CN107058266B

CN107058266B - A method of lysozyme is prepared by zymotechnique

Info

Publication number: CN107058266B
Application number: CN201710277771.9A
Authority: CN
Inventors: 潘宏涛; 卢亚萍
Original assignee: Zhejiang Aisijie Biological Science & Technology Co Ltd
Current assignee: Zhejiang Aisijie Biological Science & Technology Co Ltd
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2019-08-23
Anticipated expiration: 2037-04-25
Also published as: CN107058266A

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

A method of lysozyme is prepared by zymotechnique

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

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.