Difference between revisions of "Part:BBa K4279001"
 
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W1-lipase
 
W1-lipase
 
== Profile ==
 
== Profile ==
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Name: SP-lipase
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Name: W1-lipase
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Base Pairs: 1854 bp
 
Base Pairs: 1854 bp
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Origin: Lactiplantibacillus Plantarum, genome
 
Origin: Lactiplantibacillus Plantarum, genome
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Properties: a lipase for triacylglyceride digestion.  
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Properties: a lipase for triacylglyceride digestion.
 
== Usage and Biology ==
 
== Usage and Biology ==
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BBa_K4279000 is the coding sequence of SP-lipase. Lipase is a primary lipase critical for triacylglyceride digestion in humans and is considered a promising target for the treatment of obesity [1]. Triacylglycerol lipase is the primary lipase secreted by the pancreas, and is responsible for breaking down dietary lipids into unesterified fatty acids (FAs) and monoglycerides (MGs). Medically, lipases are targets for therapeutic intervention in the treatment of obesity. The focus of applied research with lipases has been to exploit the unusual properties of lipolytic systems for the production of chiral pharmaceuticals, improved detergents, and designer fats [2]. Obesity is a medical condition in which excess body fat accumulates to the extent that it may have a negative effect on health, leading to reduced life expectancy and/or increased health problems. Diverse approaches to the prevention and treatment of obesity have been reported [3-5]. W1-lipase (EC 3.1.1.3) is a lipase amplified from Pseudomonas sp. 7323. The SP-lipase is made up of 265 aa [6].
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BBa_K4279001 is the coding sequence of W1-lipase. Lipase is a primary lipase critical for triacylglyceride digestion in humans and is considered a promising target for the treatment of obesity [1]. Triacylglycerol lipase is the primary lipase secreted by the pancreas, and is responsible for breaking down dietary lipids into unesterified fatty acids (FAs) and monoglycerides (MGs). Medically, lipases are targets for therapeutic intervention in the treatment of obesity. The focus of applied research with lipases has been to exploit the unusual properties of lipolytic systems for the production of chiral pharmaceuticals, improved detergents, and designer fats [2]. Obesity is a medical condition in which excess body fat accumulates to the extent that it may have a negative effect on health, leading to reduced life expectancy and/or increased health problems. Diverse approaches to the prevention and treatment of obesity have been reported [3-5]. W1-lipase is a lipase amplified from Lactiplantibacillus Plantarum (LP1406), which is a gram-positive lactic acid bacteria species and exhibits ecological and metabolic adaptability and is capable of inhabiting a range of ecological niches including fermented foods, meats, plants, and the mammalian gastrointestinal tract. The W1-lipase is made up of 265 aa [6].
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[[File:BBa K4279001-figure1.png|500px|thumb|center|Figure 1. The structural modeling of an extremophilic bacterial lipase isolated from saline habitats..]]
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[[File:BBa K4279001-figure1.png|500px|thumb|center|Figure 1. The structural modeling of an extremophilic bacterial lipase isolated from saline habitats.]]
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== Construct design ==
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1. Construction of the lipase expression plasmids
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==Reference==
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The SP-lipase gene was amplified from the pseudomonas and then inserted in the XhoI and HindIII sites of pET28a (Figure 2).
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[[File:BBa K4279001- figure 2.png|500px|thumb|center|Figure 2.  W1-lipase and SP-ligase expression plasmids in this projec.]]
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[1] Paul Joyce, Catherine P. Whitby, Clive A. Prestidge, Nanostructuring Biomaterials with Specific Activities towards Digestive Enzymes for Controlled Gastrointestinal Absorption of Lipophilic Bioactive Molecules, Advances in Colloid and Interface Science,2016, 237; 52-75.
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In order to build our plasmids, plasmid pET28a was digested with XhoI and HindIII (Figure 3), and we used T4 DNA ligase to ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α competent cells and coated on the LB (Kanamycin) solid plates.
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[[File:BBa K4279001-figure3.png|500px|thumb|center|Figure 3. Gel electrophoresis of double-enzyme digested pET28a-SP-lipase.]]
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[2] Khan I, Nagarjuna R, Dutta JR, Ganesan R Enzyme-Embedded Degradation of Poly(ε-caprolactone) using Lipase-Derived from Probiotic Lactobacillus plantarum. ACS Omega. 2019, 4(2):2844-2852
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The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed (Figure 4).
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[[File:BBa K4279001- figure 4.jpg|500px|thumb|center|Figure 4. Mapped the sequencing data of the pET28a-SP-lipase.]]
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2. Protein lipase expression
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The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and cultured overnight in the medium containing resistance. When the OD600 was around 0.4-0.5, the IPTG was added to induce the expression of recombinant protein W1-lipase/SP-lipase, and then the strains were cultured at 16℃ for 20h. After that, the collected bacterial solution was cracked by Ultrasonic crushing. SDS-PAGE was used to analyze the recombinant proteins. Figure 5 showed the electrophoretic results of the protein gel.
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[3] H.L. Brockman, lipase.Encyclopedia of Biological Chemistry (Second Edition), 2013.
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[[File:BBa K4279001- figure 5.jpg|500px|thumb|center|Figure 5. SDS-PAGE detection of lipase protein.]]
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6.Lipase activity detection at different pH and temperature
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a)Standard curve measurement
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[4]. Birari RB, Bhutani KK. Pancreatic lipase inhibitors from natural sources: Unexplored potential. Drug Discov Today. 2007;12:879–889
  
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In order to measure the standard curve of the activity of lipases, we chose p-nitrophenol as the substrate and detected its absorbance value of it when adding lipases. 0.02789g of p-nitrophenol (p-np) was weighed and dissolved in 100mL of solution B, and stored in a brown reagent bottle after configuration and stored at 4°C. 0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol solution (2mmol/L) was diluted to 4mL, and the absorbance value at 410nm was measured successively. The standard curve was drawn with p-nitrophenol (0.01, 0.02, 0.03, 0.04, 0.06, 0.08, mmol/L) as the abscissa and absorbance value Y as the ordinate (Figure 6).
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[5]. Kim S, Lim SD. Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180. Food Sci Anim Resour. 2020, 40(1):87-95
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[[File:BBa K4279001-figure 6.png|500px|thumb|center|Figure 6. The standard curve of p-nitrophenol.]]
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According to the standard curve determination method, the standard curve is drawn as shown in Figure 6. Regression coefficient R2=0.9979, the results are credible.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4279001 SequenceAndFeatures</partinfo>
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[6] S.P.S. Chundawat, V. Balan, L. Da costa Sousa, B.E. Dale, Thermochemical pretreatment of lignocellulosic biomass. Bioalcohol Production. 2010, 24-72.
  
 
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Latest revision as of 09:46, 26 September 2022


W1-lipase

W1-lipase

Profile

Name: W1-lipase

Base Pairs: 1854 bp

Origin: Lactiplantibacillus Plantarum, genome

Properties: a lipase for triacylglyceride digestion.

Usage and Biology

BBa_K4279001 is the coding sequence of W1-lipase. Lipase is a primary lipase critical for triacylglyceride digestion in humans and is considered a promising target for the treatment of obesity [1]. Triacylglycerol lipase is the primary lipase secreted by the pancreas, and is responsible for breaking down dietary lipids into unesterified fatty acids (FAs) and monoglycerides (MGs). Medically, lipases are targets for therapeutic intervention in the treatment of obesity. The focus of applied research with lipases has been to exploit the unusual properties of lipolytic systems for the production of chiral pharmaceuticals, improved detergents, and designer fats [2]. Obesity is a medical condition in which excess body fat accumulates to the extent that it may have a negative effect on health, leading to reduced life expectancy and/or increased health problems. Diverse approaches to the prevention and treatment of obesity have been reported [3-5]. W1-lipase is a lipase amplified from Lactiplantibacillus Plantarum (LP1406), which is a gram-positive lactic acid bacteria species and exhibits ecological and metabolic adaptability and is capable of inhabiting a range of ecological niches including fermented foods, meats, plants, and the mammalian gastrointestinal tract. The W1-lipase is made up of 265 aa [6].

Figure 1. The structural modeling of an extremophilic bacterial lipase isolated from saline habitats.

Reference

[1] Paul Joyce, Catherine P. Whitby, Clive A. Prestidge, Nanostructuring Biomaterials with Specific Activities towards Digestive Enzymes for Controlled Gastrointestinal Absorption of Lipophilic Bioactive Molecules, Advances in Colloid and Interface Science,2016, 237; 52-75.

[2] Khan I, Nagarjuna R, Dutta JR, Ganesan R Enzyme-Embedded Degradation of Poly(ε-caprolactone) using Lipase-Derived from Probiotic Lactobacillus plantarum. ACS Omega. 2019, 4(2):2844-2852

[3] H.L. Brockman, lipase.Encyclopedia of Biological Chemistry (Second Edition), 2013.

[4]. Birari RB, Bhutani KK. Pancreatic lipase inhibitors from natural sources: Unexplored potential. Drug Discov Today. 2007;12:879–889

[5]. Kim S, Lim SD. Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180. Food Sci Anim Resour. 2020, 40(1):87-95

[6] S.P.S. Chundawat, V. Balan, L. Da costa Sousa, B.E. Dale, Thermochemical pretreatment of lignocellulosic biomass. Bioalcohol Production. 2010, 24-72.