CN106906197A - The method that zymotechnique prepares lysozyme - Google Patents
The method that zymotechnique prepares lysozyme Download PDFInfo
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- CN106906197A CN106906197A CN201710277208.1A CN201710277208A CN106906197A CN 106906197 A CN106906197 A CN 106906197A CN 201710277208 A CN201710277208 A CN 201710277208A CN 106906197 A CN106906197 A CN 106906197A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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Abstract
The present invention relates to the method that zymotechnique prepares lysozyme, comprise the following steps：Fluid nutrient medium is taken to be placed in triangular flask, thalline seed is accessed by the inoculum concentration of 1 10%v/v, shake flask fermentation is carried out, fermentation condition is 20 40 DEG C, 150 300r/min, after fermented and cultured 24h, by zymotic fluid centrifugation removal thalline, the purifying of supernatant ammonium sulfate salting-out process is taken, obtain final product lysozyme, the fluid nutrient medium includes glucose, peptone and surfactant, and the surfactant is that concentration ratio is 1:2‑2:1 fatty acid distribution of coconut oil diacetayl amide and the mixture of Tweens surfactant.The fermentation process obtains the lysozyme of greater activity by the species and concentration of surfactant in Optimal Medium.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to the method that zymotechnique prepares lysozyme.
Background technology
Lysozyme (Lysozyme), also known as cytohydrolist, can in specific manner act on bacteria cell wall peptide glycan point
Son, can be broken β between -acetylmuramic acid and acetyl glucosamine ammonia-Isosorbide-5-Nitrae glycosidic bond, make bacteria cell wall become it is loose and
Reach the effect of dissolution of bacteria.Lysozyme is distributed widely in high animal vegetable tissue and its secretion, protozoan, insect, plant
In thing and various microorganisms.
Lysozyme as one of the nonspecific immune factors in human normal body fluid and tissue, with multiple pharmacological effect,
The reaction of body panimmunity is participated in, there is antibacterial, antiviral effect, in body normal defense function and nonspecific immunity, tool
There is the important function for keeping organism physiology balance.It can improve and strengthen macrophage phagocytic and digestive function, and activation is white thin
Born of the same parents' phagocytic function, and the Neuroleptic Leukocytopenia caused by cytostatics can be improved, so as to strengthen the resistance of body.Lysozyme energy
Direct hydrolysis gram-positive bacteria, under secretory immunoglobulin A, the participation of complement, moreover it is possible to hydrolyze Gram-negative bacteria such as
Escherichia coli etc..Equally there is bacteriolysis to drug tolerant bacteria, and with spies evident in efficacy and to human body Small side effects
Point, thus be a kind of ideal medicinal enzyme.Lysozyme can with negatively charged virus protein direct effect, with DNA, RNA,
Apoprotein forms double salt, makes virally inactivated.Lysozyme can participate in mucopolysaccharide metabolism, within coordinating as enzyme antimicrobial
While clothes and externally applied drug, strength antiinflammation can be played, it can also be combined with the acidic materials of various induction inflammation loses it
Live, and the curative effect of antibiotic and other medicines can be strengthened, improve the mucopolysaccharide metabolism of periplast, so as to reach anti-inflammatory, repair
The purpose of overlying tissue.
Lysozyme is the necessary antibacterial protein of infants growth and development, putrefactive microorganisms in can suppressing enteron aisle in infants
Existence, while directly facilitate baby intestinal bacterium Bifidobacterium propagation, so as to promote baby's stomach enteral MC to form micro-
Thin curdled milk, is conducive to baby to digest and assimilate.Lysozyme can also promote the normalization of artificially feeding infant enteric bacteria group；Energy
It is enough to strengthen resistance of the factor to increase to infecting of being prevented epidemic in vivo to properdin, gamma globulin etc., especially to Premature
Youngster, the effects such as having prevention weight loss, prevention chylopoietic disease, promote body weight, so lysozyme is baby food, Ying Erpei
The good additive of square milk powder etc..In recent years, China's liquid diary product have developed rapidly, and lysozyme can be applied to be played in dairy products
Corrosion-resistant effect, is particularly suited for pasteurize milk, effectively extends storage life.Because lysozyme has certain heat-resisting quantity
Can, ultrahigh-temperature instant sterilization milk is equally applicable to, additive capacity is 300-600ppm.Its method is addition, superhigh temperature before packaging
Instantaneous sterilization milk can also be added before sterilization.There is the lysozyme of 13mg/100mL in fresh milk, content is 40mg/ in human milk
ML, adds a certain amount of lysozyme in fresh milk or milk powder, can play antisepsis.
Lysozyme is essential toolenzyme in genetic engineering, cell engineering, Fermentation Engineering.Abroad, lysozyme is more
For the extraction of bacterium substance in vivo.As long as in appropriate buffer solution, using bacteriolyze ferment treatment to sensitive thalline suspension, in conjunction with
Cell-free extract is just can obtain using means such as ultrasonic wave, refrigerated centrifuges, is further refined and be can obtain required thalline material
Such as protein, nucleic acid, enzyme and active peptides, therefore the development of biological industry will be growing day by day to the demand of lysozyme formulation.
At present, it is egg white lysozyme to be commercially available most lysozymes.With the improvement of people ' s living standards, to bacteriolyze
The demand of enzyme is increasing, depends merely on and lysozyme is extracted from egg, it is difficult to solve the demand and supply contraction.However, using micro-
Biological production lysozyme, cost is relatively low, and material-saving, environmental pollution is smaller, easily accomplishes scale production, thus exploitation is latent
Power is huge.Further, since microbe-derived lysozyme has obvious Bacteriolytic specificity, the difference according to its substrate is micro- to study
Biological cell wall construction, lysozyme is again a kind of highly useful biological tool enzyme.Most microbic muramidases are to golden yellow Portugal
Grape coccus, Gram-negative bacteria and fungi have a dissolution, and egg white lysozyme to above bacterial strain without obvious bacteriolysis, in recent years
Come, the lysozyme to multiple-microorganism source extensively and in depth study both at home and abroad.
Although the source of lysozyme is different, the chemical property of enzyme is widely different, the production optimum condition base of zymogenic bacteria
This is similar.In nutrient media components, better than inorganic nitrogen-sourced, in the medium without organic nitrogen source, meeting is direct for general organic nitrogen source
Have influence on the yield of lysozyme.Such as staphylococcus aureus strains, indispensable yeast extract, otherwise enzyme activity are just remarkably decreased.
And for example bacillus subtilis strain, its enzyme activity can be improved with meat medicinal extract.In addition, adding carbohydrate material in the medium, to enzyme activity
Property influence it is very big.Have been reported that and show, when staphylococcus aureus strains are cultivated, β-phosphoglycerol is replaced with glucose as carbon
Source, when enzyme activity reaches top, its activity reduces very fast.In culture bacillus subtilis strain, glucose has suppression
The effect of producing enzyme.When styreptomyces globispotus strain bacterial strain is cultivated, most suitable carbon source is dextrin and soluble starch.
Prior art prepares the culture medium research that bacteriolyze enzyme process uses and is limited primarily to nitrogen source, carbon for zymotechnique at present
How the selection of the compositions such as source, species and concentration for surfactant influence the activity research of lysozyme less and existing
Culture medium fermentation produces lysozyme activity limited, and causing the method for such lysozyme to be applied in the big production of actual industrialization has
Limit.
The content of the invention
The present invention obtains high living in view of the shortcomings of the prior art, providing a kind of culture medium prescription by optimization for fermentation technology
The method of property lysozyme.
The technical scheme is that：The method that zymotechnique prepares lysozyme, comprises the following steps：Take fluid nutrient medium
It is placed in triangular flask, thalline seed is accessed by the inoculum concentration of 1-10%v/v, carry out shake flask fermentation, fermentation condition is 20-40 DEG C,
150-300r/min, after fermented and cultured 24h, by zymotic fluid centrifugation removal thalline, takes the purifying of supernatant ammonium sulfate salting-out process, i.e.,
Obtain lysozyme；The fluid nutrient medium includes glucose, peptone and surfactant, and the surfactant is for concentration ratio
1:2-2:1 fatty acid distribution of coconut oil diacetayl amide and the mixture of Tweens surfactant.
Preferably, the concentration of the glucose is 10g/L, and the concentration of the peptone is 5g/L.
Preferably, the concentration range of the fatty acid distribution of coconut oil diacetayl amide and Tweens surfactant is 0.5g/L-
2g/L。
It is furthermore preferred that the concentration of the fatty acid distribution of coconut oil diacetayl amide be 1g/L, the Tweens surfactant it is dense
It is 0.5g/L to spend.
Preferably, the Tweens surfactant is Tween 80.
Preferably, the thalline is selected from streptomyces griseus, MRSE, bacillus subtilis or molten bacillus.
It is furthermore preferred that the thalline is selected from streptomyces griseus S-35, MRSE K-6-W1, bacillus subtilis K-
77 or molten bacillus S2-6b.
Preferably, the fermentation condition is 30 DEG C, 200r/min.
Preferably, the inoculum concentration is fluid nutrient medium 4%v/v.
Beneficial effects of the present invention：
The composition that inventor passes through screening and culturing medium, determines the prescription composition of glucose, peptone and surfactant, together
When Optimal Medium in surfactant species and concentration, it is final to determine that concentration ratio is 1:2-2:1 fatty acid distribution of coconut oil diethyl
The mixture of acid amides and Tweens surfactant can obtain the bacteriolyze of production greater activity as the surfactant of culture medium
Enzyme, has certain guidance meaning to the industrialization of zymotechnique production lysozyme.
Specific embodiment
First, zymotechnique prepares lysozyme
Embodiment 1：
Prepare fluid nutrient medium, wherein glucose 10g/L, peptone 5g/L, fatty acid distribution of coconut oil diacetayl amide 1g/L, tween
80 0.5g/L.Take 25mL fluid nutrient mediums to be placed in triangular flask, bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v)
Thalline seed, carry out shake flask fermentation, fermentation condition is 30 DEG C, and 200r/min after fermented and cultured 24h, obtains final product lysozyme L1.
Embodiment 2：
Prepare fluid nutrient medium, wherein glucose 10g/L, peptone 5g/L, fatty acid distribution of coconut oil diacetayl amide 0.75g/L,
Tween 80 0.75g/L.Take 25mL fluid nutrient mediums to be placed in triangular flask, bacillus subtilis are accessed by the inoculum concentration of 4% (v/v)
The thalline seed of bacterium K-77, carries out shake flask fermentation, and fermentation condition is 30 DEG C, and 200r/min after fermented and cultured 24h, obtains final product bacteriolyze
Enzyme L2.
Embodiment 3：
Fluid nutrient medium, wherein glucose 10g/L are prepared, peptone 5g/L, fatty acid distribution of coconut oil diacetayl amide 0.5g/L tell
80 1g/L of temperature.Take 25mL fluid nutrient mediums to be placed in triangular flask, bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v)
Thalline seed, carry out shake flask fermentation, fermentation condition is 30 DEG C, and 200r/min after fermented and cultured 24h, obtains final product lysozyme L3.
Embodiment 4：
Prepare fluid nutrient medium, wherein glucose 10g/L, peptone 5g/L, fatty acid distribution of coconut oil diacetayl amide 1.5g/L.Take
25mL fluid nutrient mediums are placed in triangular flask, and the thalline seed of bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v),
Shake flask fermentation is carried out, fermentation condition is 30 DEG C, and 200r/min after fermented and cultured 24h, obtains final product lysozyme L4.
Embodiment 5：
Prepare fluid nutrient medium, wherein glucose 10g/L, peptone 5g/L, Tween 80 1.5g/L.Take the training of 25mL liquid
Foster base is placed in triangular flask, and the thalline seed of bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v), carries out shaking flask hair
Ferment, fermentation condition is 30 DEG C, and 200r/min after fermented and cultured 24h, obtains final product lysozyme L5.
Embodiment 6：
Prepare fluid nutrient medium, wherein glucose 10g/L, peptone 5g/L.Take 25mL fluid nutrient mediums and be placed in triangular flask
In, the thalline seed of bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v), shake flask fermentation is carried out, fermentation condition is 30
DEG C, 200r/min after fermented and cultured 24h, obtains final product lysozyme L6.
2nd, the measure of lysozyme activity
On condensation agar plate (internal diameter contains 0.01% micrococcus lysodeikticus to fill 20mL in the flat board of 90mm), use
Punch method is determined.Fan the air device internal diameter for 7mm, the lysozyme L1-L6 testing samples 20 for adding above-described embodiment 1-6 to prepare per hole
μ l, the lysozyme of each embodiment is repeated 5 times, 30 DEG C of incubated 24h, the diameter of observed and recorded bacteriolyze circle, and by standard
The standard curve of bacteriolyze enzyme sample, converses the unit of activity (U/mL) of testing sample.
The influence of the different surfaces active species of table 1 and concentration to lysozyme activity
Result of the test shows, compared with the culture medium without surfactant, using containing surfactant coconut oil fat
The enzymatic activity of the lysozyme of the culture medium fermenting and producing of sour diacetayl amide or Tween 80 is obviously improved, it was demonstrated that fatty acid distribution of coconut oil diethyl
Acid amides or tween have facilitation to generation high activity lysozyme.Additionally, using simultaneously comprising fatty acid distribution of coconut oil diacetayl amide
More obvious to the enzymatic activity facilitation of lysozyme with the culture medium of Tween 80, particularly concentration ratio is 2:1 coconut oil fat
The culture medium of sour diacetayl amide and Tween 80 is generated and is difficult to expected excellent effect.
Above-described embodiment is only the preferred embodiment of the present invention, it should be pointed out that come for one of ordinary skill in the art
Say, under the premise of not departing from the present invention, can also do some improvement, these also should be regarded as within protection scope of the present invention.
Claims (9)
1. the method that zymotechnique prepares lysozyme, comprises the following steps：Take fluid nutrient medium to be placed in triangular flask, by 1-10%
The inoculum concentration of v/v accesses thalline seed, carries out shake flask fermentation, and fermentation condition is 20-40 DEG C, 150-300r/min, fermented and cultured
After 24h, by zymotic fluid centrifugation removal thalline, the purifying of supernatant ammonium sulfate salting-out process is taken, obtains final product lysozyme, it is characterised in that
The fluid nutrient medium includes glucose, peptone and surfactant, and the surfactant is that concentration ratio is 1:2-2:1
The mixture of fatty acid distribution of coconut oil diacetayl amide and Tweens surfactant.
2. the method for preparing lysozyme according to claim 1, it is characterised in that the concentration of the glucose is 10g/L,
The concentration of the peptone is 5g/L.
3. the method for preparing lysozyme according to claim 1, it is characterised in that the fatty acid distribution of coconut oil diacetayl amide and
The concentration range of Tweens surfactant is 0.5g/L-2g/L.
4. the method for preparing lysozyme according to claim 3, it is characterised in that the fatty acid distribution of coconut oil diacetayl amide
Concentration is 1g/L, and the concentration of the Tweens surfactant is 0.5g/L.
5. the method for preparing lysozyme according to claim any one of 1-4, it is characterised in that live on the Tweens surface
Property agent be Tween 80.
6. the method for preparing lysozyme according to claim 1, it is characterised in that the thalline be selected from streptomyces griseus,
MRSE, bacillus subtilis or molten bacillus.
7. the method for preparing lysozyme according to claim 6, it is characterised in that the thalline is selected from streptomyces griseus S-
35th, MRSE K-6-W1, bacillus subtilis K-77 or molten bacillus S2-6b.
8. the method for preparing lysozyme according to claim 1, it is characterised in that the fermentation condition is 30 DEG C, 200r/
min。
9. the method for preparing lysozyme according to claim 1, it is characterised in that the inoculum concentration is fluid nutrient medium
4%v/v.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110403848A (en) * | 2018-04-28 | 2019-11-05 | 好维股份有限公司 | A kind of oral care product with antibacterial action |
| CN113897344A (en) * | 2021-11-03 | 2022-01-07 | 浙江莱康生物工程有限公司 | Lysozyme composition with anti-inflammatory effect |
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| CN1687397A (en) * | 2004-12-21 | 2005-10-26 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN104263709A (en) * | 2014-09-26 | 2015-01-07 | 天津生机集团股份有限公司 | Egg-white lysozyme and preparation method thereof |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1687397A (en) * | 2004-12-21 | 2005-10-26 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN104263709A (en) * | 2014-09-26 | 2015-01-07 | 天津生机集团股份有限公司 | Egg-white lysozyme and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110403848A (en) * | 2018-04-28 | 2019-11-05 | 好维股份有限公司 | A kind of oral care product with antibacterial action |
| CN113897344A (en) * | 2021-11-03 | 2022-01-07 | 浙江莱康生物工程有限公司 | Lysozyme composition with anti-inflammatory effect |
| CN113897344B (en) * | 2021-11-03 | 2023-09-08 | 浙江莱康生物工程有限公司 | Lysozyme composition with anti-inflammatory effect |
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Application publication date: 20170630 Assignee: Zhejiang Laikang Bioengineering Co., Ltd Assignor: ZHEJIANG AEGIS BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd. Contract record no.: X2020980002388 Denomination of invention: Method for preparing lysozyme by means of fermentation process Granted publication date: 20190621 License type: Common License Record date: 20200521 |