CN1206353C - Preparation method of beta cyclodextrin carthworm plasmin modification enzyme - Google Patents

Abstract

The present invention uses earthworm fibrinolysin as a raw material and uses beta-cyclodextrin for chemical modification. The stability of modification enzyme (beta-CDEFE) is enhanced. According to the verification of pharmacokinetics experiment, the distribution phase half life of beta-CDEFE in vivo is 1h and is 1.52 times of that of EFE. The affinity to a substrate (casein) of the beta-CDEFE is 3.5 times of that of the EFE.

Description

The preparation method of beta cyclodextrin carthworm plasmin modification enzyme

Technical field

The present invention relates to the preparation of modifying enzyme, especially (β-CD) modifies the beta-cyclodextrin earthworm fibrinolysin modifying enzyme that earthworm fibrinolysin (EFE) back forms (β-CDEFE) to beta-cyclodextrin.

Background technology

Thrombus disease, cardiovascular disorder are the multiple common diseases of a class serious harm human health.Though medicines such as at present commercially available fiber eliminating enzyme, Ahylysantinfarctase, urokinase have been used for clinical, but poor stability, easy inactivation, in the body residence time short, be difficult for entering focus cell (tissue), cost an arm and a leg, easily cause clinical scarce limits such as hemorrhage, do not reach generally and apply.Seeking effective medicine is the focus that domestic and international medical circle is paid close attention to.Earthworm is commonly called as earthworm, year history surplus in the of existing 1200 at home of being used as medicine, and use till today.In the vermis, the main effective constituent for the treatment of above-mentioned disease is earthworm plasmin (EFE).There are above-mentioned disadvantage at present commercially available " 912 " capsule, earthworm capsule, thrombolysis capsule etc. equally clinical, and based on oral, curative effect is lower.General zymin is used for: (1) long half time in vivo; (2) non-immunogenicity; (3) easily enter cell and tissue.For this reason, seek the focus that new thrombolysis medicine source and novel form thereof are current domestic and international research.

Summary of the invention

The objective of the invention is to seek that a kind of wide material sources, preparation are easy, the source new drugs of the dissolving fibrin of good stability, applied range, to adapt to needs of economic development.

Earthworm is world's environmental protection animal, is not only the valuable ingredients of traditional Chinese medicine, good new egg-white food and feed, and in soil improvement, eliminate public hazards, preserve the ecological environment on, in the performance special role of aspects such as material cycle, species diversity.This discovery, medicine new resources, new variety have not only been increased, and the deep development that drives vermiculture industry, processing industry and research thereof had remarkable economical and social benefit, simultaneously to shaking off poverty and setting out on the road to prosperity, cultivate "three highs" agriculture, improveing aspect such as ecotope crucial meaning is also arranged.

We began the earthworm fibrinolytic enzyme is studied 90 years last century.In recent years, with beta-cyclodextrin the earthworm fibrinolytic enzyme is carried out chemically modified, (β-Huan hu jing yin rong xian mei xiu shi mei, English name are abbreviated as β-CDEFE) to prepare the beta-cyclodextrin earthworm fibrinolytic enzyme modifying enzyme of high vigor

The present invention is achieved in that

(the preparation of β-CDEFE) of beta-cyclodextrin earthworm fibrinolytic enzyme modifying enzyme

Earthworm fibrinolysin is added in the beta-cyclodextrin solution, evenly after, in 30-50 ℃ of water bath with thermostatic control, intermittently stirred 40-70 minute, stirred 8-14 hour down at 32 ℃ again, reacted solution is beta-cyclodextrin earthworm fibrinolysin modifying enzyme.

The ratio of beta-cyclodextrin and earthworm fibrinolysin is 1: 4 (volume ratio) in the reaction.

Beta-cyclodextrin concentration is to the influence of modification effect

Beta-cyclodextrin is joined by concentration gradient in the earthworm fibrinolysin solution of the certain enzyme concn of 4ml (as 600U/ml), and 45 ℃ of waters bath with thermostatic control 1 hour are intermittently stirred, then cool to room temperature.Ultrasonication 10 minutes, room temperature lower magnetic force agitator stirs 10s, repeats this process 2-3 time, again in stirred overnight at room temperature, obtains beta-cyclodextrin fibrinolysin solution.Survey its corresponding activity change, zymoprotein concentration is 1.5mg/ml, is 100% with the vigor that does not add beta-cyclodextrin enzyme liquid.Result such as following table 1:

Beta-cyclodextrin (mg/ml) 0.5 13579

The relative vigor of modifying enzyme (%) 86.36 86.50 85.90 86.75 86.10 85.80

Table 1

By table 1 as seen, different beta-cyclodextrin concentration is little to the activity influence of earthworm fibrinolysin, is best with 5mg/ml.

The solution enzyme concn is to the influence of modification effect

With beta-cyclodextrin concentration is that 5mg/ml joins the enzyme concn gradient from the 4ml enzyme liquid of 300-600U/ml, and 45 ℃ of waters bath with thermostatic control 1 hour are intermittently stirred, then cool to room temperature.Ultrasonication 10 minutes, room temperature lower magnetic force agitator stirs 10s, repeats this process 2-3 time, again in stirred overnight at room temperature, obtains beta-cyclodextrin fibrinolysin solution.Surveying its corresponding activity change, is 100% with the enzyme liquid of without polishing dose earthworm fibrinolysin respective concentration, and beta-cyclodextrin concentration is 5mg/ml.Result such as following table 2:

Solution enzyme concn (U/ml) 300 400 500 600

The relative vigor of modifying enzyme (%) 86.15 85.10 86.25 88.00

Table 2

By table 2 as seen, the solution enzyme concn is little to the modification effect influence, and concentration is higher, and its effect is better.Below Shi Yan enzyme concn is 500U/ml.

The rate of recovery of present method reaches 85%.Product is a liquid enzyme formulation.Powdered after the freeze-drying is the solid zymin, and these goods are soluble in water, and the aqueous solution is colourless, transparent, has protein properties.

The property feature of beta-cyclodextrin earthworm fibrinolysin modifying enzyme

1. physical property: certified products is colourless, tasteless, nontoxic, transparent aqueous solution, and agreeing has protein properties.Solid zymin after the freeze-drying is a white powder, and moisture content and ash (conventional method) are respectively less than 7% and 5%.

2. enzymatic property:

This enzyme stability is good, and at room temperature vigor is stable, can at room temperature operate.

The Km of beta-cyclodextrin earthworm fibrinolysin modifying enzyme (Michaelis-Menton constant, substrate casein) is 0.028% (mass percent); Be 1/3.5 of EFE (0.098%).Being beta-cyclodextrin earthworm fibrinolysin modifying enzyme has improved 3.5 times to the avidity of substrate than EFE.

Beta-cyclodextrin earthworm earthworm fibrinolysin modifying enzyme is respectively pH5.8～10.6 and pH10.0 to stability and the reaction optimal pH of pH; Respectively than protoenzyme (pH5.8～10.6 pH7.8) increase.

The thermostability of beta-cyclodextrin albumen earthworm fibrinolysin modifying enzyme and effect optimum temperuture, enzyme activity vigor in 30～60 ℃ of scopes is stable, increases than (30～55 ℃) of EFE; Its optimum temperuture is 60 ℃, and also (55 ℃) than EFE increase.

The beta-cyclodextrin earthworm fibrinolysin modifying enzyme distribution phase transformation period in vivo is 1h, is 1.52 times of EFE.

Beta-cyclodextrin earthworm fibrinolysin modifying enzyme vigor is 818U/ml, is 1788.8U/mg protein than vigor.

Beta-cyclodextrin earthworm fibrinolysin modifying enzyme and earthworm fibrinolysin vitality test:

1. with the casein substrate

Take by weighing the 1g casein, add about 80ml 0.02mol/L Ph7.8 phosphoric acid buffer (PBS), after water-bath (60 ℃) dissolving, use with a kind of damping fluid and be settled to 100ml.Draw enzyme liquid 0.5ml in 37 ℃ of insulation 10min, add the casein solution 2ml of same insulation then, shake all, after 37 ℃ of water-baths accurately are incubated 15min, add 2ml 10% trichloroacetic acid solution termination reaction immediately.Leave standstill 30min under the room temperature, filter, filtrate is measured light absorption value down in the 280nm wavelength.Under above-mentioned condition, light absorption value (A _280nm) the enzyme amount that changes 0.001 unit is 1 enzyme activity position (U).

2. be substrate with benzoyl-own ester of L-arginine (BAEE)

Taking by weighing 27.4mgBAEE is dissolved in PBS (concentration is 0.4 * 10 ^-3Mol/L), get 2ml BAEE in cuvette, add enzyme liquid 30～50 μ l to be measured, mixing is measured optical density(OD) (A down in the 253nm wavelength immediately _253nm) increased value.With under these conditions, the enzyme amount that every min increases by 0.001 ODU is 1 BAEE enzyme activity unit (U).

Protein content determination

Pressing the Bradford method, is standard protein with the bovine serum albumin.Preparation protein is from the standard water solution of 0～900 μ g/ml, above-mentioned each concentration standard protein soln of joining is drawn 0.1 respectively in test tube, each adds 5 Xylene Brilliant Cyanine G G-250 solution, after shaking up, at 595nm, under the wavelength, be the blank pipe zeroing of do of zero (replacing protein soln) with 0.1ml distilled water with the protein concn.Measure and respectively manage A _595nm, be X-coordinate with the protein concn, A _595nmFor ordinate zou is made typical curve.The A of working sample in kind _595nm, check in the protein concn of sample solution from typical curve.

Advantage of the present invention:

(1) basic material of the present invention is an earthworm, and raw material sources are easy, cheapness, and can drive the comprehensive utilization of earthworm;

(2) the explained hereafter facility investment is few;

(3) modifying enzyme thermostability and pH stability all are better than protoenzyme;

(4) modification is comparatively simple;

(5) modifying enzyme vigor height reaches 1788.8U/mg protein.

Embodiment

Drawing 1ml concentration is β-CD solution (PBS preparation) of 5mg/ml, joins among the EFE that 4 concentration are 500U mixing.45 ℃ of water bath with thermostatic control 1h intermittently stir.Cool to room temperature at room temperature stirs 12h then, obtains beta-cyclodextrin earthworm fibrinolysin modifying enzyme liquid enzyme formulation, and vigor reclaims 86%.After the freeze-drying, powdered, water soluble, the aqueous solution are colourless, transparent, have proteinic feature.

Claims (1)

1. the preparation method of beta cyclodextrin carthworm plasmin modification enzyme, it is characterized in that earthworm fibrinolysin is added in the beta-cyclodextrin solution, evenly, in 30～50 ℃ of waters bath with thermostatic control, intermittently stirred 40-70 minute, stirred 8-14 hour down at 32 ℃, reacted solution is beta cyclodextrin carthworm plasmin modification enzyme again;

The ratio of beta-cyclodextrin and earthworm fibrinolysin is 1: 4 (volume ratio) in the reaction;

The reaction density of beta-cyclodextrin is 0.5-9mg/ml;

The reaction density of earthworm fibrinolysin is 300-600U/ml.