CN1026898C - Process for producing lysozyme - Google Patents
Process for producing lysozyme Download PDFInfo
- Publication number
- CN1026898C CN1026898C CN 89108802 CN89108802A CN1026898C CN 1026898 C CN1026898 C CN 1026898C CN 89108802 CN89108802 CN 89108802 CN 89108802 A CN89108802 A CN 89108802A CN 1026898 C CN1026898 C CN 1026898C
- Authority
- CN
- China
- Prior art keywords
- pupa
- diacetylmuramidase
- cellulose
- component
- lysozyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a method for using tussah pupa as the raw material to produce a lysozyme. The tussah pupa has plentiful lysozymes, and the enzyme activity is enhanced by more than 10 times after artificial induction. Pupa blood is collected, heated, subjected to hromatography by a DEAE-cellulose and chitin enclosed cellulose column, concentrated in vacuum after being eluted, dialyzed and dried in vacuum to obtain the lysozyme, and the recovery rate of the lysozyme is high. The relevant cellulose can be regenerated and reused. The technology of the method can be linked with other technologies (such as the extraction of pupa protein, etc.) of the comprehensive utilization of the tussah pupa.
Description
The present invention relates to the production method of N,O-Diacetylmuramidase, improvements over the prior art, and N,O-Diacetylmuramidase is the enzyme of special dissolution of bacteria.At present, it is raw material that Ovum Gallus domesticus album is adopted in the preparation of N,O-Diacetylmuramidase, adopts salting-out process or alcohol precipitation to remove Deproteinization in process of production, or with resin chromatography purification technique.Therefore, increased the desalination operation and in elution process with the egg white Macrodilution, expend the energy when making drying.
The present invention is raw material with the cocoon chrysalis, and silkworm chrysalis improves the vigor of N,O-Diacetylmuramidase through the artificial induction; Heating removes albumen, and DEAE-Mierocrystalline cellulose chromatography separates, the Mierocrystalline cellulose chromatography purifying of chitin parcel, and vacuum-drying forms.N,O-Diacetylmuramidase yield height, technology is simple, and is with low cost, and the Mierocrystalline cellulose chromatography can semi-automation be regulated and control the renewable use of Mierocrystalline cellulose.Cocoon chrysalis also can be produced the product that economic worth is arranged in a large number except that extracting N,O-Diacetylmuramidase, as pupal fat, pupa albumen etc., its technology overlaps.
Production Flow Chart such as block diagram 1.
One. the collection of cocoon chrysalis:, supply to use behind dead, the broken pupal cell of rejecting with the silkworm chrysalis behind the living pupa and cocoon-reeling; Cut the cocoon pupa and should keep pupal cell intact, injured or damage broken person discard.
Two, artificial induction: induction method has three kinds.
1. inject intestinal bacteria: bacterial classification is respectively Ecoli K
12D
31Or HB
101, dosage is 10
8Bacterium/ml, every pupa inject 50 μ l, and place more than 72 hours for using at 25 ℃ the injection back.
2. warm air is induced: the pupa of will living is put in 50 ℃～55 ℃ hot air boxs and was handled 30～60 minutes, handles the back and places 48～72 hours for using at 25 ℃.
3. ultrasonic wave is induced: the pupa of will living immerses in the flowing water, handles 20,000 hertz of frequencies, 150 watts of power, time 5-10 minute with ultrasonic generator.Handle the back 25 ℃ rev/mins centrifugal, collect pupa blood, remaining pupal cell is for comprehensive utilization.
Four. pupa blood removes albumen: adds phenylthiourea 0.02%～0.05%, mix fast in the water-bath to stir and be heated to 60 ℃, and speed is chilled to room temperature, the centrifugal albumen precipitation of removing, collecting clear liquid is the N,O-Diacetylmuramidase component.Remaining pupa blood residue is for comprehensive utilization.
Five. adjust pH: the N,O-Diacetylmuramidase component adds equal-volume 0.1M, and the pH8.0 phosphate buffered saline buffer carries out column chromatography after stirring.
Six .DEAE-cellulose chromatographies: DEAE-cellulose ion-exchange chromatography, activation routinely, dress post.Column diameter be 1: 10～12 at high proportion, make in the post earlier fully saturatedly in the post with ealkaline buffer, can select 0.1M for use, pH8.0 phosphoric acid salt will above-mentioned N,O-Diacetylmuramidase component injects in the post and do ion-exchange, control flow velocity 2ml/cm
2Minute, use 0.1M, pH8.0 phosphate buffered saline buffer wash-out N,O-Diacetylmuramidase component.Regeneration was for using after the DEAE-cellulose column reclaimed the pupa haemproteins.
Seven. chitin parcel cellulose chromatography: adorn post behind the chitin parcel cellulose activation, use earlier 0.1M, the pH8.0 phosphate buffered saline buffer is with after fully saturated in the post, be washed till slight alkalinity with deionized water again, make affinity chromatography, control flow velocity 2ml/cm in the N,O-Diacetylmuramidase component injection chitin parcel cellulose column with wash-out behind the DEAE-cellulose chromatography
2Minute, treat abundant affine absorption after, use 0.1M, pH2～2.5 acetate buffer wash-out N,O-Diacetylmuramidase components, chitin parcel is cellulose regenerated for using.
Eight. vacuum concentration, dialysis and drying: collect the N,O-Diacetylmuramidase component, at 60 ℃, 600mmHg is concentrated to thick with the film under vacuum thickener down, move in the dialysis tubing, remove freshen 4 ℃ of following flowing water dialysis, content is at 60 ℃, vacuum-drying under the 600mmHg is the N,O-Diacetylmuramidase goods.
The cocoon chrysalis 1kg that lives makes raw material, with intestinal bacteria K
12D
31Be inoculated in liquid LB substratum (NaCl10g adds water to 1 liter for peptone 10g, yeast extract 5g, pH7.2) in, 37 ℃ of shaking culture are crossed liquid, reach 10
8The level of bacterium/ml, every pupa are injected 50 μ l, are positioned over 25 ℃, 72 hours.
Silkworm chrysalis puncture back centrifuging and taking pupa blood, 3000 rev/mins of centrifugal basket driers centrifugal 10 minutes, are collected the about 150ml of pupa blood.Add 0.02% phenylthiourea, be heated to 60 ℃ in stirred in water bath, speed is chilled to room temperature, centrifugal 3000 rev/mins.Clear liquid was the N,O-Diacetylmuramidase component to remove Deproteinization in 10 minutes.
Add equal-volume 0.1M in the N,O-Diacetylmuramidase component, the pH8.0 phosphate buffered saline buffer is used the DEAE-cellulose chromatography, collects effusive N,O-Diacetylmuramidase component, and about 250ml makes affinity chromatography with chitin parcel Mierocrystalline cellulose, uses 0.1M in the post, and the pH8.0 phosphate buffered saline buffer is saturated.Behind the adsorption chromatography, use 0.1M, pH2.0～2.5 acetate buffer wash-outs, the N,O-Diacetylmuramidase component of collecting wash-out, about 150ml.
At 60 ℃, the 600mmHg vacuum concentration desalts with the semi-permeable membranes dialysis to thick with the N,O-Diacetylmuramidase component, and content is drained under the 600mmHg at 60 ℃, is finished product, and about 100mg, enzyme activity reach 20000 units/mg protein level.
Example two
The cocoon chrysalis 1kg that lives makes raw material, silkworm chrysalis is put in 52 ℃ of baking ovens handled 30 minutes, puts 25 ℃, 48 hours.
Silkworm chrysalis puncture back centrifuging and taking pupa blood is collected the about 150ml of pupa blood, adds 0.02% phenylthiourea, is heated with stirring to 60 ℃ in water-soluble, and speed is chilled to room temperature, and centrifugal 3000 rev/mins, clear liquid was the N,O-Diacetylmuramidase component to remove Deproteinization in 10 minutes.
Add equal-volume 0.1M in the N,O-Diacetylmuramidase component, the pH8.0 phosphate buffered saline buffer is used the DEAE-cellulose ion-exchange column chromatography, collects effusive N,O-Diacetylmuramidase component, about 250ml.
Commercially available chitin 50g fully soaks with 40%NaOH liquid, smashs to pieces, and 37 ℃ of dippings 3 hours, water flushing, suction filtration is converted into chitin deacetylase to remove surplus alkali, to make.Under alkalescence (pH10) condition, add the 100g Mierocrystalline cellulose, fully mix and stir, transfer to acidity with glacial acetic acid then, make the chitin precipitation, spent acid is removed in washing, use 0.1M, the pH8.0 phosphate buffered saline buffer soaks, and behind the dress post affinity chromatography is wherein carried out in the N,O-Diacetylmuramidase component injection of DEAE-Mierocrystalline cellulose wash-out, use 0.1MpH2～2.5 acetate buffer wash-out N,O-Diacetylmuramidase components at last, collect 150ml approximately.
At 60 ℃, the 600mmHg vacuum concentration desalts with the semi-permeable membranes dialysis to thick with the N,O-Diacetylmuramidase component, and content is drained under the 600mmHg at 60 ℃, is finished product, gets 85mg approximately, and enzyme activity reaches 18000 units/mg protein level.
Reference
1.Emel′yanenko，P.A.，Stayukova，L.G.，1983，Extraction of lysozyme from colostrum.USSRpatent，SU 1041937Al，CA，99（25）208831y.
2.Eisai Co Ltd，1986，Production of lysozyme，Japan Patent，JP 83201986A2，CA，100（15）117182k.
3.Hasegawa，Mineo，Ozaki，Kitao，1983，Apparatus for collecting lysozyme from egg white byabsorption.South Africa Patent ZA 8208806A，CA，100（15）117181j.
Claims (9)
1, process for producing lysozyme is characterized in that:
(1) with the cocoon chrysalis is raw material;
(2) silkworm chrysalis improves the vigor of enzyme in the pupa blood through the artificial induction;
(3) collect pupa blood, the heating of pupa blood removes albumen and gets the N,O-Diacetylmuramidase component;
(4) the N,O-Diacetylmuramidase component gets crude zyme preparation through DEAE-cellulose ion exchange column under alkaline condition;
(5) crude zyme preparation is through chitin parcel Mierocrystalline cellulose affinity chromatography column purification, and zymin vacuum concentration drying obtains product behind the purifying.
2,, it is characterized in that said artificial induction's silkworm chrysalis is selected from following three kinds of methods according to the said process for producing lysozyme of claim 1:
(1) the injection intestinal bacteria are induced: bacterial classification is respectively EcoliK
12D
31Or HB
101, dosage is 10
8Individual bacterium/ml, every pupa injection 50ml, the injection back is placed more than 72 hours at 25 ℃;
(2) warm air is induced; The pupa of will living is put in 50 ℃ of-55 ℃ of hot air boxs and was handled 30-60 minute, handles the back at 25 ℃, places 48-72 hour;
(3) ultrasonic wave is induced: the pupa of will living immerses in the mobile water, is 20,000 hertz with frequency, and 150 watts of ultrasonic generators of power were handled 5-10 minute, handles the back at 25 ℃, places 72-96 hour.
3, according to the said process for producing lysozyme of claim 1, it is characterized in that in the pupa blood adding phenylthiourea 0.02%-0.05%, and in water-bath, be heated to 60 ℃, make albumen precipitation, after centrifugal the N,O-Diacetylmuramidase component.
4,, it is characterized in that in centrifugal N,O-Diacetylmuramidase component after removing albumen, adding isopyknic 0.1M, the phosphoric acid salt of pH8.0 according to the said process for producing lysozyme of claim 3.
5,, it is characterized in that the N,O-Diacetylmuramidase component does ion-exchange through DEAE-cellulose ion exchange column according to claim 1,4 said process for producing lysozyme.
6, according to the said process for producing lysozyme of claim 5, it is characterized in that DETE-cellulose column 0.1M, pH8.0 phosphoric acid salt institute is saturated.
7,, it is characterized in that crude zyme preparation is through chitin parcel cellulose column purifying according to the said process for producing lysozyme of claim 1.
8, according to the said process for producing lysozyme of claim 7, it is characterized in that chitin parcel cellulose column 0.1M, pH8.0 phosphoric acid salt institute is saturated.
9, according to the said process for producing lysozyme of claim 1, it is characterized in that the N,O-Diacetylmuramidase component behind chitin parcel cellulose column purifying, at 60 ℃, vacuum-drying gets product under the 600mmHg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 89108802 CN1026898C (en) | 1989-11-22 | 1989-11-22 | Process for producing lysozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 89108802 CN1026898C (en) | 1989-11-22 | 1989-11-22 | Process for producing lysozyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1043740A CN1043740A (en) | 1990-07-11 |
| CN1026898C true CN1026898C (en) | 1994-12-07 |
Family
ID=4857731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 89108802 Expired - Fee Related CN1026898C (en) | 1989-11-22 | 1989-11-22 | Process for producing lysozyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1026898C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064082C (en) * | 1994-06-14 | 2001-04-04 | 北京市联合大学 | Bioenzyme used for dismounting pictures and the method therefor |
| CN1108376C (en) * | 2000-05-17 | 2003-05-14 | 华南农业大学 | Process for preparing gel medium of lysozyme affinity chromatography |
| CA2859164A1 (en) * | 2011-12-21 | 2013-06-27 | Colgate-Palmolive Company | Oral care compositions |
-
1989
- 1989-11-22 CN CN 89108802 patent/CN1026898C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1043740A (en) | 1990-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111235203B (en) | Production method of clam active peptide | |
| CN101307348B (en) | Method for preparing undenatured collagen from fish scale of fresh water fish | |
| CN101544999B (en) | Method for producing and purifying high purity and low molecular weight sodium heparin | |
| CN1915437A (en) | Technique for preparing sponge produced from collagen | |
| CN112062834B (en) | Deep sea fish skin collagen peptide and extraction and preparation method thereof | |
| CN107266609B (en) | Method for extracting pomegranate peel residue pectin by electrolytic water | |
| CN101147611A (en) | Method for processing clearing type pineapple juice | |
| CN103361393A (en) | Preparation method of andrias oligopeptide | |
| CN113234181B (en) | Preparation method of chondroitin sulfate | |
| CN1137998C (en) | Zymolysis process of producing heparin sodium | |
| CN1026898C (en) | Process for producing lysozyme | |
| CN113563459A (en) | Protein extraction process in production process of collagen peptide | |
| CN1342714A (en) | Process for extracting heparin sodium by integrated biologic method | |
| CN106317259A (en) | Joint production technology for extracting heparin sodium and protein peptide powder from pork lung | |
| CN1654670A (en) | Preparation method of chondroitin sulfate | |
| CN117247445A (en) | Purification method for collagen peptide production | |
| CN114807284A (en) | Preparation method of high-purity small-molecule fish skin collagen peptide | |
| CN110105385A (en) | A kind of sea cucumber waste liquid comprehensive utilzation technique | |
| CN109480082A (en) | A kind of method that shellfish feeds bait and its reduces heavy metal in body of shellfish | |
| CN108179167A (en) | A kind of industrialized producing technology of wood frog body protein oligopeptide | |
| RU2088112C1 (en) | Method of preparing the primary beet pectin | |
| CN115717136A (en) | Extraction and production process of pepsin | |
| CN104450841A (en) | Method for producing chondroitin sulfate and co-producing hydrolyzed collagen from fish cartilage | |
| CN103333939A (en) | Method for preparing gelatin from bone collagen fibers by alkali-enzyme composite degradation | |
| CN1039844A (en) | From silkworm chrysalis, extract the method for amino acid composite liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |