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Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme

A technology of gallic acid and esterase, which is applied in the field of preparation and catalytic conversion of enzymes, can solve the problems of low hydrolysis rate, low enzyme output, easy oxidation, etc., and achieve high conversion rate, simple operation and strong controllability

Inactive Publication Date: 2010-11-17
DONGHUA UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, conventional chemical methods such as acid or alkali hydrolysis have disadvantages such as extremely low hydrolysis rate, extremely unstable hydrolyzed products or easily oxidized during the reaction process.
In addition, according to literature reports, pig liver esterase cannot hydrolyze EGCG, and no other commercially available esterase or lipase can hydrolyze EGCG
Only Tannase (Tannase) can hydrolyze EGCG into non-ester type catechin EGC, but the production of this enzyme is small and the price is high

Method used

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  • Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme
  • Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme
  • Induction preparation of EGCG esterase and method for producing EGC and gallic acid by using the enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Seed culture of Aspergillus niger ATCC 46890

[0033] Aspergillus niger seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N; use Aspergillus niger ATCC 46890 as the production strain, at pH 5.0, 30°C, 200r / Min conditions for 36 hours.

[0034] 2. Fermentation of Aspergillus niger to produce enzymes

[0035] Aspergillus niger enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N, add EGCG to a final concentration of 2.5g / L, pH5.0; at 4% inoculum size , at 30°C and 160r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0036] 3. Enzymatic hydrolysis of EGCG

[0037] Reaction system: 10g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hydrolase solutio...

Embodiment 2

[0042] 1. Seed culture of Aspergillus niger MC 57

[0043] Aspergillus niger seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N; Aspergillus niger MC 57 was used as the production strain, pH 5.0, 30°C, 200rpm culture 36 hours.

[0044] 2. Fermentation of Aspergillus niger to produce enzymes

[0045] Aspergillus niger enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N, add EGCG to a final concentration of 5.0g / L, pH5.0; at 8% inoculum size , at 30°C and 160r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0046] 3. Enzymatic hydrolysis of EGCG

[0047] Reaction system: 15g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hydrolase solution blank system: pH5....

Embodiment 3

[0050] 1. Seed culture of Aspergillus niger ATCC 46890

[0051] Aspergillus niger seed medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N; use Aspergillus nigerATCC 46890 as the production strain, at pH 5.0, 30°C, 200r / min for 36 hours.

[0052] 2. Fermentation of Aspergillus niger to produce enzymes

[0053] Aspergillus niger enzyme production medium: 1% glucose, 0.1% peptone, 0.05% citric acid, 0.015% Tween 80, 2% Vogel's medium N, add EGCG to a final concentration of 10.0g / L, pH5.0; at 8% inoculum size , at 30°C and 130r / min for 72h to induce enzyme production. The fermented supernatant liquid collected by filtration or centrifugation is the EGCG hydrolase liquid. In the experiment, the fermentation culture without adding EGCG was used as the control.

[0054] 3. Enzymatic hydrolysis of EGCG

[0055] Reaction system: 30g / L EGCG substrate 2mL, EGCG hydrolase solution 2mL, pH5.2 acetate buffer 2mL; EGCG hydrolase solution blank sys...

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Abstract

The invention relates to a method for inducing aspergillus niger to generate EGCG (epigallocatechin-3-gallate) esterase with EGCG, comprising the steps that: (1) an aspergillus niger seed culture medium is cultivated for 36 hours at 25-30 DEG C and 100-250r / min; (2) the aspergillus niger seed culture medium with a inoculated dose of 4-10 percent is transferred to a fermented culture medium that contains the EGCG and cultivated for 72 hours at 25-30 DEG C and 100-250r / min, and filtered and centrifugated to get enzyme liquid; then the enzyme is used for hydrolyzing the EGCG so as to transfer the EGCG into EGC (epigallocatechin) and ellagic acid, comprising: (1) an EGCG enzyme hydrolysis reaction system, an enzyme liquid blank system and a substrate blank system are constructed; (2) the reaction system and the blank systems are reacted for 0-48 hours at 25-60 DEG C or until the hydrolysis reaction is almost over; (3) high performance liquid and thin layer chromatography are used for analyzing the substrate and the products. The invention has high EGC transfer ratio and yield and the products are stable and easy to be separated, besides, the invention has the advantages of safety, environment protective, simple operation and strong controllability, thereby being applicable to mass scale production.

Description

technical field [0001] The invention belongs to the field of enzyme preparation and catalytic transformation of enzyme, in particular relates to the induction preparation of an EGCG esterase and a method for producing EGC and gallic acid by using the enzyme. Background technique [0002] Tea catechins are the main physiologically active components in tea. Tea catechins consist of epi-gallocatechin-3-gallate (EGCG), epicatechin-3-gallate (epicatechin-3- gallate, ECG), gallocatechin-3-gallate (gallocatechin-3-gallate, GCG), epicatechin (epicatechin, also known as cis-catechin, EC), epigallocatechin (epigallocatechin, also known as cis- gallocatechin, EGC) and other components. Catechin monomers are highly effective in anti-oxidation, scavenging free radicals, anti-tumor, anti-mutation, anti-aging, anti-virus, anti-allergy, anti-bacterial and anti-inflammatory, enhancing immune function, anti-radiation, lowering blood pressure, and lowering blood fat. It has a good effect on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/18C12P7/42C12P7/62C12R1/685
Inventor 洪枫钟坤
Owner DONGHUA UNIV
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