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Lipase

A technology of lipase and transformant, which is applied in the field of lipase to prepare the lipase, can solve the problems of high production cost, low lipase production efficiency, lipase production technology needs to be improved, and achieve high enzymatic activity and high production efficiency effect

Active Publication Date: 2011-07-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current production efficiency of lipase is still low and the production cost is high
Therefore, the existing lipase production technology still needs to be improved

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1: Construction of expression vector pEASY-BTL2-M@BL21(DE3) and acquisition of Escherichia coli transformants

[0169] The BTL2-M gene is fully artificially synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd. according to the nucleotide sequence of SEQ ID NO:2.

[0170] Afterwards, using the upstream primer BTL2-M_F: 5'-GCGGCATCCCCGCGCGCCA-3' (SEQ ID NO: 4) and the downstream primer BTL2-M_R: 5'-TTACGGGCGCAAACTCGCCA-3' (SEQ ID NO: 5), the synthetic BTL2 -M performs chain polymerization to amplify the BTL2-M gene. After agarose nucleic acid electrophoresis separation and purification of the PCR amplification product, TA cloning was performed with the pEASY-E2 expression vector of Quanshijin Company. Ligate 1 microliter of pEASY-E2 (carrying penicillin resistance marker) and 3 microliters of PCR product at room temperature for 15 minutes, then transfer to 50 microliters of Escherichia coli competent cells BL21(DE3), and place in ice bath for 20 min...

Embodiment 2

[0171] Example 2: Construction of pEASY-BTL2-O@BL21(DE3) recombinant vector and acquisition of Escherichia coli transformants

[0172] The BTL2-O gene is fully artificially synthesized by Shanghai Xuguan Biotechnology Development Co., Ltd. Using the upstream primer BTL2-O_F: 5'-GCGGCATCCCCACGCGCCA-3' (SEQ ID NO: 6) and the downstream primer BTL2-OR: 5'-TTAAGGCCGCAAACTCGCC-3' (SEQ ID NO: 7) to synthesize BTL2-O The gene was subjected to chain polymerization to amplify the BTL2-O gene. After separation and purification of PCR amplification products by agarose nucleic acid electrophoresis, TA cloning was carried out with the pEASY-EV2 expression vector (carrying penicillin resistance marker) of Quanshijin Company. Ligate 1 microliter of pEASY-EV2 and 3 microliters of PCR products at room temperature for 15 minutes, then transfer to 50 microliters of Escherichia coli competent cells BL21(DE3), let it stand in an ice bath for 20 minutes, then quickly transfer to a water bath at 42...

Embodiment 3

[0173] Example 3: expression of lipase in pEASY-BTL2-M@BL21(DE3) shake flask

[0174]Inoculate pEASY-BTL2-M@BL21(DE3) into a test tube containing 5 mL of LB medium, and culture overnight in a shaker at 37 degrees Celsius and 200 rpm. Then, transfer 1 ml of the culture solution to a 250 ml shake flask containing 100 ml of LB medium, and culture it in a shaker at 200 rpm at 37 degrees Celsius for about 2 hours until the OD 600nm =0.6, IPTG was added to a final concentration of 0.4 mM to induce BTL2-M gene expression. Six hours after induction, the samples were ultrasonically crushed, and the lipase activity was measured. The enzyme activity of lipase to hydrolyze butyric acid triglyceride was 300 U / mL.

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Abstract

The invention discloses a lipase, which is formed by an amino acid sequence shown as the SEQ ID No.1. The invention further discloses a polynucleotide encoding the lipase, and an expression vector and a transformant containing the polynucleotide, as well as a method for preparing the lipase. The lipase according to the embodiment of the invention has activity which is significantly greater than that of the existing lipase, and the preparation method of the lipase according to the embodiment of the invention has higher yield.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to lipase, a polynucleotide encoding the lipase, an expression vector containing the polynucleotide, a transformant, and a method for preparing the lipase. Background technique [0002] Lipase refers to the lipase that hydrolyzes the ester bonds in carboxylic acid triglycerides. It mainly catalyzes triglycerides into diglycerides, monoglycerides, fatty acids and glycerol. It has been more than 100 years since the discovery of rabbit pancreatic lipase in 1834. Lipase has the biological function of catalyzing hydrophobic oils into water-soluble fatty acids and glycerol, maintains high catalytic activity and strong stability in inorganic solvents, and has broad-spectrum / special specificity for substrates , endowing lipase with great application value in industries such as food oil processing industry, detergent industry, synthesis of ester bond compounds and synthesis of chiral dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N15/09
Inventor 欧先金刘德华杜伟
Owner TSINGHUA UNIV
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