New application of GDSL protein to preparation of lipase

A technology of lipase and protein, which is applied in the field of lipase preparation, can solve the problems that cannot meet the requirements of lipase, and achieve the effects of good pH stability, great economic value, and high enzyme activity

Inactive Publication Date: 2013-01-16
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The thermal stability of lipase has always been a bottleneck in the research, production and application of lipase. Judging from the current status of lipase research, only improving the properties of lipase through protein engineering is far from meeting the requirements for lipase in various fields.

Method used

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  • New application of GDSL protein to preparation of lipase
  • New application of GDSL protein to preparation of lipase
  • New application of GDSL protein to preparation of lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the acquisition of wild bacteria and the discovery of lipase

[0041] 1. Screening of lipase-producing strains

[0042] Primary screening medium (g / L): (NH 4 ) 2 SO 4 1g, NH 4 NO 3 1g, NaCl 1g, MgSO 4 ·7H 2 O 0.1g, K 2 HPO 3 1g, FeSO 4 ·7H 2 O 0.01g, Na 2 CO 3 Adjust the pH to 8.0, add 1.0% olive oil, and sterilize.

[0043] Liquid fermentation medium (g / L): soluble starch 10g, (NH 4 ) 2 SO 4 5g,K 2 HPO 3 1g, corn syrup powder 20g, soybean cake powder 20g, triolein 10g, pH 8.0.

[0044] Preliminary screening: The pond sediment samples were taken from the polyculture fish pond of Zhejiang Jiaxing Xinxin Feed Co., Ltd. The sampling time was July 2009, and the samples were stored at 4°C. Dilute the pond bottom sludge sample tenfold with sterile PBS buffer, take 100 microliters and spread it on the primary screening medium, and place it at 60°C for cultivation. The lipase-producing strain can degrade olive oil and produce a transparent cir...

Embodiment 2

[0055] The preparation of embodiment 2, GDSL protein

[0056] 1. Construction of recombinant expression vector

[0057] 1. Synthesize the double-stranded DNA molecule shown in sequence 3 of the sequence listing.

[0058] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of B2F and B2R to obtain a PCR amplification product.

[0059] B2F:5'-GAC GAATTC ATGAGACGCGGTATTGTAAGCAC-3' (the underline marks the restriction endonuclease EcoRI restriction endonuclease recognition sequence);

[0060] B2R:5'-GAC GCGGCCGC TTGTTTGTCCTCCTCCGTCCAC-3' (the restriction endonuclease NotI recognition sequence is underlined).

[0061] PCR reaction conditions: pre-denaturation at 95°C for 5min; 30 cycles of 94°C for 30sec, 60°C for 30sec, and 72°C for 1min; finally, extension at 72°C for 10min.

[0062] 3. Digest the PCR amplified product in step 2 with restriction endonucleases EcoRI and NotI, and recover the di...

Embodiment 3

[0090] Embodiment 3, GDSL protein is identified as the property of lipase

[0091] Various experiments in Example 3 were carried out using the GDSL protein solution prepared in Step 4 of Example 2.

[0092] 1. Determination of optimum pH and pH stability

[0093] 1. Optimal pH

[0094] Detect the optimum pH of the GDSL protein solution. For the method, refer to step 3 of Example 2, 5. The only difference is that different buffers are used as solution B, and the following buffers are used respectively: 50mmol / L Gly-HCl of pH 2.0-3.6 Buffer, 50mmol / L HAc-NaAc buffer at pH 3.6-5.0, 50mmol / L citric acid-Na at pH 5.0-8.0 2 HPO 4 Buffer, 50mmol / L Tris-HCl buffer at pH 8.0-9.0 and 50mmol / L Gly-NaOH buffer at pH 9.0-12.0.

[0095] Using citric acid-Na at pH 7.5 2 HPO 4 When the buffer is used as solution B, the enzyme activity is the highest, and the highest enzyme activity is taken as 100%, and the relative enzyme activity under the condition of using other buffers as solution ...

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Abstract

The invention discloses new application of GDSL protein to preparation of lipase. The invention provides application of the GDSL protein to (1) preparation of lipases or (2) degradation of a substrate of the lipase. The GDSL protein is the following protein in (a) or (b): (a) the protein comprises the amino acid sequence shown as the sequence 2; and (b) the protein is formed in the way that the amino acid sequence shown as the sequence 2 is subjected to substitution and / or deletion and / or addition of one or more amino acid residues, has lipase activity and is derived from the sequence 2. The GDSL protein has lipase activity. The application of the GDSL protein as the lipase has the advantages of higher reaction temperature, high heat stability, action under the alkaline condition, high pH stability, effects on the substrates of C2-C12 and high enzyme activity. The invention opens up a new way for the process flow of the lipase and has a great economic value.

Description

technical field [0001] The invention relates to the new application of GDSL protein in the preparation of lipase. Background technique [0002] Lipase (1ipase, triacylglycerolacylhydrolases, EC3.1.1.3) is triacylglycerol acyl hydrolase, which is one of the most important enzymes in industrial enzyme preparations. It can catalyze the hydrolysis of natural substrate oil at the oil-water interface to generate fatty acids, Glycerin and monoglycerides or diglycerides can also catalyze various chemical reactions such as transesterification, transesterification, ester synthesis, alcoholysis, acidolysis, and ammonolysis, and usually have the advantages of high catalytic efficiency and simple catalytic conditions. [0003] At present, most of the lipases used in industry are extracellular enzymes produced by microorganisms. In terms of reaction temperature, most of them are mesophilic enzymes (the optimum reaction temperature is 37-40°C), and the reaction is limited at high temperatu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N1/21C12N15/55C12P7/22C12P7/40C12P7/54C12P7/52C12P7/64C12R1/19
Inventor 周志刚何夙旭徐俐杨雅麟张美超李青
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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