Overview

Lactose intolerance is a prevalent condition characterized by the inability to digest or absorb dairy products, with symptoms including diarrhea, nausea, and abdominal pain (Mayo Clinic). This digestive impairment stems from the body’s failure to break down lactose, a disaccharide found in dairy. Lactase, the enzyme responsible for hydrolyzing lactose into galactose and glucose, is deficient in individuals with lactose intolerance, leading to the onset of symptoms. Current commercial solutions primarily involve incorporating galactosidases into dairy products. Specifically, β-T-galactosidase—an enzyme that catalyzes the breakdown of lactose into galactose and glucose—has been used to mitigate lactose intolerance. However, this approach faces significant limitations in widespread adoption: the glucose generated by β-T-galactosidase activity makes these modified dairy products unsuitable for consumers with strict glucose intake restrictions, such as individuals with diabetes. To address this gap, we developed a synergistic dual-enzyme system comprising β-T-galactosidase and glucose oxidase. The former hydrolyzes lactose, while the latter oxidizes the resulting glucose to eliminate its presence. To implement this system, we utilized the β-T-Gal gene and the GOD gene for the synthesis of β-T-galactosidase and glucose oxidase, respectively. The success of our engineering efforts was validated through Design-Build-Test-Learn (DBTL) cycles, encompassing plasmid engineering, protein expression and purification, and dairy product (milk) optimization.

Part 1 pPICZαA-β-T-Gal

Objective:

Engineer the yeast Pichia pastoris to express and purify recombinant β-T-galactosidase (β-T-Gal), and validate the lactose-hydrolyzing activity of the purified enzyme.

Design:

Construction of the pPICZαA-β-T-Gal Plasmid

The pPICZαA-β-T-Gal plasmid consists of two key components: the pPICZαA vector and the β-T-Gal gene. The pPICZαA vector is a commonly used vector for yeast expression. The β-T-Gal gene encodes β-T-galactosidase, which facilitates the hydrolysis of lactose into galactose and glucose. Homologous recombination was employed to construct the pPICZαA-β-T-Gal plasmid.

The plasmid consists of the β-T-Gal gene, AOX1 promoter and terminator, and two endonuclease binding sites for EcoRI and SalI. The AOX1 promoter is activated by the inducer methanol, resulting in transcriptional induction. The two endonuclease binding sites for ECORI and SalI enable homologous recombination of β-T-Gal and the vector. Another part is the α-factor secretion signal, which helps mature and process the synthesized protein.

Fig 1. Plasmid map of pPICZαA-β-T-Gal

Build:

The recombinant expression plasmid pPICZαA-β-T-Galactosidase was successfully constructed for heterologous expression in Pichia pastoris. The gene encoding the synthetic β-T-Galactosidase was amplified via PCR, generating a distinct fragment of approximately 2209 bp. As shown in Fig. 2, agarose gel electrophoresis analysis of the PCR product revealed a single, clear band that aligned with the expected molecular size, confirming the specificity and success of the amplification. The correct fragment was subsequently ligated into the pPICZαA vector for downstream applications.

Fig 2. Electrophoresis result of β-T-Gal

Following transformation into E. coli DH5α, positive clones were selected from the zeocin-resistant colonies (Fig. 3A) and subjected to colony PCR for initial verification. The analysis revealed a specific amplification product of approximately 2209 bp, which corresponds to the expected size of the β-T-Gal gene (Fig. 3B). Subsequent DNA sequencing of the plasmid from a positive clone confirmed the precise insertion of the target sequence into the pPICZαA vector without mutations, thereby validating the successful construction of the pPICZαA-β-T-Gal plasmid (Fig. 3C).

Fig 3. pPICZαA-β-T-Gal Recombinant Plasmid Identification Map

Test:

Before yeast transformation, the recombinant plasmids were linearized via restriction digestion. Fig. 4 presents the electrophoresis result of the linearized DNA, with bands matching the expected size (~5693 bp for pPICZαA-β-T-Gal).

Fig 4. Electrophoresis Result of Linearized pPICZαA-β-T-Gal

The linearized plasmid was transformed into Pichia pastoris cells pre-cultured in YPD medium. Positive transformants were selected on YPD plates containing zeocin, as shown in Fig. 5A, indicating successful integration of the expression cassette into the yeast genome. To further verify this, genomic DNA was extracted from the transformants and analyzed by PCR. As demonstrated in Fig. 5B, a DNA fragment of approximately 2209 bp, consistent with the expected size of the β-T-Gal gene, was amplified, confirming the stable incorporation of the target expression cassette into the yeast chromosome.

Fig 5. Validation of yeast transformation results

The expression and purification of β-T-Galactosidase were assessed by SDS-PAGE. As depicted in Fig. 6, the purified protein preparation exhibited a single, distinct band migrating at a position consistent with the theoretical molecular weight of ~80 kDa when compared to the protein ladder, confirming the integrity and homogeneity of the recombinant product.

Fig 6. β-T-Gal Protein Expression Map

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. 7, 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 7. Results of β-T-Gal Hydrolysis Activity Test

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 8 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 8. 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.

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. 9). This confirms that the synergistic effect of β-T-Gal and GOD is feasible in fresh milk.

Fig 9. TLC Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity

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 2 and Table 3), and the relevant results are shown in Fig. 10.

Table 2. 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 3. 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 10. 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.

Learn:

Refining the expression of β-T-Gal in Pichia pastoris revealed that procedural rigor, specifically temperature control and aseptic technique, is fundamental to experimental success. Building on this foundation, the expression construct itself will be optimized through two targeted strategies: codon optimization to align with host tRNA pools, and rational engineering of the α-factor secretion signal to enhance the efficiency of protein translocation and secretion, with the ultimate goal of maximizing functional enzyme yield.

Part 2 pPICZαA-GOD

Objective:

Engineer Pichia pastoris to express and purify glucose oxidase (GOD), and validate its glucose-reducing activity.

Design:

Construction of the pPICZαA-GOD Plasmid

To achieve efficient expression of glucose oxidase in Pichia pastoris, this study constructed the recombinant expression plasmid pPICZαA-GOD based on the established pPICZαA vector system. This plasmid adopts a modular design philosophy, continuing the construction strategy consistent with our group's previous work: the vector backbone is a high-efficiency expression vector for Pichia pastoris, and the target gene GOD encodes glucose oxidase, which catalyzes the oxidation of glucose to produce hydrogen peroxide and D-gluconic acid-δ-lactone. This reaction is the key step in glucose clearance within the dual-enzyme system.

The core functional elements of the plasmid were systematically engineered to include: - A methanol-inducible expression unit comprising the AOX1 promoter and terminator - EcoRⅠ and SalⅠ restriction sites for homologous recombination - An α-factor signal peptide directing protein secretion - A 6×His tag for convenient purification. The synergistic interaction of these elements ensures efficient secretory expression and convenient purification of the recombinant GOD protein in Pichia pastoris GS115.

The ultimate goal of this design is to achieve co-expression of GOD and β-T-Gal via this plasmid, establishing a dual-enzyme system capable of continuously catalyzing lactose degradation and glucose clearance. This provides critical technological support for developing low-lactose dairy products.

Fig 11. Plasmid map of pPICZαA-GOD

Build:

The recombinant expression plasmid pPICZαA-Glucose Oxidase (pPICZαA-GOD) was constructed using homologous recombination. The gene encoding glucose oxidase (GOD) was amplified by polymerase chain reaction (PCR), resulting in a DNA fragment of approximately 1284 bp, which corresponds to the expected length of the full-length coding sequence. As shown in Fig. 12, agarose gel electrophoresis analysis of the PCR product displayed a single, clear band of the correct size, confirming the successful and specific amplification of the target gene prior to its insertion into the linearized pPICZαA vector.

Fig 12. Electrophoresis result of GOD

The recombinant plasmid was transformed into E. coli DH5α competent cells, and transformants were selected on zeocin-containing plates, as shown by the single colonies in Fig. 13A. Multiple colonies were initially screened by colony PCR to identify those carrying an insert of the expected size. A positive clone identified by PCR was subsequently subjected to DNA sequencing. The sequencing results confirmed that the glucose oxidase (GOD) gene was accurately inserted into the pPICZαA vector without mutations, thereby verifying the successful construction of the pPICZαA-Glucose Oxidase plasmid.

Fig 13. pPICZαA-GOD Recombinant Plasmid Identification Map

Test:

Before transforming into yeast, the recombinant plasmids were linearized by restriction digestion. Fig 14 shows the electrophoresis result of the linearized DNA, with bands of the expected size (~4778 bp for pPICZαA-GOD).

Fig 14. Electrophoresis result of pPICZαA-GOD after DNA linearization

The linearized pPICZαA-GOD plasmid was transformed into Pichia pastoris host cells pre-cultured in YPD medium. Successful transformants were selected on YPD plates supplemented with zeocin, with colony growth evident in Fig. 15A, indicating positive integration of the expression cassette into the yeast genome. To molecularly confirm the integration, genomic DNA was extracted from selected transformants and analyzed by PCR using gene-specific primers. As shown in Fig. 15B, an amplification product of approximately 1294 bp, corresponding to the expected size of the GOD gene fragment, was detected, verifying the precise and stable incorporation of the GOD expression cassette into the yeast chromosome.

Fig 15. Validation of yeast transformation results

The transformed yeast strains were cultured under inducing conditions to express the recombinant GOD protein. Subsequently, the secreted protein was purified from the culture supernatant using C-terminal His-tag technology. Analysis of the purification process by SDS-PAGE revealed a predominant band at approximately 48 kDa, which aligns with the expected molecular weight of the glycosylated GOD protein. The electrophoretic profile demonstrates successful purification, with the target protein band being the major component in the elution fraction, indicating a high purity level suitable for subsequent functional characterization.

Fig 16. Result of the GOD Protein purification

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 was measured for reaction absorbance using a Multiskan™ FC microplate reader.

Table 4. 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 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 17. Results of the GOD Hydrolysis Activity Test

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. This confirms that the synergistic effect of β-T-Gal and GOD is feasible in fresh milk.

Fig 18. TLC Results of Dual-Enzyme (β-T-Gal + GOD) Synergistic Activity

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, and the relevant results are shown in Fig. 19.

Table 5. 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 6. 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 19. 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.

Learn:

In the construction and expression practice of the pPICZαA-GOD plasmid, we have summarized the following key insights: Adopting transformation and purification protocols consistent with the pPICZαA-β-T-Gal system significantly enhances the standardization of experimental procedures and the reproducibility of results. Using the identical pPICZαA vector backbone to express different enzyme proteins provides systematic comparability for evaluating the expression levels and secretion efficiency of the two recombinant proteins. Future optimization will focus on analyzing expression differences between β-T-Gal and GOD under identical induction conditions to balance the yield ratios of components within the dual-enzyme system. Furthermore, systematic optimization of key parameters such as methanol concentration and induction duration will be pursued to further enhance the enzymatic yield of recombinant GOD.

Next Steps:

Although this study yielded encouraging results, several limitations exist that point to directions for future research. Currently, TLC analysis can only provide qualitative and semi-quantitative analysis of the products. Subsequent studies could achieve precise quantification of the target prebiotics by spotting GOS, glucose, and glucuronic acid standards as internal references. Furthermore, this study tested only the synergistic effect of a 1:1 mixture of β-T-Gal and GOD. Optimizing enzyme ratios (e.g., 2:1, 1:2) may further enhance overall system efficiency and reduce costs. Simultaneously, refining the temperature gradient (e.g., 35–50°C) would aid in more precisely identifying the optimal temperature point. Finally, assessing the stability and shelf life of the developed dairy product, followed by clinical trials to validate its efficacy in alleviating lactose intolerance symptoms, represents a critical step in translating laboratory findings into practical applications.