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Lipase sv-lip5 and its application in the hydrolysis of astaxanthin ester

A technology of sv-lip5 and astaxanthin ester, which is applied in the fields of application, hydrolase, enzyme, etc., can solve problems such as difficult industrial production, low enzyme catalytic efficiency, and unknown biological safety of Pseudomonas aeruginosa, and achieves the goal of using High efficiency, high hydrolysis rate, high yield of astaxanthin

Active Publication Date: 2022-04-08
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current reports, most studies use commercial enzymes or lipases obtained from different expression systems for the hydrolysis of astaxanthin esters, but these enzymes have low catalytic efficiency, and the catalytic efficiency of extracellular crude extracts is the highest, but The biological safety of Pseudomonas aeruginosa is unknown and it is difficult to use in industrial production

Method used

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  • Lipase sv-lip5 and its application in the hydrolysis of astaxanthin ester
  • Lipase sv-lip5 and its application in the hydrolysis of astaxanthin ester
  • Lipase sv-lip5 and its application in the hydrolysis of astaxanthin ester

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Obtaining of lipase Sv-lip5

[0036] In order to find highly active lipase, the present invention purchased Streptomyces lividans ( Streptomyces violascens , No. SDUM420004), through previous experimental research, it was found that its culture medium can detect high enzyme activity of lipase and esterase. In order to discover the highly active lipase or esterase, the whole genome was sequenced. According to the gene annotation information obtained by sequencing, a potential target gene fragment expressing lipase is screened out. The nucleotide sequence of the gene fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the expressed protein is shown in SEQ ID NO: 1. indicated that it may have lipase activity, named lipase Sv-lip5.

[0037] The gene sequence of lipase Sv-lip5 was analyzed using the method reported in the literature, and the evolutionary tree of lipase Sv-lip5 and other family lipolytic enzymes was constructed using MEGA 9.0 softwa...

Embodiment 2

[0039] Example 2 Study on enzymatic properties of lipase Sv-lip5

[0040] (1) Optimal temperature of lipase Sv-lip5

[0041] The optimum temperature of lipase Sv-lip5 was determined by detecting the enzyme activity at different temperatures (25°C, 30°C, 35°C, 40°C, 50°C, 55°C, 60°C). The activity at the optimum temperature was defined as 100%, and the activity at other temperatures was expressed as a percentage of the highest activity. by the result ( figure 1 ) It can be seen that when the temperature is lower than 45 °C, the enzyme activity gradually increases with the increase of temperature, and reaches the maximum value at 45 °C, and when the temperature is greater than 45 °C, the enzyme activity gradually decreases with the increase of temperature, When the temperature is higher than 60°C, the lipase Sv-lip5 has almost no enzymatic activity. Therefore, the optimum temperature of lipase Sv-lip5 is 45°C.

[0042] (2) Temperature stability of lipase Sv-lip5

[0043] Acco...

Embodiment 3

[0052] Example 3 Lipase Sv-lip5 catalyzes the hydrolysis of astaxanthin ester to efficiently prepare free astaxanthin

[0053] The lipase Sv-lip5 enzyme powder prepared in Example 1 was used for the hydrolysis of astaxanthin ester. The reaction system was as follows: 5 mL Tris-HCl buffer (100 mM, pH 9.0), 200 mg astaxanthin ester, 11 U fat Enzyme Sv-lip5 (to p The definition of the hydrolysis activity of NPP), the reaction mixture was placed in a water bath shaker at 40 °C for 12 h, and the production of free astaxanthin was detected by liquid chromatography.

[0054] The results of liquid chromatography were as Figure 7 , 8 , 9, and 10, Figure 7 The middle is the comparison of the reaction solution substances before and after the reaction, and the results show that the lipase Sv-lip5 has a strong hydrolysis effect on astaxanthin esters, and can hydrolyze most of the astaxanthin esters into astaxanthin. Figure 8 , 9 , 10 are the optimization of the reaction pH, the vol...

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Abstract

The invention discloses a lipase Sv-lip5 and its application in hydrolyzing astaxanthin ester, belonging to the technical field of functional enzyme screening. The amino acid sequence of the lipase Sv-lip5 is shown in SEQ ID NO:1. The gene encoding lipase Sv-lip5 has a nucleotide sequence as shown in SEQ ID NO:2. Application of the lipase Sv-lip5 in hydrolyzing astaxanthin ester or preparing free astaxanthin. The invention also provides a method for hydrolyzing astaxanthin ester or preparing free astaxanthin. The present invention also provides a recombinant expression vector and a recombinant engineering bacterium comprising a gene encoding lipase Sv-lip5. The lipase Sv-lip5 of the present invention has astaxanthin ester hydrolysis activity, can break the ester bond in astaxanthin ester, realize the efficient preparation of free astaxanthin, and solve the technical problem of low catalytic efficiency of existing enzymes. The invention is of great significance to the industrial application of astaxanthin ester.

Description

technical field [0001] The invention relates to a lipase Sv-lip5 and its application in hydrolyzing astaxanthin ester, belonging to the technical field of functional enzyme screening. Background technique [0002] Free astaxanthin is a fat-soluble ketone carotenoid, which has powerful anti-oxidation, anti-inflammatory and immunomodulatory effects, and has great application potential in the treatment of cardiovascular and cerebrovascular diseases, diabetes, cancer and immune system diseases . [0003] The main form of astaxanthin in nature is fatty acid esters combined with fatty acids. The diversity of fatty acids makes the composition of astaxanthin esters more complex, and the low water solubility and low bioavailability of astaxanthin esters also limit its application in the food industry. Astaxanthin cannot be synthesized in most animals and can only be obtained from other sources such as food. Therefore, it is necessary to hydrolyze astaxanthin esters into astaxanthi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/75C12N1/21C12P23/00C12R1/125
CPCC12N9/20C12N15/75C12P23/00C12Y301/01003C12N2800/101
Inventor 毛相朝闫娇孙建安高坤鹏余可欣
Owner OCEAN UNIV OF CHINA
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