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Tea polyphenol oxidase isozyme monomers PP01 and PP02 and preparation method thereof

A polyphenol oxidase isoenzyme, PP01 technology, applied in the field of enzyme preparation, can solve the problems of low purification multiple, small amount of protein in the uploaded sample, long operation time, etc. Lost, short operating time effect

Inactive Publication Date: 2015-04-08
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The latest separation and preparation technology of tea polyphenol oxidase isozymes reported at home and abroad also has the disadvantages of low purification factor, small amount of uploaded sample protein, and long operating time due to the limitation of research methods and technical means. The enzyme is only an isoenzyme with a certain relative molecular weight, and no clear isoenzyme monomer such as amino acid sequence structure, molecular weight, and isoelectric point can be obtained

Method used

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  • Tea polyphenol oxidase isozyme monomers PP01 and PP02 and preparation method thereof
  • Tea polyphenol oxidase isozyme monomers PP01 and PP02 and preparation method thereof
  • Tea polyphenol oxidase isozyme monomers PP01 and PP02 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] (1) Preparation of crude enzyme solution

[0062] Weigh 50g fresh leaves of Zhenghe Dabai tea tree with one bud and two leaves, add -20°C pre-cooled acetone 150mL tissue homogenate for 2.5min, then vacuum filter and wash with 1000mL -20°C acetone (80% volume concentration) until rinse The solution was colorless, and finally added 200 mL of -20°C acetone (100% volume concentration) to rinse, and freeze-dried the tea residue at -30°C for 24 hours to obtain acetone powder. Add 180mL of 0.1M citric acid-phosphate buffer solution (containing 10% glycerol, 0.2gVc, 1mM EDTA, 6g PVPP) of 0.1M pH=5.6 to the dry acetone powder obtained, homogenate at 4°C, and then place Extract at 4°C for 1 hour, shake 4 times during the period, take the extract and filter it with four layers of gauze, centrifuge at 8000r / min for 20 minutes, collect the supernatant, which is the crude enzyme solution.

[0063] (2) Ammonium sulfate fractional precipitation

[0064] Slowly add 22.14g of anhydrous...

Embodiment 2

[0072] (1) Preparation of crude enzyme solution

[0073] Weigh 60g of fresh leaves of Fuyun No. 6 tea tree with one bud and two leaves, add -25°C pre-cooled acetone 200mL tissue homogenate for 2min, then use 1400mL -20°C acetone (80% volume concentration) to vacuum filter and wash until the The washing solution was colorless, and finally added 200 mL of -20°C acetone (100% volume concentration) for rinsing, and the tea residue was freeze-dried at -25°C for 26 hours to obtain acetone powder. Add 190mL of 0.1M citric acid-phosphate buffer solution (containing 9% glycerol, 0.24gVc, 1mM EDTA, 10g PVPP) of 0.1M pH=5.6 to the dry acetone powder obtained, homogenate at 4°C, and then place Extract at 4°C for 2 hours, shake 6 times during the period, take the extract and filter it with four layers of gauze, centrifuge at 10000r / min for 18 minutes, collect the supernatant, which is the crude enzyme solution.

[0074] (2) Ammonium sulfate fractional precipitation

[0075] Slowly add 24...

Embodiment 3

[0083] (1) Preparation of crude enzyme solution

[0084] Weigh 70g fresh leaves of Taoyuan big-leaf tea tree with one bud and two leaves, add -30°C pre-cooled acetone 220mL tissue homogenate for 3 minutes, then vacuum filter and rinse with 1500mL -25°C acetone (80% volume concentration) until rinse The liquid was colorless, and finally added 320 mL of -25°C acetone (100% volume concentration) to rinse, and freeze-dried the tea residue at -30°C for 30 hours to obtain acetone powder. Add 200mL of 0.1M citric acid-phosphate buffer solution (containing 9% glycerol, 0.22gVc, 1mM EDTA, 8g PVPP) of 0.1M pH=5.6 to the obtained dry acetone powder, homogenate at 4°C, and then place Extract at 4°C for 3 hours, shake 8 times during the period, take the extract, filter it through four layers of gauze, centrifuge at 12000r / min for 15min, collect the supernatant, which is the crude enzyme solution.

[0085] (2) Ammonium sulfate fractional precipitation

[0086] Slowly add 22.96g of anhydro...

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Abstract

The invention discloses tea polyphenol oxidase isozyme monomers PP01 and PP02 and a preparation method thereof. With respect to the technical states that existing tea polyphenol oxidase is low in purification factor, low in loading quantity of protein samples and long in operation time, the obtained isozyme is the isozyme with one relative molecular weight, and the isozyme monomer with specific amino acid sequence structure, molecular weight, isoelectric point, enzymatic property and the like cannot be obtained, two tea polyphenol oxidase isozyme monomers with specific specific amino acid sequence structures, molecular weight, isoelectric points, enzymatic properties and the like are separated and prepared by sequentially adopting the technologies and optimization processes such as enrichment of zymoprotein with acetone powder, fractional precipitation of ammonium sulfate, strong anion exchange chromatography, gel medium chromatography, molecular identification and enzymatic property analysis. The method has the advantages of low cost, large preparation amount, high pertinence, short operation cycle and the like, and is of important theoretical and practical significance on deepening of the separation and purification technology of tea polyphenol oxidase isozyme, promoting of directional enzymatic synthesis of a theaflavin component by virtue of the tea polyphenol oxidase isozyme monomers, development and utilization of advantaged isozyme of tea polyphenol oxidase, isozyme immobilization, enzyme protein structure analysis, enzyme gene cloning, functional verification and the like.

Description

technical field [0001] The invention belongs to enzyme preparation technology, and in particular relates to a tea polyphenol oxidase isoenzyme monomer P01 and PP02, and a preparation method of the two isoenzyme monomers. Background technique [0002] Polyphenol Oxidase (Polyphenol Oxidase, PPO) is an oxidoreductase that is ubiquitous in organisms and has a variety of isoenzymes and different physiological functions. There were significant differences in polyphenol oxidase isozymes. Wang Kunbo conducted electrophoresis analysis on polyphenol oxidase isoenzymes from different sources, and found that there were 11 isoenzymes in pears, 5 in tea, 5 in mushrooms, 4 in apples, and only 1 in laccase. During the research of black tea processing technology, Liu Zhonghua found that the number of PPO isozyme enzyme bands and the enzyme activity of the same enzyme band at different processing stages were different, and the substrate specificity was also different. Buzen et al. separate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
CPCC12N9/0059C12Y110/03001
Inventor 肖文军滕杰龚志华邓燕莉
Owner HUNAN AGRICULTURAL UNIV
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