CN107058266B - A method of lysozyme is prepared by zymotechnique - Google Patents
A method of lysozyme is prepared by zymotechnique Download PDFInfo
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- CN107058266B CN107058266B CN201710277771.9A CN201710277771A CN107058266B CN 107058266 B CN107058266 B CN 107058266B CN 201710277771 A CN201710277771 A CN 201710277771A CN 107058266 B CN107058266 B CN 107058266B
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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 a kind of methods for preparing lysozyme by zymotechnique, the following steps are included: fluid nutrient medium is taken to be placed in triangular flask, thallus 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, fermented and cultured for 24 hours after, fermentation liquid is centrifuged removal thallus, supernatant is taken to be purified with ammonium sulfate salting-out process, up to lysozyme, the fluid nutrient medium includes glucose, peptone and inorganic salts, and the inorganic salts are concentration than the MgSO for 1:4-4:14And ZnCl2Mixture.The fermentation process obtains the lysozyme of greater activity by the type and concentration of inorganic salts in Optimal Medium.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to a method of lysozyme is prepared by zymotechnique.
Background technique
Lysozyme (Lysozyme), also known as cytohydrolist can act on bacteria cell wall peptide glycan point in specific manner
Son can make β between -acetylmuramic acid and acetyl glucosamine ammonia-Isosorbide-5-Nitrae glycosidic bond fracture, make bacteria cell wall become it is loose and
Achieve the effect that dissolution of bacteria.Lysozyme is distributed widely in high animal vegetable tissue and its secretion, protozoan, insect, plant
In object and various microorganisms.
Lysozyme has multiple pharmacological effect as one of the nonspecific immune factors in human normal body fluid and tissue,
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 enhance macrophage phagocytosis and digestive function, activate white thin
Born of the same parents' phagocytic function, and oligoleukocythemia caused by cytostatics can be improved, to enhance the resistance of body.Lysozyme energy
Direct hydrolysis gram-positive bacteria, secretory immunoglobulin A, complement participation under, moreover it is possible to hydrolyze Gram-negative bacteria such as
Escherichia coli etc..Equally there is bacteriolysis to drug tolerant bacteria, and there is spy significant in efficacy and to human body Small side effects
Point, thus be a kind of ideal medicinal enzyme.Lysozyme can directly be acted on negatively charged virus protein, with DNA, RNA,
Apoprotein forms double salt, makes virally inactivated.Lysozyme can participate in mucopolysaccharide metabolism, in cooperation as enzyme antimicrobial
While clothes and externally applied drug, strength antiinflammation can be played, it can also make its mistake in conjunction with the various acidic materials for inducing inflammation
It is living, and the curative effect of antibiotic and other medicines can be enhanced, improve the mucopolysaccharide metabolism of periplast, to reach anti-inflammatory, repair
The purpose of overlying tissue.
Lysozyme is the necessary antibacterial protein of infants growth and development, can inhibit putrefactive microorganisms in enteron aisle in infants
Existence, while baby intestinal bacterium Bifidobacterium proliferation is directly facilitated, so that it is micro- to promote in baby's stomach and intestine milk casein to be formed
Thin curdled milk is conducive to baby's digestion and absorption.Lysozyme can also promote the normalization of artificially feeding infant enteric bacteria group;Energy
Enough factors of reinforcing preventing epidemic to properdin, gamma globulin etc. in vivo are to increase the resistance to infection, especially to Premature
Youngster, have prevention weight loss, prevention chylopoietic disease, promote weight and other effects, 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 in dairy products play
Corrosion-resistant effect is particularly suitable for pasteurize milk, effectively extends storage life.Since lysozyme has certain heat-resisting quantity
Can, it is equally applicable to ultrahigh-temperature instant sterilization milk, additive capacity 300-600ppm.Its method is addition before packaging, superhigh temperature
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
A certain amount of lysozyme is added in mL in fresh milk or milk powder, can play antisepsis.
Lysozyme is genetic engineering, cell engineering, essential toolenzyme in Fermentation Engineering.At abroad, lysozyme is more
Extraction for bacterium substance in vivo.As long as to sensitive thallus suspension in appropriate buffer, with bacteriolyze enzymatic treatment, in conjunction with
Cell-free extract just can be obtained using means such as ultrasonic wave, refrigerated centrifuges, further required thallus substance can be obtained in purification
Such as protein, nucleic acid, enzyme and active peptides, therefore demand of the development of biological industry to lysozyme formulation will be growing day by day.
Currently, being commercially available most lysozymes is egg white lysozyme.As the improvement of people's living standards, to bacteriolyze
The demand of enzyme is increasing, depends merely on and extracts lysozyme from egg white, is difficult to solve the demand and supply contraction.However, using micro-
Biological production lysozyme, cost is relatively low, saves material, and environmental pollution is smaller, large-scale production easy to accomplish, thus develops latent
Power is huge.In addition, being studied according to the difference of its substrate micro- since microbe-derived lysozyme has apparent Bacteriolytic specificity
Biological cell wall construction, lysozyme are a kind of highly useful biological tool enzyme again.Most microbic muramidases are to golden yellow Portugal
Grape coccus, Gram-negative bacteria and fungi have a dissolution, and egg white lysozyme to the above bacterial strain without obvious bacteriolysis, in recent years
Come, both at home and abroad the lysozyme in multiple-microorganism source in depth study extensively.
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, general organic nitrogen source, can be direct in the medium without organic nitrogen source better than inorganic nitrogen-sourced
Influence the yield of lysozyme.Such as staphylococcus aureus strains, indispensable yeast extract, otherwise enzyme activity is just remarkably decreased.
Its enzyme activity can be improved with meat medicinal extract in for another example bacillus subtilis strain.In addition, glucide is added in the medium, to enzyme activity
Property influence it is very big.It has been reported that and shows when cultivating staphylococcus aureus strains, glucose is used to replace β-phosphoglycerol as carbon
Source, when enzyme activity reaches top, activity reduces very fast.In culture bacillus subtilis strain, glucose has inhibition
The effect of producing enzyme.When cultivating styreptomyces globispotus strain bacterial strain, most suitable carbon source is dextrin and soluble starch.
The prior art prepares the culture medium research that bacteriolyze enzyme process uses for zymotechnique at present and is limited primarily to nitrogen source, carbon
The selection of the ingredients such as source, the activity research how type and concentration for inorganic salts influence lysozyme is less, and existing culture
Base fermentation generation lysozyme activity is limited, causes the method for such lysozyme in actual industrial mass production using limited.
Summary of the invention
The present invention is in view of the shortcomings of the prior art, provide a kind of high living by the culture medium prescription acquisition in optimization for fermentation technology
The method of property lysozyme.
The technical scheme is that a kind of method for preparing lysozyme by zymotechnique, comprising the following steps: take liquid
Body culture medium is placed in triangular flask, is accessed thallus seed by the inoculum concentration of 1-10%v/v, is carried out shake flask fermentation, fermentation condition is
20-40 DEG C, 150-300r/min, fermented and cultured for 24 hours after, by fermentation liquid be centrifuged removal thallus, take supernatant ammonium sulfate precipitation
Method purifies to get lysozyme;The fluid nutrient medium includes glucose, peptone and inorganic salts, and the inorganic salts are concentration ratio
For the MgSO of 1:4-4:14And ZnCl2Mixture.
Preferably, the concentration of the glucose is 10g/L, and the concentration of the peptone is 5g/L.
Preferably, the MgSO4And ZnCl2Concentration range be 1g/L-4g/L.
It is furthermore preferred that the MgSO4Concentration be 4g/L, the ZnCl2Concentration be 1g/L.
Preferably, the thallus is selected from streptomyces griseus, staphylococcus epidermis, bacillus subtilis or molten bacillus.
It is furthermore preferred that the thallus is selected from streptomyces griseus S-35, staphylococcus epidermis 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:
Inventor determines the composition of glucose, peptone and inorganic salts, while excellent by the ingredient of screening and culturing medium
Change the type and concentration of inorganic salts in culture medium, it is final to determine concentration than the MgSO for 1:4-4:14And ZnCl2Mixture is as training
The inorganic salts for supporting base can get the lysozyme of production greater activity, has to the industrialization of zymotechnique production lysozyme and centainly refers to
Lead meaning.
Specific embodiment
One, zymotechnique prepares lysozyme
Embodiment 1:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L, MgSO44g/L, ZnCl21g/L.Take 25mL
Fluid nutrient medium is placed in triangular flask, by the thallus seed of the inoculum concentration access bacillus subtilis K-77 of 4% (v/v), is carried out
Shake flask fermentation, fermentation condition be 30 DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L1.
Embodiment 2:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L, MgSO42.5g/L, ZnCl22.5g/L.It takes
25mL fluid nutrient medium is placed in triangular flask, and the thallus seed of bacillus subtilis K-77 is accessed by the inoculum concentration of 4% (v/v),
Carry out shake flask fermentation, fermentation condition be 30 DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L2.
Embodiment 3:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L, MgSO41g/L, ZnCl24g/L.Take 25mL
Fluid nutrient medium is placed in triangular flask, by the thallus seed of the inoculum concentration access bacillus subtilis K-77 of 4% (v/v), is carried out
Shake flask fermentation, fermentation condition be 30 DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L3.
Embodiment 4:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L, MgSO45g/L.Take 25mL fluid nutrient medium
It is placed in triangular flask, by the thallus seed of the inoculum concentration access bacillus subtilis K-77 of 4% (v/v), carries out shake flask fermentation, hair
Ferment condition be 30 DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L4.
Embodiment 5:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L, ZnCl25g/L.Take 25mL fluid nutrient medium
It is placed in triangular flask, by the thallus seed of the inoculum concentration access bacillus subtilis K-77 of 4% (v/v), carries out shake flask fermentation, hair
Ferment condition be 30 DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L5.
Embodiment 6:
Fluid nutrient medium is prepared, wherein glucose 10g/L, peptone 5g/L.25mL fluid nutrient medium is taken to be placed in triangular flask
In, by the thallus seed of the inoculum concentration access bacillus subtilis K-77 of 4% (v/v), carry out shake flask fermentation, fermentation condition 30
DEG C, 200r/min, fermented and cultured for 24 hours after to get lysozyme L6.
Two, the measurement of lysozyme activity
(20mL is filled in the plate that internal diameter is 90mm and contains 0.01% micrococcus lysodeikticus), use on condensation agar plate
Punch method measurement.Fan the air device internal diameter be 7mm, every hole be added above-described embodiment 1-6 preparation lysozyme L1-L6 sample to be tested 20
μ l, the lysozyme of each embodiment are repeated 5 times, and 30 DEG C of constant temperature incubations for 24 hours, observe and record the diameter of bacteriolyze circle, and pass through standard
The standard curve of bacteriolyze enzyme sample converses the unit of activity (U/mL) of sample to be tested.
The influence of table 1 different Inorganic Salts and concentration to lysozyme activity
Test result show with without inorganic salts culture medium compared with, using contain inorganic salts MgSO4Or ZnCl2Culture
The enzymatic activity of the lysozyme of base fermenting and producing is obviously improved, it was demonstrated that MgSO4And ZnCl2There is promotion to high activity lysozyme is generated
Effect.In addition, using MgSO is included4And ZnCl2Culture medium it is more obvious to the enzymatic activity facilitation of lysozyme, especially
Concentration is than the MgSO for 4:14And ZnCl2Culture medium produce and be difficult to expected excellent effect.Above-described embodiment is only the present invention
Preferred embodiment, it should be pointed out that for those of ordinary skill in the art, under the premise of not departing from the present invention, also
Several improvement can be done, within these also should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of method for preparing lysozyme by zymotechnique, comprising the following steps: take fluid nutrient medium to be placed in triangular flask,
Thallus seed is accessed by the inoculum concentration of 1-10%v/v, carries out shake flask fermentation, fermentation condition is 20-40 DEG C, 150-300r/min,
Fermented and cultured for 24 hours after, fermentation liquid is centrifuged removal thallus, takes the purifying of supernatant ammonium sulfate salting-out process to get lysozyme, it is special
Sign is that the fluid nutrient medium includes glucose, peptone and inorganic salts, and the inorganic salts are concentration than the MgSO for 4:14
And ZnCl2Mixture, the thallus are bacillus subtilis K-77.
2. the method according to claim 1 for preparing lysozyme, which is characterized in that the concentration of the glucose is 10g/L,
The concentration of the peptone is 5g/L.
3. the method according to claim 1 for preparing lysozyme, which is characterized in that the MgSO4And ZnCl2Concentration model
Enclosing is 1g/L-4g/L.
4. the method according to claim 3 for preparing lysozyme, which is characterized in that the MgSO4Concentration be 4g/L, institute
State ZnCl2Concentration be 1g/L.
5. the method according to claim 1 for preparing lysozyme, which is characterized in that the fermentation condition is 30 DEG C, 200r/
min。
6. the method according to claim 1 for preparing lysozyme, which is characterized in that the inoculum concentration is fluid nutrient medium
4%v/v.
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CN102102095A (en) * | 2010-11-30 | 2011-06-22 | 浙江大学 | Method for preparing lysozyme by fermenting marine streptomyces |
CN103382462A (en) * | 2013-05-09 | 2013-11-06 | 中国科学院天津工业生物技术研究所 | Bacillus licheniformis lysozyme and its production method and application thereof |
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CN102102095A (en) * | 2010-11-30 | 2011-06-22 | 浙江大学 | Method for preparing lysozyme by fermenting marine streptomyces |
CN103382462A (en) * | 2013-05-09 | 2013-11-06 | 中国科学院天津工业生物技术研究所 | Bacillus licheniformis lysozyme and its production method and application thereof |
Non-Patent Citations (1)
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海洋细菌S-12-86的产溶菌酶条件;张琇 等;《中国水产科学》;20070531;第14卷(第3期);第425-429页 |
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Application publication date: 20170818 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 fermentation process Granted publication date: 20190823 License type: Common License Record date: 20200521 |
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