Thermally stable lipase as well as preparation method and applications thereof

A technology of lipase and thermostabilization, applied in the field of enzyme engineering, can solve problems such as the design of protein dynamic stability and the lack of a deep understanding of protein unfolding kinetics

Active Publication Date: 2016-09-21
GUANGDONG RUISHENG TECH GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] None of the above has a deep understanding of protein unfolding kinetics, nor does it involve the design of protein dynamic stability

Method used

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  • Thermally stable lipase as well as preparation method and applications thereof
  • Thermally stable lipase as well as preparation method and applications thereof
  • Thermally stable lipase as well as preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Establishment of unfolding model of Yarrowia lipolytica lipase 2 and screening of disulfide bonds.

[0064] (1) Search and download the crystal structure of Lip2 (PDB ID: 3O0D) from the RCSB PDB crystal database with an accuracy of The crystal is a heptamer, and each polymer has different degrees of missing atoms. Therefore, the complete A-chain of the skeleton atoms is selected as the initial model, and the missing side chain amino acids of the A-chain are repaired using the Swiss-pdb Viewer software, and Disulfide is used to byDesign software, the Cys120-Cys123 disulfide bond in the A chain is reconstructed, and all the crystal water of the A chain is retained in the simulation. Except for the HIS289 in the center of the triplet, which uses the HID protonation state, the rest of the histidines are set to the default pH of Gromacs The protonation state of =7.00 obtains the pretreatment model;

[0065] (2) Use the GROMACS 5.04 software to carry out three-d...

Embodiment 2

[0101] The construction of embodiment 2 lipase mutant expression plasmid

[0102] Using the Yarrowia lipolytica lipase 2 sequence (Genbank ID: AJ012632.1) as the target fragment, with EcoRI and NotI as the restriction enzyme sites, the whole gene was synthesized and constructed pPICZαA-Lip2, through two consecutive reverse The mutation PCR method introduces disulfide bond mutation, and the primers used for point mutation are shown in Table 2.

[0103] Table 2 Summary of mutation primers

[0104]

[0105]

[0106] Note: The bold slash is the mutation site

[0107] The PCR amplification conditions are: 94°C for 2min; 94°C for 10s, 66°C for 30s, 68°C for 5min, 10 cycles. The reaction system is shown in Table 3 below.

[0108] Table 3 PCR reaction system

[0109]

[0110] The amplified product was digested with DnpI enzyme to digest the template, and after agarose gel electrophoresis to detect the size of the mutant band, it was ligated and circularized overnight with...

Embodiment 3

[0111]Example 3: Electrotransformation of Pichia pastoris with linearized plasmid, screening of transformants and screening of enzyme production

[0112] After the positive transformants with correct sequencing were expanded overnight in LLB liquid medium, the plasmid was extracted, linearized with PmeI, purified and recovered, and a total of 5 μg of the plasmid linearized product was mixed with X33 Pichia pastoris for electroporation transformation. Competent preparation of Pichia pastoris refers to the operation manual of Invitrogen Company. The electroporation program was set according to the parameters recommended by Bio-Rad.

[0113] Add 1 mL of 1mol / L sorbitol solution immediately after electroporation, incubate and recover the bacterial solution at 30°C for 1 hour, and spread it evenly on the YPDS+Zeocin (Zeocin concentration is 200 μg / ml) resistance plate for screening; after culturing for 3 days, put The grown single colony was picked on the BMMY-rhodamine B plate fo...

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Abstract

The invention discloses a thermally stable lipase as well as a preparation method and applications of the thermally stable lipase. The lipase is S2-210 lipase with the amino acid sequence shown as SEQ ID NO.1, S8-214 lipase with the amino acid sequence shown as SEQ ID NO.2, S14-216 lipase with the amino acid sequence shown as SEQ ID NO.3, or S191-241 lipase with the amino acid sequence shown as SEQ ID NO.4. According to the thermally stable lipase and the preparation method thereof, the unfolding process of Yarrowia lipolytica lipase 2 is stimulated through the long-time high-temperature molecular dynamics simulation, and the main area of protein unfolding and the critical steps for forming the wet molten-globule state are analyzed, so that the key site for transforming the kinetic stability are effectively screened. The thermally stable lipase is heat-resisting, is long in half-life period, and is particularly suitable for being applied in the industry.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a thermostable lipase, a preparation method and application thereof. Background technique [0002] Lipase (EC 3.1.1.3), the full name of triacylglycerol hydrolase, is widely used in feed industry, food processing, cosmetics, detergents, biomedicine and bioenergy. [0003] Candida lipolytica (Yarrowia lipolytica) is an unconventional yeast that belongs to food-safe yeast. At present, through continuous gene interference analysis, it has been found that it can encode 16 lipases Lip2, Lip4, Lip5, Lip7-19 . The homology of these lipases is different, and the properties are quite different. Among them, Yarrowia lipolytica lipase 2 (Lip2) is the main extracellular lipase, which has high catalytic activity on medium and long-chain fatty acid triglycerides (C12-C16), and is widely used in oil hydrolysis, sewage Treatment, food processing, bioenergy, chemical synthesis, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81
CPCC12N9/20C12Y301/01003
Inventor 管武太吴炜坤李力浪
Owner GUANGDONG RUISHENG TECH GRP CO LTD
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