CN1276426A - Radish lysozyme and its preparing process - Google Patents

Abstract

A process for extracting radish lysozyme using radish leaves as raw material includes homogenizing, leaching, filtering, affinity adsorption, charging in column, washing, affinity eluting, collecting lysozyme liquid, dialysis, concentrating and freeze drying. It features high enzyme activity up to 40000-70000 U/mg, broad spectrum and high kill action on G bacteria. The active center of protein and enzyme containing Trp, His, Glu and Asp residues.

Description

RAPHANUS SATIVUA LYSOZYME and preparation method thereof

The present invention relates to the preparation of N,O-Diacetylmuramidase, relate in particular to the preparation of RAPHANUS SATIVUA LYSOZYME.

N,O-Diacetylmuramidase generally is used for the sterilization, anticorrosion and fresh-keeping of food service industry, break in fermentation industry and bionic thalline, in pharmaceutically clinical treatment and diagnosis, the overseas utilization N,O-Diacetylmuramidase is made medicine, be used for the treatment of hemorrhage, blood urine, bloody sputum, chronic nasal sinus and diseases such as nasal sinusitis, preventing dental caries, and the clinical diagnosis of disease such as leukemia, pulmonary tuberculosis, renal failure.The application of N,O-Diacetylmuramidase more and more widely, domestic N,O-Diacetylmuramidase production can be satisfied the domestic market needs far away, part N,O-Diacetylmuramidase and preparation thereof need import.

Present commercially available N,O-Diacetylmuramidase, main is feedstock production with the Ovum Gallus domesticus album, and its technology of preparing complexity, raw materials cost height, product enzyme activity are low, and the egg source is limited, and contends over raw materials with food.The N,O-Diacetylmuramidase of producing with Ovum Gallus domesticus album is only to G ⁺Bacterium has killing action, and to G ^-Bacterium, fungi are inoperative.

At present no matter pharmaceutically, or on the crop protection to make the control fungi-medicine with N,O-Diacetylmuramidase few.As paddy rice 100 multiple diseases, wherein be more than 90% by fungus-caused.Therefore, along with development of high-tech in food, medicine and the plant protection, seek new enzyme source and must go with new enzyme thing.

The object of the invention is to seek a kind of bacteriolyze wide spectrum, good stability, preparation is easy, effect reaches applied range well N,O-Diacetylmuramidase, to adapt to needs of economic development.

We once generally investigated the lysozyme content of more than 50 section's 120 various plants in South China in the period of 1989-1991, found that lysozyme content is the highest in the radish.Thereby found RAPHANUS SATIVUA LYSOZYME (Chinese RAPHANUS SATIVUA LYSOZYME by name; The Chinese phonetic alphabet is Luo bo rong junmei; English Raphanus sativua lysozyme by name).

This enzyme does not at home and abroad appear in the newspapers and the approved products listing as yet.The property feature of RAPHANUS SATIVUA LYSOZYME

The physical property certified products is pale powder, and is nontoxic, tasteless, soluble in water, and aqueous solution crystalline form is a rectangle.Have protein properties, it is pure to reach electrophoresis (SDS-polyacrylamide electrophoresis).The moisture of solid zymin (conventional method)＜10%, ash content＜7%.Zymologic property

The relative molecular mass of enzyme is 11.3KD.The molecular composition of enzyme is made up of with the peptide chain that peptide bond connects 108 amino acid whose residues, is single composition, monomeric enzyme.

The zymoprotein iso-electric point is 9.0 (pI=9.0).

The ph stability of enzyme and effect optimal pH are respectively pH3.5～7.5 and pH5.8～6.6.

Thermostability and effect optimum temperuture are respectively 25～65 ℃ and 50～55 ℃.

Storage stability: the solution enzyme is 200 days transformation period at room temperature.The solid zymin year rate of disintegration is 9.8%.

Km (micrococcus lysodeikticus) 40 μ g/mL.

Activation energy is 2.92 * 10 ⁴J/mol.

The active centre of enzyme is Trp, His, Glu, Asp residue.

Activity inhibitor: Gly formicester (1000mmol/L), histamine (0.2mol/L), N-acetylglucosamine (0.9mol/L), Ca ²⁺(100mmol/L), Fe ²⁺(0.05mmol/L).

Enzyme activity (tiring) 4-7 ten thousand U/mg protein.

RAPHANUS SATIVUA LYSOZYME has 108 amino acid whose residues, and Trp, His, Glu and Asp residue are contained in the active centre of enzyme, and these are different with other N,O-Diacetylmuramidase, and wherein the Trp residue may participate in the formation of " enzyme-substrate complex "; Through " induced-fit " and " substrate distortion ", His, Glu and Asp residue when having water to exist, may participate in electronics, proton in the catalytic process transmission and to substrate enforcement katalysis.RAPHANUS SATIVUA LYSOZYME amino acid composition sees Table 1

Table 1 RAPHANUS SATIVUA LYSOZYME amino acid is formed

Amino acid molecular is counted the amino acid molecular number

Lys 3 Cys 2

His 1 Val 5

Ary 6 Met 1

Asp 13 Ile 5

Thr 6 Leu 6

Ser 9 Tyr 3

Glu 9 Trp 2

Pro 6 Phe 5

Gly 17 Ala 10

Add up to 108 RAPHANUS SATIVUA LYSOZYME activity determination methods

With the micrococcus lysodeikticus is substrate, adds 0.066mol/L phosphoric acid buffer (pH6.2) and grinds to be made into and select suspension liquid (A _450nmBe about 0.7).Draw 2.5mL substrate suspension liquid down at 30 ℃, add the rapid mixing of 0.1mL enzyme liquid, measure the optical density(OD) drop-out value down, in being reacted into linear scope, calculate enzymic activity in the 450nm wavelength.Under these conditions, the enzyme amount with enzyme minute 0.001 optical density value of decline is an activity unit (U).Correct photoabsorption to 0 point with distilled water, and calculate enzyme activity by following formula:

In the formula, Δ A _450nm=Δ A _450nmSample/minute-Δ _A450nmBlank/minute, blank with damping fluid replacement enzyme liquid.The measuring method of protein content

Press the Bradford method, with the bovine serum albumin is standard protein, be made into the standardized solution of protein concentration from 0-900 μ g/mL, above-mentioned each concentration standard protein solution of joining is drawn 0.1mL respectively to add in the test tube, respectively add 5mL Coomassie brilliant blue G250 solution again, after shaking up, under the 595nm wavelength, with protein concentration is 0 to serve as blank pipe zeroing, measures the A of each pipe _595nm, be X-coordinate with the protein concentration, A _595nmFor ordinate zou is made typical curve.A with same method working sample solution _595nm, can check in the protein concentration of sample solution from the typical curve.Amino acid whose mensuration amino acid is formed the employing automatic analyzer for amino acids and is measured.RAPHANUS SATIVUA LYSOZYME target level of product quality enzyme activity reclaims 61-65%, and purifying multiple 77-310 times, enzyme is than 4200-71100U/mg protein alive.The preparation of RAPHANUS SATIVUA LYSOZYME (one) is a raw material with fresh Folium Raphani or piece root, raw material is clean, airing.(2) raw material homogenate: in raw material, add 0.5-1 times of volume material amount through 4 ℃ of precoolings

0.066mol/L, pH.6.2 phosphoric acid buffer, homogenate.(3) filter, collect supernatant liquor: filter with nylon cloth, 4000 rev/mins of filtrates are centrifugal

(room temperature) 15 minutes collects supernatant liquor, detects enzyme activity, the enzyme ratio of supernatant liquor and lives

Power and protein content.(4) affine absorption: in supernatant liquor, add the affinity chromatography medium of 1.5 times of volumes, in 4

℃ whip attachment 1 hour.It number is 00114272.0 that chromatography media is used Chinese patent application

Method be prepared.(5) dress post and washing: will adsorb the medium upper prop of enzyme, and use 5mmol/L NaHCO ₃Molten

Liquid wherein contains 0.2mol/L NaCl and washs to A _280nmLess than 0.1.(6) affinity elution: with 0.1mol/L HAC wash-out, collect the enzymic activity peak, be pure enzyme liquid,

Detect volume enzyme activity, specific activity of enzyme and the protein content of collecting enzyme liquid, and calculate

Enzyme activity reclaims.(7) dialysis, concentrated and freeze-drying: 1. with the elutriant 0.066mol/L of above-mentioned collection, the pH.6.2 phosphoric acid buffer was dialysed more than 12 hours, and ultrafiltration and concentration adds protective material and is lyophilized into lyophilized powder to original volume 1/3.

In the film washing liquid of above-mentioned collection, add protective material and sanitas, make the 69091V/mg protein enzyme solution.

Protective material and sanitas are: NaF, glycerine, ethanol, phenylformic acid, protective material, sanitas, enzyme liquid ratio of mixture (percent by volume) are: NaF (0.01mol/L) is: 0.125%, and glycerine is: 20%, ethanol is: 5%, phenylformic acid is: 0.05%, enzyme liquid is: 74.825%.Or glycerine is: 20%, phenylformic acid is: 0.05%, enzyme liquid is: 79.95% (percent by volume).Advantage of the present invention: one. the present invention is a raw material with root, the leaf of radish, and raw material sources are easy, inexpensive, and the development and use that adapt to agricultural byproducts are not contended over raw materials with food service industry again.Two. explained hereafter facility investment of the present invention is few, and technology is simple.Three. this product relative molecular weight is little, is the basic protein that a polypeptide chain is formed, and is single composition, monomeric enzyme, and effect need not participate in by cofactor.The product stability height.Trp, His, Glu and Asp residue are contained in the product activity center, and these are different with other N,O-Diacetylmuramidase.Four. fungicidal spectrum is wide, not only to G ⁺Bacterium has killing action, to G ^-Bacterium has killing action equally, and fungi is had fabulous bacteriostatic action, and insect is also had certain lethal effect.These are also different with other N,O-Diacetylmuramidase.Five. enzyme activity is up to 4-7 ten thousand U/mg protein.

Example:

Get the fresh turnip leaves of 174g, clean, shred, add the 0.066mol/L of 200mL through 4 ℃ of precoolings, the pH.6.2 phosphoric acid buffer, homogenate is filtered with nylon cloth, filtrate 4000 rev/mins centrifugal (room temperature) 15 minutes, supernatant liquor 210mL, what add 1.5 times of volumes uses 5mmol/L NaHCO ₃Pretreated affinity chromatography medium, in 4 ℃ of whip attachment 45 minutes, dress post (30cm * 20cm), use 5mmol/L NaHCO ₃Solution wherein contains 0.2mol/L NaCl, washs to A _280nmLess than 0.1, use 0.1mol/L HAC wash-out again, Fraction Collector is collected, flow velocity is 1 pipe/5 minutes, the 5mL/ pipe is collected enzymic activity and is partly measured, and the result is as follows: step cumulative volume total protein total activity enzyme reclaims productive rate than purifying multiple vigor alive

(mL) (mg) (U) (U/mg) (*) (%) slightly put forward 210 220 120,120 545 1 100 1265 affinity chromatographys 50 1.85 78,000 42,162 77.4 65 10.6

With 0.066mol/L, the dialysis of PH6.2 phosphoric acid buffer 12 hours, ultrafiltration and concentration was got 10mL and is added 2.5mL glycerine, 7uL phenylformic acid to 15mL with above-mentioned 50mL elutriant, the Freeze Drying Equipment freeze-drying, 0.8671mg lyophilized powder, specific activity of enzyme are 40285V/mg albumen.

Getting 5mL in addition adds 8uLNaF (12.5mmol/L), 1.3mL glycerine, 0.3mL ethanol, 3uL phenylformic acid, promptly can be made into the 69091V/mg protein enzyme solution.

Claims (2)

1. RAPHANUS SATIVUA LYSOZYME, the physical property that it is characterized in that this enzyme is that pale powder is nontoxic, and is tasteless, soluble in water, aqueous solution crystalline form is a rectangle, has protein properties, it is pure to reach electrophoresis (SDS-polyacrylamide electrophoresis), the moisture of solid zymin (conventional method)＜10%, ash content＜7%; Its zymologic property:

The relative molecular mass of enzyme is 11.3KD, and the molecular composition of enzyme is made up of with the peptide chain that peptide bond connects 108 amino acid whose residues, is single composition, monomeric enzyme;

The zymoprotein iso-electric point is 9.0 (pI=9.0),

The ph stability of enzyme and effect optimal pH are respectively pH3.5～7.5 and pH5.8～6.6;

Thermostability and effect optimum temperuture are respectively 25～65 ℃ and 50～55 ℃;

Storage stability: the solution enzyme is 200 days transformation period at room temperature, and the solid zymin year rate of disintegration is 9.8%;

Km (micrococcus lysodeikticus) 40 μ g/mL;

Activation energy is 2.92 * 10 ⁴J/mol;

The active centre of enzyme is Trp, His, Glu, Asp residue;

Activity inhibitor: Gly formicester (1000mmol/L), histamine (0.2mol/L), N-acetylglucosamine (0.9mol/L), Ca ²⁺(100mmol/L), Fe ^2-(0.05mmol/L);

Enzyme activity (tiring) 4-7 ten thousand U/mg protein;

2. the preparation method of RAPHANUS SATIVUA LYSOZYME, it is characterized in that: (one) is raw material with fresh Folium Raphani or piece root, with raw material clean, airing: the homogenate of (two) raw material: in raw material, add 0.5-1 times of volume material amount through 4 ℃ of precoolings

0.066mol/L, pH.6.2 phosphoric acid buffer, homogenate; (3) filter, collect supernatant liquor: filter with nylon cloth, 4000 rev/mins of filtrates are centrifugal

(room temperature) 15 minutes collects supernatant liquor; (4) affine absorption: in the affinity chromatography medium of 1.5 times of volumes of supernatant liquor adding, in 4 ℃

Whip attachment 1 hour; (5) dress post and washing: will adsorb the medium upper prop of enzyme, and use 5mmol/L NaHCO ₃Molten

Liquid wherein contains 0.2mol/L NaCl and washs to A _280nmLess than 0.1; (6) affinity elution: use the 0.1mol/LHAC wash-out, collect the enzymic activity peak, be pure enzyme liquid; (7) dialysis, concentrated and freeze-drying:

1. with the elutriant 0.066mol/L of above-mentioned collection, the pH.6.2 phosphoric acid buffer was dialysed more than 12 hours, and ultrafiltration and concentration adds protective material and is lyophilized into lyophilized powder to original volume 1/3;

2. with adding protective material and sanitas in the elutriant of above-mentioned collection, make enzyme solution;