Part:BBa_K5093001
EC.2.4.1.9
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 801
Illegal BglII site found at 809
Illegal XhoI site found at 2395 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 759
- 1000COMPATIBLE WITH RFC[1000]
EC 2.4.1.9 Gene
Base Pairs: 2400bp
Origin: Lactobacillus johnsonii [1], synthesized
Properties
EC 2.4.1.9 codes for the enzyme inulosucrase, which transfers a fructose group from the disaccharide sucrose to a growing inulin chain, a fiber, to produce glucose and inulin. The reaction is illustrated in Figure 2.
The new essential part, EC 2.4.1.9, originated in Lactobacillus johnsonii [1] and is 2400 bp long, as shown in Figure 1. The biotech company GeneScript artificially synthesized the gene samples we used. EC 2.4.1.9 codes for the enzyme inulosucrase, which transfers a fructose group from sucrose, a disaccharide, to a growing inulin chain, producing glucose and inulin. The reaction is illustrated in Figure 2.
Use and Biology
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% glucose and 50% inulin.
Inulin is a soluble dietary fiber (SDF) and has been approved by the Food and Drug Administration to facilitate the nutritional values of foods. The gut microbiota can efficiently ferment it. 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 [3–5]. Furthermore, dietary fiber could lower blood lipid concentration, reducing the risk of hyperlipidemia [6]. Research has also shown inulin calcium-ion-absorption-facilitating and antioxidant properties [7].
Inulin also has wide applications in pharmacy. Because it cannot be digested or fermented in the initial portion of the human alimentary canal and quickly reaches the distal portion of the colon, inulin can be utilized as a drug carrier, especially for colon diseases. Furthermore, its structure and properties enable it to be a stabilizing agent and cryoprotectant [7].
Most of the attention on inulosucrase is due to the functions of its product, inulin. However, as our project aims to synthesize inulin in the human gut, its effect on its substrate, sucrase, is also vital. It reduces the absorption of fructose by adding it to inulin 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 [8–18], without diminishing the sweetness of food and drink.
Obtaining, Amplifying, and Identifying the Gene
The biotechnology company GeneScript 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 2400 bp. The bands representing EC 2.4.1.9 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 inulosucrase 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 88 kDa. This demonstrates that inulosucrase 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).
[3] Guan Z-W, Yu E-Z, Feng Q. Soluble Dietary Fiber, One of the Most Important Nutrients for the Gut Microbiota. Molecules 2021;26:6802. https://doi.org/10.3390/molecules26226802.
[4] Nie Y, Luo F. Dietary Fiber: An Opportunity for a Global Control of Hyperlipidemia. Oxid Med Cell Longev 2021;2021:5542342. https://doi.org/10.1155/2021/5542342.
[5] Te Morenga LA, Howatson AJ, Jones RM, Mann J. Dietary sugars and cardiometabolic risk: systematic review and meta-analyses of randomized controlled trials of the effects on blood pressure and lipids. The American Journal of Clinical Nutrition 2014;100:65–79. https://doi.org/10.3945/ajcn.113.081521.
[6] Stanhope KL, Schwarz JM, Keim NL, Griffen SC, Bremer AA, Graham JL, et al. Consuming fructose-sweetened beverages, not glucose-sweetened ones, increases visceral adiposity and lipids and decreases insulin sensitivity in overweight/obese humans. J Clin Invest 2009;119:1322–34. https://doi.org/10.1172/JCI37385.
[7] Alexander Bentley R, Ruck DJ, Fouts HN. U.S. obesity as a delayed effect of excess sugar. Econ Hum Biol 2020;36:100818. https://doi.org/10.1016/j.ehb.2019.100818.
[8] Blüher M. Obesity: global epidemiology and pathogenesis. Nat Rev Endocrinol 2019;15:288–98. https://doi.org/10.1038/s41574-019-0176-8.
[9] Yu Z, Ley SH, Sun Q, Hu FB, Malik VS. Cross-sectional association between sugar-sweetened beverage intake and cardiometabolic biomarkers in US women. Br J Nutr 2018;119:570–80. https://doi.org/10.1017/S0007114517003841.
[10] Jebril M, Liu X, Shi Z, Mazidi M, Altaher A, Wang Y. Prevalence of Type 2 Diabetes and Its Association with Added Sugar Intake in Citizens and Refugees Aged 40 or Older in the Gaza Strip, Palestine. International Journal of Environmental Research and Public Health 2020;17:8594. https://doi.org/10.3390/ijerph17228594.
[11] Basu S, Yoffe P, Hills N, Lustig RH. The Relationship of Sugar to Population-Level Diabetes Prevalence: An Econometric Analysis of Repeated Cross-Sectional Data. PLOS ONE 2013;8:e57873. https://doi.org/10.1371/journal.pone.0057873.
[12] Maersk M, Belza A, Stødkilde-Jørgensen H, Ringgaard S, Chabanova E, Thomsen H, et al. Sucrose-sweetened beverages increase fat storage in the liver, muscle, and visceral fat depot: a 6-mo randomized intervention study. The American Journal of Clinical Nutrition 2012;95:283–9. https://doi.org/10.3945/ajcn.111.022533.
[13] Li Y, Hruby A, Bernstein AM, Ley SH, Wang DD, Chiuve SE, et al. Saturated Fats Compared With Unsaturated Fats and Sources of Carbohydrates about Risk of Coronary Heart Disease: A Prospective Cohort Study. J Am Coll Cardiol 2015;66:1538–48. https://doi.org/10.1016/j.jacc.2015.07.055.
[14] Pacheco LS, LaceyJr JV, Martinez ME, Lemus H, Araneta MRG, Sears DD, et al. Sugar‐Sweetened Beverage Intake and Cardiovascular Disease Risk in the California Teachers Study. Journal of the American Heart Association 2020. https://doi.org/10.1161/JAHA.119.014883.
[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.
[16] Adeva-Andany MM, Martínez-Rodríguez J, González-Lucán M, Fernández-Fernández C, Castro-Quintela E. Insulin resistance is a cardiovascular risk factor in humans. Diabetes & Metabolic Syndrome: Clinical Research & Reviews 2019;13:1449–55. https://doi.org/10.1016/j.dsx.2019.02.023.
[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.
[18] Janzi S, Ramne S, González-Padilla E, Johnson L, Sonestedt E. Associations Between Added Sugar Intake and Risk of Four Different Cardiovascular Diseases in a Swedish Population-Based Prospective Cohort Study. Front Nutr 2020;7. https://doi.org/10.3389/fnut.2020.603653.
| None |