Part:BBa_K5093000
EC.2.4.1.5
short description
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 883
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4004
Illegal BamHI site found at 1496
Illegal XhoI site found at 4435 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
EC 2.4.1.5 Gene
Base Pairs: 4440bp
Origin: Leuconostoc citreum [1], synthesized
Properties
EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.
This new essential part, EC 2.4.1.5, originated in Leuconostoc citreum [1] and is 4440 bp long, as shown in Figure 1. The biotech company Gene Script artificially synthesized the gene samples we used. EC 2.4.1.5 codes for the enzyme dextransucrase, which transfers a D-glucosyl group from sucrose, a disaccharide, to a growing dextran chain, a fiber, by an alpha (1→6) linkage. The reaction is illustrated in Figure 2.
Use of Dextransucrase
Sucrose is usually broken down into two monosaccharides, glucose and fructose, which are absorbed and metabolized. However, the protein that the gene codes for converts this disaccharide into 50% fructose and 50% dextran. This is the primary approach to dextran synthesis.
Dextran is a versatile substance. As a soluble dietary fiber, it can be efficiently fermented by the gut microbiota. High diversity of the gut microbiota has been proven to reduce the incidence of many chronic diseases, including inflammatory bowel disease, colorectal cancer, obesity, and T2DM [3]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [4].
Its medical uses include its antithrombotic and anticoagulant activities and its role in maintaining blood osmolarity to replace expensive plasma proteins. Its excellent biological compatibility and degradability have made it a promising drug administration vector. Dextran is also a food additive with great stabilizing, thickening, and emulsifying abilities. In cosmetics, dextran and its derivatives also function as lubricants, humectants, and thickeners in skin and hair care products. Dextran could also facilitate imaging technologies and the manufacture of paper.
The majority of attention on dextransucrase is associated with the functions of dextran. However, as our project aims to synthesize dextran in the human gut, its effect on its substrate, sucrase, is also vital. It reduces the absorption of glucose by adding them to dextran chains, and hence the chance of getting obese or contracting non-communicable diseases such as hyperglycemia, dyslipidemia, diabetes, coronary heart disease, non-alcoholic fatty liver disease, and stroke [5–18], without diminishing the sweetness of food and drink.
Obtaining, Amplifying, and Identifying the Gene
The biotechnology company Gene Script synthesizes the DNA molecules containing our desired gene. Then, they are cut with restriction endonucleases NdeI and XhoI and amplified using polymerase chain reaction (PCR). The gene's length is 4440 bp. The bands representing EC 2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in Figure 3, indicating that the cutting and amplifying are successful.
Protein Purification and SDS-PAGE
Nickel affinity chromatography effectively purifies dextransucrase because the protein contains a 6×his tag. We got a more precise result with little interference by non-specifically bound proteins. Figure 4 shows only one band with a molecular weight of 167 kDa. This demonstrates that dextransucrase is successfully expressed and purified.
References
[1] NCBI. Dextransucrase 2024. https://www.ncbi.nlm.nih.gov/protein/BAF96719.1 (accessed July 25, 2024).
[2] BRENDA. BRENDA:EC2.4.1.5 2023. https://www.brenda-enzymes.org/enzyme.php?ecno=2.4.1.5 (accessed June 6, 2024).
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[15] Saadatagah S, Pasha AK, Alhalabi L, Sandhyavenu H, Farwati M, Smith CY, et al. Coronary Heart Disease Risk Associated with Primary Isolated Hypertriglyceridemia; a Population-Based Study. J Am Heart Assoc 2021;10:e019343. https://doi.org/10.1161/JAHA.120.019343.
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[17] Park WY, Yiannakou I, Petersen JM, Hoffmann U, Ma J, Long MT. Sugar-Sweetened Beverage, Diet Soda, and Nonalcoholic Fatty Liver Disease Over 6 Years: The Framingham Heart Study. Clinical Gastroenterology and Hepatology 2022;20:2524-2532.e2. https://doi.org/10.1016/j.cgh.2021.11.001.
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