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Partial glyceride lipase mutant and application thereof

A technology of partial glycerides and mutants, which is applied in the field of preparation of partial glyceride lipase mutants, can solve the problems of easy inactivation of enzymes, complicated side reactions, and low yield of products, so as to improve the half-life, avoid side reactions, The effect of reducing separation and purification steps

Active Publication Date: 2018-10-12
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

This patented technology improves on existing methods for producing fatty acids through oxiranases that use specific catalysts or other techniques like enzymes. It also increases its ability to resist attack from certain chemical substances used during manufacturing processes while maintaining their functionality over time. Overall, this new method allows for efficient production at reduced costs compared to previous ways.

Problems solved by technology

This patented technical problem addressed in this patents relating to chemistry processes for producing specific substances like epoxies involves generating free radicals during these procedures due to decomposition caused by harsh environments and limited availability. These techniques involve modifying existing ones through gene manipulation technology called bioengineering, resulting in improved performance over time.

Method used

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  • Partial glyceride lipase mutant and application thereof
  • Partial glyceride lipase mutant and application thereof
  • Partial glyceride lipase mutant and application thereof

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Embodiment 1

[0032] The construction of embodiment 1 partial glyceride lipase mutant

[0033] Using the partial glyceride lipase plasmid pGAPZαA-PCL derived from Penicillium as a template, the mutant primers were designed, and the plasmid containing the mutant gene was amplified by PCR. After the PCR amplification product was confirmed by 1% agarose gel electrophoresis, an appropriate enzyme digestion system was designed, and DpnI was added to remove the original template with methylation. After digestion for 4 hours, the digested product was purified by PCR, and the purified product was confirmed by nucleic acid electrophoresis. The obtained purified product was mixed with Escherichia coli competent cells DH5α, placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and quickly placed on ice for 5 minutes. Add the competent cells obtained in the previous step to 1ml LB medium and activate at 37°C and 180rpm for 50min. After activation, the competent cells were centrifuged at ...

Embodiment 2

[0047] Embodiment 2 Expression and protein purification of partial glyceride lipase mutant

[0048] Expression of the mutant: the plasmid of the partial glyceride lipase mutant was linearized by DNA restriction endonuclease BlnI at 37°C, and the digested product was confirmed by nucleic acid electrophoresis after 5 hours of digestion. After the mutant plasmid was cut completely, the resulting enzyme-digested product was purified by PCR, and finally eluted with sterile water. The purified product was electrotransferred into Pichia pastoris X-33, and the transformation solution was spread on a YPD plate containing 100 μg / mL bleomycin (Zeocin), cultured at 37°C for 48 hours, and the single colony grown was the expression strain. A single colony of the partial glyceride lipase mutant was picked and inoculated in 50ml YPD medium for 24 hours at 37°C and 200rpm, and then further inoculated into 500ml YPD medium for expansion. After 72 hours, the fermented culture solution was centr...

Embodiment 3

[0055] Embodiment 3 Determination of thermal stability of partial glyceride lipase mutant

[0056] The PCL purified in Example 2 and its mutants Y84R, M209A, I260E and I260R were incubated at 45°C at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 , 12h sampling, placed on ice to prevent further loss of enzyme activity, accurate determination of residual enzyme activity. In this experiment, the method of measuring the content of peroxyacid was used to measure the peroxidase activity of partial glyceride lipase PCL and its mutants.

[0057] Definition of enzyme activity: Under certain conditions, the amount of enzyme required to oxidize 1 μmol MCD per minute is an enzyme activity unit, expressed in U. It is known that MCD has a maximum absorbance value at 290nm, and the corresponding enzyme activity can be calculated by comparing the detected absorbance value with the MCD standard curve.

[0058] The reaction system is: 0.1M pentanoic acid pH5.0 buffer (containing 180μM MCD, 9...

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Abstract

The invention discloses a partial glyceride lipase mutant. The mutant is obtained through the site-directed mutagenesis of a partial glyceride lipase derived from penicillium; one or more following mutation modes are adopted for a mutation site of the partial glyceride lipase, wherein tyrosine at an 84th site of the protein is mutated to arginine, methionine at a 209th site of the protein is mutated to alanine, and isoleucine at a 260th site of the protein is mutated to glutamic acid or arginine. By means of the mutant, the thermal stability and hydrogen peroxide tolerance of partial glyceridelipase are greatly improved. In addition, compared with a wild partial glyceride lipase, the capability of catalyzing ester to be subjected to an epoxidation reaction by the obtained mutant is remarkably improved, the yield of target products is high, and the mutant has significant advantages in industrial application.

Description

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Claims

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Application Information

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Owner SOUTH CHINA UNIV OF TECH
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