A tea tree mushroom fibrinolytic enzyme and preparation method thereof

A technology of fibrinolytic enzyme and tea tree mushroom, which is applied in the field of bioengineering, can solve the problems of reducing the activity of fibrinolytic enzyme, reducing the fibrinolytic effect, and many steps of separation and purification.

Active Publication Date: 2020-10-20
QIQIHAR UNIVERSITY
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  • Abstract
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Problems solved by technology

These four kinds of plasmin are all produced by fungal fermentation, and there are more fungal fermentation metabolites, resulting in more steps for subsequent separation and purification of plasmin, too many separation and purification steps will reduce the activity of plasmin, As a result, the fibrinolytic effect has declined. In recent years, the applicant has been working hard to find a new source of plasmin with uncomplicated components, which is easy for subsequent separation and purification.
The applicant team found through screening that most edible fungi contain fibrinolytic enzymes, among which the content of fibrinolytic enzymes in tea tree mushroom is higher, reaching 2780.05±34.32U / g (calculated by dry weight). It is worth noting that domestic and foreign There is no report about extracting plasmin from tea tree mushroom in related research

Method used

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  • A tea tree mushroom fibrinolytic enzyme and preparation method thereof
  • A tea tree mushroom fibrinolytic enzyme and preparation method thereof

Examples

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preparation example Construction

[0022] The present invention also provides a method for preparing the tea tree mushroom fibrinolytic enzyme described in the above technical solution, comprising: extracting the fruiting body of tea tree mushroom with physiological saline to obtain an extract; performing solid-liquid separation on the extract to obtain the crude The enzyme liquid is separated by salting out, gel chromatography, weak cation exchange chromatography, hydrophobic interaction chromatography and strong cation exchange chromatography in sequence to obtain tea tree mushroom fibrinolytic enzyme.

[0023] In the present invention, the fruiting bodies of Agrocybe abaculi are preferably obtained by drying and then crushing the fruiting bodies of Agrocybe. In the present invention, there is no special limitation on the crushing method, and conventional crushing methods can be used. In the present invention, there is no special limitation on the source of the fruiting bodies of the tea tree mushroom, and co...

Embodiment 1

[0033] Preparation of tea tree mushroom fibrinolytic enzyme

[0034] 1) Preparation of crude enzyme solution: Agrocybe sporocarp is air-dried and crushed, and the powder of Agrocybe sporocarp is added to 0.9% normal saline at a ratio of 1:10 (w / v), extracted at 4°C for 6 hours, 4°C, 10000r / Centrifuge for 15 minutes, and take the supernatant for later use;

[0035] 2) Take the centrifuged supernatant and adjust its ammonium sulfate saturation to 80%, salt out overnight at 4°C, centrifuge at 4°C and 10,000r / min for 15min, discard the supernatant, and use 50mL 0.02mol / L PBS for precipitation (pH6.0) buffer solution dissolved, 4 ℃ standby;

[0036] 3), Gel chromatography desalting and decolorization separation: the enzyme solution after salting out is separated by Sephadex G-25 gel chromatography column, the eluent is 0.02mol / L PBS (pH6.0) buffer solution, and the flow rate is 6mL / min , to collect the active components;

[0037] 4), Weak cation exchange chromatography separati...

Embodiment 2

[0041] The properties of the tea tree mushroom fibrinolytic enzyme in Example 1 after separation and purification were measured. The related enzymatic properties were determined by fibrin plate method.

[0042] The tea tree mushroom fibrinolytic enzyme prepared in Example 1 is composed of two subunits, and the molecular weights of the two subunits are 31.4kDa and 21.2kDa respectively; the optimum temperature for action is 47°C; it can be preserved better at the physiological pH of the human body Active, more stable in neutral alkaline environment than in acidic environment; Fe 3+ 、K + and Zn 2+ It has obvious protective effect on the enzyme, while different concentrations of Fe 2+ It showed obvious inhibitory effect on tea tree mushroom fibrinolytic enzyme. The enzyme can not only directly degrade fibrin, but also activate plasminogen to degrade fibrin indirectly; it can degrade the α, β and γ chains of human fibrinogen in sequence; the Edman degradation method measured th...

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Abstract

The invention provides a tea tree mushroom fibrinolytic enzyme and a preparation method thereof, and relates to the technical field of bioengineering. The tea tree mushroom fibrinolytic enzyme is composed of two subunits A and B, and the molecular weight of the subunit A is 31.4kDa; the subunit The molecular weight of B is 21.2kDa; the twelve amino acid sequence of the N-terminal of subunit A is shown in SEQ ID No.1; the twelve amino acid sequence of the N-terminal of subunit B is shown in SEQ ID No.2. The optimal action temperature of plasmin provided by the invention is 47°C; it can preserve better activity at the physiological pH of human body, and different concentrations of Fe 2+ It has obvious inhibitory effect on tea tree mushroom fibrinolytic enzyme; it has good thrombolytic performance, good safety in use, and low production cost, which provides a certain research basis for the application in the preparation of thrombolytic drugs and / or functional foods , also provides a new way for the comprehensive utilization of tea tree mushroom.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a tea tree mushroom fibrinolytic enzyme and a preparation method thereof. Background technique [0002] Thrombotic diseases are a serious threat to human life and health, and their disability and mortality rates are increasing year by year, which is one of the main causes of human death today. Thrombolytic therapy is currently the main method for the treatment and prevention of thrombotic diseases. The thrombolytic drugs currently used in clinical treatment are effective, but there are still many defects and are expensive. Therefore, it is an urgent need for modern medicine to develop new thrombolytic drugs that are efficient, fast, prevent re-embolization and reduce side effects. The development of new thrombolytic substances from various biological resources in nature is an important way to develop thrombolytic agents. [0003] Tea tree mushroom (Agrocybe aegerita) belo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/58
CPCC12N9/58C12Y304/24072C12Y304/21007
Inventor 刘晓兰李冠龙郑喜群邓永平
Owner QIQIHAR UNIVERSITY
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