In our project, we developed a capsule containing two catalytic enzymes dextransucrase and inulosucrase that are coded by genes EC.2.4.1.5 and EC.2.4.1.9 to catalyse the conversion from sucrose to two types of soluble dietary fibres (SDFs) dextran and inulin respectively as shown in Figure 1. Therefore, individuals may enjoy their diets without change in flavor while reducing the human body's effective absorption of sugars in food and increasing fibre intake. With no change to the sugar content of the food, this initiative intends to promote bowel movement, regulate gut bacteria, and lower the quantity of sugar absorbed by the human body into the bloodstream.

Figure 1 The overview flowchart of our experiment

We designed 2 plasmids: pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9. The DNA fragments EC.2.4.1.5 and EC.2.4.1.9 are amplified from the genome of Leuconostoc citreum[1]and the genome of Lactobacillus johnsonii[2].

 

1.        Identification and pre-modification of target genes

To construct our plasmids, we let the company synthesise the DNA fragments. To excise two bands of interest (EC.2.4.1.5, EC.2.4.1.9), we used restriction endonuclease NdeⅠ and XhoⅠ to cut the DNA fragments and the pET28a vector.          

Theoretically, the gene fragment lengths for EC.2.4.1.5 and EC.2.4.1.9 are 4431bp and 2391bp, respectively. After PCR amplification, the TAE agarose gel electrophoresis results (Figure 2) proved the theoretical values, and we could continue our experiment. After DNA extraction from the gel, T4 ligase was used to link the vector and the target genes (EC.2.4.1.5 and EC.2.4.1.9) to produce the recombinant plasmid.

Figure 2. The identification of plasmid pET-28a, gene fragments EC.2.4.1.5 and EC.2.4.1.9 by restriction enzymes digestion and PCR amplification.

M: Maker; EC.2.4.1.5: Gene fragment EC.2.4.1.5 that digested by Restriction endonuclease NdeⅠ and XhoⅠ. EC.2.4.1.9: Gene fragment EC.2.4.1.9 that digested by Restriction endonuclease NdeⅠ and XhoⅠ. 

 

2.        Plasmid profile

At the same time, Snapgene was used to create the profiles of the plasmids pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9 so that parts of the plasmids, including the target gene, restriction gene, and multiple cloning site, were demonstrated and understood.

Figure 3. Plasmid profile of pET28a-EC.2.4.1.5(left) and pET28a-EC.2.4.1.9(right).

 

3.        Plasmid transformation

In this part, we transformed the plasmids constructed into DH5α E.coli and replicated them. After extracting and verifying the plasmids, we transformed the plasmids into the bacteria again.

The recombinant plasmid was transformed into E.coli (DH5α), and we incubated them overnight at 37°C after streak inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in Figure 4. Afterward, we picked 3 individual colonies from each petri dish and extracted their plasmids. A gel electrophoresis was run to confirm that the extracted plasmids were the ones we required. As EC.2.4.1.5 and EC.2.4.1.9 have DNA fragment lengths of 4431bp and 2391bp, our results show that we get the desired plasmids. The DNA sequencing diagram shows the desired lengths and sequencing for pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9 (Figure 5) indicates the plasmid construction of pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9 are successful.

Figure 4. Incubate the plasmids containing strains and gel electrophoresis. A: Gel electrophoresis was used to confirm that the extracted plasmids were the ones we required. B: pET28a-EC.2.4.1.5 containing strain. C: pET28a-EC.2.4.1.9 containing strain.

Figure 5. The DNA sequencing diagram for pET28a-EC.2.4.1.9

1.        Expression of protein

The recombinant plasmid was first transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies of each desired plasmid were selected for PCR identification of transformants to confirm that the colonies have positive results (shown in Figure 6) and then transferred into 1L fresh LB (Kana+) culture medium for the scale-up cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.5 and EC.2.4.1.9 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and centrifugation.

 

Figure 6. DNA gel electrophoresis to verify the construction of correct recombinant plasmids from E.coli BL21(DE3). A: DNA gel electrophoresis verification of genes. B: E.coli BL21(DE3) that contain EC.2.4.1.5 and EC.2.4.1.9

 

2.        Protein purification

Nickel affinity chromatography is used to purify the protein sample we got for a clearer SDS-PAGE result with little interference by non-specifically bound proteins. In Figure 7, the bands with sizes of 167 kDa and 88 kDa for dextransucrase and inulosucrase demonstrate that the expected proteins are expressed and collected. The target proteins (dextransucrase and inulosucrase) are successfully expressed and confirmed by the target bands shown in the supernatant and flowthrough part. In the wash part, some target proteins are washed away with other non-specific proteins, and in the elution part, a single and clear band illustrating the target protein with the expected size for both dextransucrase and inulosucrase, which have sizes of 167 kDa and 88 kDa.

 

Figure 7 Results of SDS-PAGE of supernatant, flowthrough, wash, and elution parts for the protein purification of dextransucrase and inulosucrase extracted from EC.2.4.1.5 and EC.2.4.1.9 in E.coli BL21(DE3)

Both enzymes convert sucrose to 50% monosaccharides and 50% SDFs, while a lower proportion of monosaccharides could be achieved by combining the two enzymes in an ideal ratio. Thin-layer chromatography and high-performance liquid chromatography (HPLC) were carried out to find the optimum ratio and conditions.

 

1.        Thin-layer chromatography

In a standard 2% sucrose solution, a certain amount of two recombinant enzyme solutions with the ratio of 1:1, 1:2, and 2:1 between concentrations of dextransucrase and inulosucrase was added, and the reaction was shaken at 37°C for 1h. After the reaction, thin-layer chromatography detected the sucrose changes in the reaction solution and the glucose content generated qualitatively. Figure 8 shows that the group with the least sucrose concentration undigested has a ratio of 2:1 between dextransucrase and inulosucrase.

 

Figure 8. Thin-layer chromatography is used to identify the efficiency of different recombinant enzyme solutions. 1: The recombinant enzyme solution with a ratio of 1:1 between dextransucrase and inulosucrase. 2: The recombinant enzyme solution with a ratio of 1:2 between dextransucrase and inulosucrase. 3: The recombinant enzyme solution with a ratio of 2:1 between dextransucrase and inulosucrase.

 

2.        High-performance liquid chromatography

In addition, sugarcane juice was used as a type of simulated food suspension to test enzyme function in real conditions. The experiment took place under 37℃ to simulate human body temperature. A 1:1 ratio of concentration between dextransucrase and inulosucrase is used with masses of 1mg, 5mg, 10mg, 15mg, and 20 mg of each type of enzyme per 100 g sugarcane juice for 5 minutes, 10 mins, 15 mins, 30 mins, and 60 mins. Once the reaction time is reached, the solution is immediately heated to 65℃ to denature the enzymes. Afterward, HPLC is carried out to analyse the amount of sucrose and glucose contained in the solution, the raw data are listed below (Table 1). We used Origin Pro to process data, forming 3D mappings about glucose and sucrose concentrations in various conditions (Figure 8). By analysing the raw data and the 3D mapping, we could conclude that in general, with a longer reaction time, catalysing tends to become more complete, which means higher glucose concentration while lower sucrose concentration. In speaking of each enzyme's sample size, except for the extremely high remaining sucrose with 1mg of dextransucrase and inulosucrase, it is difficult to summarise the overall effects of the change in glucose and sucrose concentrations. A low sucrose concentration (0g/100g) was left in the solution when 20 mg of each enzyme was left in the solution and reacted for 60 mins, but a higher glucose concentration was formed (2.9g/100g).

Table 1 The amount of sucrose and glucose contained in 100 g sugarcane solution with the existence of dextransucrase and inulosucrase at 37℃.

 

However, when the sample size is 10 mg for each enzyme, noticeably low remaining sucrose (0.088g/100g) and relatively low glucose production (2.6g/100g) are observed when the reaction time is 15 mins. In conclusion, we determined that allowing 10 mg of each enzyme to react in sugarcane juice for 15 mins is ideal to guarantee little sucrose sustain and glucose synthesis at 37°C.

Figure 9 3D mapping of glucose(right) and sucrose(left) concentrations in various conditions under 37°C.

Subsequently, we may start the construction of the capsule to protect two protein-based enzymes from denaturation by the low pH of gastric juice by adopting the microencapsulation technique, in which enzymes are coated in small spheres. The capsule made of calcium chloride and sodium alginate provides high strength, elasticity, and thermal stability to resist pepsin and hydrochloric acid in the stomach [3]. Moreover, the effect of recombinant enzymes in the actual environment of the gastrointestinal tract should also be tested in the future with distinct approaches, including the in vitro method, animal experiments, and human clinical trials.

1.    ‘dextransucrase [Leuconostoc citreum] - Protein - NCBI’. Accessed: Jul. 26, 2024. [Online].
2.    ‘glycoside hydrolase family 68 protein [Lactobacillus johnsonii] - Protein - NCBI’. Accessed: Jul. 26, 2024. [Online]
3.    YANG B. et al., ‘Preparation and properties of lipase microcapsules,’ China Dairy Industry, vol. 51, no. 9, pp. 10-14+64, 2023.