresh milk. Thin Layer Chromatography (TLC) results demonstrated that, under the synergistic action of the two enzymes, lactose was hydrolyzed into glucose and galactooligosaccharides (GOS). Moreover, the hydrolysis effect in fresh milk was identical to that in the pure lactose solution (Fig. 12). This confirms that the synergistic effect of β-T-Gal and GOD is feasible in fresh milk.

Fig 12. TLC Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity 

 3.5 Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC

To quantitatively compare the decomposition efficiency of lactose in milk under the synergistic action of dual enzymes (β-T-Gal and GOD), we employed High-Performance Liquid Chromatography (HPLC) to dynamically monitor the decrease in lactose concentration and the increase in glucose concentration in milk during the reaction. The experimental setup was as follows: β-T-Gal and GOD were mixed at a 1:1 ratio and added to the milk system; the reaction was initiated under three temperature conditions (25°C, 37°C, and 42°C), and samples were taken at 10-minute, 20-minute, and 60-minute intervals after the start of the reaction. The concentrations of lactose and glucose at each time point were determined by HPLC (Table 3 and Table 4), and the relevant results are shown in Fig. 13. 

Table 3. Lactose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	3.5	2.7	2.4
10	2.6	2.1	2.5
20	2.5	1.7	1.4
60	2.1	2.1	1.2

 

Table 4. Glucose Concentration Detected by HPLC

Time (min)	25℃	37℃	42℃
0	0	0	0
10	0	2	3.5
20	0	3.4	6.1
60	3.6	5.6	6.7

 In terms of the effect of temperature on reaction efficiency, both the lactose decomposition rate and glucose production rate at 42°C were significantly higher than those at 25°C and 37°C. This indicates that 42°C is the optimal reaction temperature for the dual-enzyme synergistic system to catalyze lactose decomposition in milk. A further analysis of the reaction kinetic characteristics at the optimal temperature (42°C) revealed that the first 20 minutes of the reaction were the stage with the fastest glucose production rate. This phenomenon suggests that during the initial stage of dual-enzyme synergy (the first 20 minutes), the binding efficiency between the enzymes and the substrate (lactose) is the highest, and the catalytic activity is at its peak, allowing lactose to be rapidly decomposed into glucose. However, as the reaction proceeds, the glucose production rate gradually slows down, which may be attributed to the decrease in substrate (lactose) concentration, the feedback inhibition caused by the accumulation of the product (glucose), or a slight decline in enzyme activity over the reaction time.

Fig 13. Quantitative Analysis of Dual-Enzyme Synergistic Products by HPLC 

In summary, this study determined that the optimal reaction conditions for dual-enzyme synergistic lactose decomposition are 42°C, identifying the initial 20 minutes as the critical phase for high-efficiency conversion. During this period, enzyme-substrate binding is highly efficient, catalytic activity reaches its peak, and rapid lactose degradation is achievable. Therefore, controlling the reaction at 42°C for 20 minutes represents an optimized strategy balancing efficiency and energy consumption.

Discussion

This study successfully constructed the recombinant expression plasmids pPICZαA-β-T-Gal and pPICZαA-GOD, and achieved the recombinant expression and purification of both enzymes in Pichia pastoris. Functional validation results demonstrated that the purified β-T-Gal and GOD possess high catalytic activity. Furthermore, the dual-enzyme system exhibited significant synergistic effects within a milk matrix, efficiently decomposing lactose into glucose and generating galacto-oligosaccharides (GOS) with prebiotic potential. This research provides crucial enzyme resources and process parameters for developing functional dairy products tailored for lactose-intolerant individuals.

Firstly, protein expression and purification form the foundation for studying enzymatic properties. SDS-PAGE analysis revealed clear bands at the expected molecular weights (Fig. 8), confirming the successful acquisition of highly pure β-T-Gal and GOD. Subsequent enzyme activity assays further verified the functional integrity of the purified proteins. The hydrolysis activity curve of β-T-Gal (Fig. 9) showed a good linear relationship between its catalytic activity and enzyme concentration, which is consistent with classical enzyme kinetics theory, indicating the enzyme's stability under the tested conditions without significant aggregation or inactivation. Similarly, the GOD activity test (Fig. 10), while not perfectly linear, demonstrated a significant increase in absorbance with rising enzyme concentration, unequivocally confirming its ability to oxidize glucose. This non-linear relationship might stem from substrate diffusion limitations or slight product inhibition effects in high-concentration enzyme solutions, which are common phenomena in coupled enzyme reaction systems.

Secondly, TLC analysis revealed the diversity in β-T-Gal's catalytic behavior, demonstrating both hydrolytic and transglycosylase activities. At low lactose concentrations, hydrolysis dominated, yielding primarily glucose and galactose (Fig. 11). However, when the lactose concentration increased to 5%, the generation of GOS was clearly observed (Fig. 11), and its yield increased with increasing substrate concentration. This phenomenon aligns with the transglycosylation reaction mechanism of β-T-galactosidase: under high substrate concentrations, the glycosylated intermediate formed by the enzyme and lactose is more likely to be attacked by another lactose molecule rather than a water molecule, leading to GOS formation. This finding holds significant application value, as GOS is a recognized prebiotic that can selectively promote the proliferation of beneficial bacteria in the gut. This implies that our dual-enzyme system can not only eliminate lactose but also enrich the product with beneficial components in situ, thereby enhancing its added value.

Most crucially, HPLC quantitative analysis confirmed the synergistic action of β-T-Gal and GOD in the milk system and identified the optimal reaction conditions. The catalytic efficiency of the dual-enzyme system at 42°C was significantly higher than at 25°C and 37°C (Fig. 13), indicating that 42°C is the ideal temperature point balancing the optimal activities of both enzymes. The reaction kinetic curve showed a rapid increase in glucose production within the first 20 minutes, followed by a slowed rate. This typical biphasic kinetic profile can be attributed to several factors: the high initial rate is due to abundant lactose substrate and enzymes being at their peak activity; as the reaction progresses, substrate depletion and product (glucose) accumulation may cause feedback inhibition on both GOD and β-T-Gal, while the enzyme molecules themselves might also experience slight inactivation due to prolonged thermal incubation. Therefore, controlling the reaction at 42°C for 20 minutes represents an ideal process window for achieving high-efficiency lactose decomposition while balancing energy consumption.

Although this study yielded encouraging results, certain limitations exist, pointing the way for future research. The current TLC analysis allows only qualitative/semi-quantitative assessment of products. Subsequent studies should include authentic standards of GOS, glucose, and glucuronic acid as internal references for precise quantification of the target prebiotics. Furthermore, this study tested the synergy of β-T-Gal and GOD only at a 1:1 ratio. Optimizing the enzyme ratio (e.g., 2:1, 1:2) could potentially further enhance the overall system efficiency and reduce costs. Simultaneously, refining the temperature gradient (e.g., 35-50°C) would help pinpoint the optimal temperature more accurately. Finally, evaluating the stability and shelf-life of the developed dairy product and ultimately validating its efficacy in alleviating lactose intolerance symptoms through clinical trials are critical steps for translating these laboratory findings into a practical product. 

References

[1]  U.S. Department of Health and Human Services. (2018, February). Definition & facts for lactose intolerance. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)