Overview

In this study, we contributed 4 new parts to iGEM, encompassing 2 basic parts and 2 composite parts. Leveraging these 4 new parts and 4 previously existing basic parts, we successfully constructed recombinant plasmids for expressing two core functional enzymes—β-T-galactosidase (key for lactose hydrolysis) and glucose oxidase (critical for glucose elimination)—and validated their activity in the Pichia pastoris expression system. These engineered components form the technical foundation of our dual-enzyme system, which addresses the limitation of traditional lactose intolerance solutions.

Building on these parts, we further optimized the application of the dual-enzyme system in dairy product modification, achieving efficient lactose degradation while reducing glucose content, and even promoting the accumulation of galacto-oligosaccharides (GOS)—a prebiotic with potential health benefits. This solution offers a novel, scalable, and consumer-friendly approach to improving dairy product accessibility for lactose-intolerant populations, making a meaningful contribution to food science and public health by expanding dietary options and enhancing nutritional intake for vulnerable groups.

The relevant parts are listed in the table below. All 8 parts included in the table provide a complete and reusable part toolkit for future research on enzyme expression and dairy product engineering.

Table1. All parts in this project

Part Number	Part Name	Part Type	Part Function
BBa_K945000	AOX1 promoter	basic part	Regulatory
BBa_K863203	AOX1 terminator	basic part	Terminator
BBa_K3141000	pPICZαA	basic part	Plasmid_Backbone
BBa_K3584001	His tag-Lac operate-T7 tag	basic part	Tag
BBa_252LAY4D	Beta-T-Galactosidase	basic part	Coding
BBa_258NI5BN	Glucose oxidase	basic part	Coding
BBa_25RILBZU	pPLCZαA-Beta-T-Galactosidase	composite part	Plasmid
BBa_25DDF4OS	pPLCZɑA-Glucose oxidase	composite part	Plasmid

Best Basic Part: Beta-T-Galactosidase

Base Pairs: 2209 bp

Origin: Kibdelosporangium sp. MJ126-NF4

Usage and Biology: The dual catalytic activities of glycosyltransferase-type β-T-galactosidase are defined as follows: (1) Hydrolytic activity: Using water molecules as acceptors, it cleaves the β-1,4-glycosidic bond in lactose, yielding glucose and galactose (lactose + H₂O → glucose + galactose). (2) Glycosyltransferase activity: Using other sugar molecules as substrates, the enzyme first forms a covalent intermediate with lactose, then transfers the galactose moiety to the recipient sugar, synthesizing a new glycosidic bond and yielding galactosyl oligosaccharides (GOS). Glycosyltransferase activity is key to achieving high-value conversion with this enzyme.

The value of glycosyltransferase-type β-T-galactosidase lies in its controllable catalytic directionality. By precisely regulating reaction conditions (e.g., substrate concentration), its catalytic pathway can be guided: at low substrate concentrations, it favors lactose hydrolysis; at high substrate concentrations, its glycosyltransferase activity is significantly enhanced, enabling efficient synthesis of prebiotic oligosaccharides (GOS). 

Contribution Design

For the genetic design of β-T-Gal, we adopted a synthetic biology strategy. Its sequence (2199 bp, encoding 783 amino acids) was newly optimized rather than directly derived from traditional microbial sources of β-T-galactosidase. His tag (BBa_K3584001) was incorporated at the C-terminus to facilitate purification. The sequence was optimized for enhanced expression in the Pichia pastoris expression system. Subsequent thin-layer chromatography experiments confirmed that this enzyme exhibits over 90% lactose degradation efficiency at 40°C, demonstrating its structural and functional innovation.

 Fig 1. Gene map of β-T-Gal

Experiments Approach

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 

Before yeast transformation, the recombinant plasmids were linearized via restriction digestion. Fig. 8 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 

Functional Test

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 2, three parallel experiments were conducted at each concentration to ensure data reliability. 

Table 2. 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 3 and Table 4), and the relevant results are shown in Fig. 10. 

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 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.

Summary

We successfully obtained highly active microbial-derived recombinant glycosyltransferase-type β-T-galactosidase (β-T-Gal) and glucose oxidase (GOD) through a heterologous recombinant expression system. Building upon this foundation, we innovatively applied the synergistic catalysis of these two enzymes to develop a novel dairy processing technology. This process not only efficiently degrades lactose to alleviate lactose intolerance but also synthesizes galacto-oligosaccharides (GOS) through β-T-Gal's glycosyltransferase activity. Simultaneously, glucose is oxidatively removed using GOD, ultimately yielding functional dairy products with triple health benefits: low lactose, low glucose, and high GOS dietary fiber. This represents a technological leap from simple lactose hydrolysis to comprehensive nutritional fortification.