CN106367459A - Method for preparing oligomeric hyaluronic acid with different molecular weights - Google Patents
Method for preparing oligomeric hyaluronic acid with different molecular weights Download PDFInfo
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Abstract
The invention discloses a method for preparing oligomeric hyaluronic acid with different molecular weights, and belongs to the technical field of bioengineering. The method has the advantages that HA producing recombinant bacillus subtilis E168T/pP43-DU-PBMS is used as a starting strain, hyaluronidase from leech is added in HA fermentation procedures and is combined with sucrose fed-batch fermentation, accordingly, hyaluronic acid with low molecular weights and oligosaccharides with molecular weights ranging from 1*10<3> Da to 1*10<5> Da can be prepared by means of fermentation coupling enzymatic hydrolysis, and the yields of HA with low molecular weights and the oligosaccharides of the HA can be increased; certain foundations can be laid for efficiently preparing the hyaluronic acid with the low molecular weights and the oligosaccharides, and the method is suitable for industrial production application.
Description
Technical field
The present invention relates to a kind of method preparing different molecular weight oligomerization hyaluronic acid, belong to technical field of bioengineering.
Background technology
Hyaluronic acid (hyaluronicacid, ha), is a kind of macromolecule viscous polysaccharide, with its unique molecular structure and
Physicochemical property, so as to have good moisture retention, viscoelasticity, permeability and ductility, is moisturizing in the nature having now been found that
Property best material, no any immunogenicity and toxicity simultaneously, be widely used in the industries such as cosmetics, food and medicine neck
Domain.Hyaluronic acid shows multiple important physiological functions with its unique molecular structure and physicochemical property in body, such as moistens
Sliding joint, adjusts the permeability of blood vessel wall, regulatory protein matter, Water-Electrolyte diffusion and operating, promotes wound healing etc..
Recent study discovery (salwowska, n.m, bebenek, k.a, zadlo, d.a, wcislo-dziadecka,
D.l, j cosmetdermatol, 2016), the ha of low-molecular-weight is (less than 1 × 105)) and hyaluronic acid oligosaccharide have uniqueness
Biological function.Research shows, molecular size range is larger on the biological activity impact of ha, and the ha of different molecular weight ranges but shows
Go out distinct physiologic function (salwowska, n.m, bebenek, k.a, zadlo, d.a, wcislo-dziadecka,
D.l, j cosmetdermatol, 2016).Ha (the mr > 1 × 10 of high molecular6) due to having preferable viscoelasticity, moisturizing
Property, suppression inflammatory reaction, the function such as lubrication, can be applicable to high-end cosmetic industry, ophthalmologic operation adhered elastomer and joint intracavity note
Penetrate treatment.The ha of intermediate molecular weight is (between 1 × 105-106) there is good moisture retention, lubrication and medicament slow release effect, extensively
For cosmetics, eye drop, skin burn concrescence and post-operation adhesion preventing.The ha of low-molecular-weight is (less than 1 × 105) and oligomerization transparent
Matter acid, shows very strong biological activity, has suppression tumor diffusion, promotes wound healing, promote bone and angiogenesis, exempt from
The effects such as epidemic disease regulation, and be easy to penetrate in corium, it is the activator of immunocyte, cytokine.Therefore, small molecule hyalomitome
Acid has broad application prospects in health care of food, cosmetics and clinical treatment field.
Research with small molecule ha and ha oligosaccharide is rapidly growing with application, in recent years, starts both at home and abroad to pay attention to the fall of ha
Solution and its preparation research of catabolite.At present, the method for ha degraded mainly has mechanical degradation, chemical degradation, biodegradation 3 big
Class.Physical degradation methods mainly have heating, mechanical shear stress, ultraviolet, ultrasound wave,60The factors such as co irradiates, gamma-radiation radiation
Ha all can be made to degrade.But, the ha molecular weight of Physical preparation is higher than 10,000, and the molecular weight ranges distribution of generation is larger, produces
Product less stable.The chemical degradation method of ha has Hydrolyze method and oxidation degradation method, and Hydrolyze method divides acid hydrolysis (hcl) and basic hydrolysiss
(naoh) oxidant that, oxidative degradation is commonly used is sodium hypochlorite (naclo) and hydrogen peroxide (h2o2).Although using chemical degradation
The relative molecular mass of method catabolite can be controlled by changing soda acid or the addition of oxidant and response time, fall
Solution cost is relatively low, and but chemical degradation method introduces chemical reagent it is easy to large-scale production, and reaction condition is complicated, easily gives ha property
Produce impact and the purification of product brings difficulty, and produce substantial amounts of industrial wastewater.Biological enzyme degraded is ha in hyaluronic acid
In the presence of enzyme (haase), β-glucosides bond fission is degraded.Enzymatic isolation method is gentle biodegrading process, not only can prepare little point
Sub- ha, and the oligosaccharide of chain a length of 4～22 can be obtained using the method, and can be obtained single after gel chromatography separation
Ha oligosaccharide.But, the hyaluronidase of current commercialization is typically to extract preparation from animal tissue, expensive, is not suitable for
Industrial applications.And in prior art, apply the engineering bacteria of expression hyaluronidase and hyaluronan synthase simultaneously to produce small molecule
There is the unmanageable shortcoming of enzyme digestion reaction in the method for ha in actual production.Therefore, a kind of low cost, process is simple, are suitable for
The method of small molecule ha of industrialization large-scale production has important application potential.
Content of the invention
It is an object of the invention to provide a kind of method preparing different molecular weight oligomerization hyaluronic acid, it is initial in fermentation
When to adjust dissolved oxygen be 100%, be reduced in dissolved oxygen in fermentation process≤30% when to fermentation liquid in add 10-105u/
Ml hyaluronidase；Described microorganism be produce high molecular weight hyaluronic acid genetic engineering bacterium, including recombined bacillus subtilis,
Restructuring Corynebacterium glutamicum, recombination bacillus coli；The described fermentation medium phosphate buffered saline of 50mm ph 7.0,
Its composition includes: yeast extract 20-30g/l, peptone 1.5-3g/l, mgso41.2～2.0g/l, sucrose 20-40g/l；
Using xylose as derivant, the ph controlling fermentation liquid is 6.0-8.0 to described sweat；25-40 DEG C of fermentation temperature；Described interpolation
Hyaluronidase is to be added in fermentation system dissolved oxygen≤30%.
In one embodiment of the invention, described microorganism is recombined bacillus subtilis e168t/pp43-du-
pbms.
In one embodiment of the invention, described hyaluronidase is Hirudo hyaluronidase (genbank accession number
For ahv78514.1).
In one embodiment of the invention, described fermentation is carried out in the fermentation medium；Described fermentation culture
The base phosphate buffered saline of 50mm ph 7.0, its composition includes: yeast extract 20g/l, peptone 2g/l, mgso4
1.5g/l, sucrose 20-40g/l.
In one embodiment of the invention, described fermentation is to carry out in microbial inoculant to fermentation medium sending out
Ferment.
In one embodiment of the invention, 2h adds the xylose of 20g/l as derivant after inoculation.
In one embodiment of the invention, the ph of described fermentation processes fermentation liquid is 7.0.
In one embodiment of the invention, described fermentation processes temperature is 37 DEG C.
In one embodiment of the invention, the fermentation time of described sweat is 14-28h.
In one embodiment of the invention, carry out feed supplement in described sweat, control sucrose concentration is 5～10g/
l.
Beneficial effect: the invention provides a kind of different molecular weight oligomerization hyaluronic acid directly prepared during the fermentation
Method, by adding enzyme liquid during strain fermentation, solves the recombined bacillus subtilis fermentation later stage due to the accumulation of ha
The problem that viscosity increases, increased the dissolved oxygen of fermentation liquid and improves the yield of ha；Secondly, enzyme hydrolysiss preparation in fermentation liquid
Oligomerization hyaluronan molecule amount is less than 105Da, and primary product is oligosaccharide it is easy to purification reclaims.This invention operating process letter
Single, reaction condition is gentle, specificity is high and product purity high, industrially for preparing oligomerization hyaluronic acid and its redundant organism
Have potential and be widely worth very much.
Brief description
Fig. 1 produces ha time chart for recombined bacillus subtilis batch fermentation；
Fig. 2 is the impact graph of a relation that training strategy and hyaluronidase interpolation time accumulate to ha；A, in sweat
8h adds hyaluronidase batch fermentation time history；B, in sweat, 8h adds hyaluronidase fed-batch fermentation
Time history；C, in sweat, 4h adds hyaluronidase fed-batch fermentation time history；D, fermented
In journey, 4h adds hyaluronidase fed-batch fermentation time history；
Fig. 3 measures the molecular weight of tunning micromolecule hyaluronic acid for efficient liquid phase gel exclusion chromatography.
Specific embodiment
Seed culture medium (g/l): yeast extract 5, peptone 10, nacl 10, xylose 20, glucose 10, mgso4
1.5.
Fermentation medium (g/l): yeast extract 20, peptone 2, mgso41.5,50mm phosphate buffers, ph
7.0, sucrose 20-40,
Embodiment 1 recombined bacillus subtilis batch fermentation
Bacterial strain used by the present invention is recombined bacillus subtilis e168t/pp43-du-pbms (production of
specific-molecular-weight hyaluronan by metabolically engineered bacillus
Build in subtilis 168, publication date is on 2 3rd, 2016), bacterial strain first line growth, picking on lb culture medium flat plate
Monoclonal recombinant bacterial strain is inoculated in containing 20g/l xylose, the glucose of 10g/l, the mgso of 1.5g/l4With the card of 50 μ g/ml that
The lb fluid medium of mycin, is placed in 37 DEG C of 200rpm overnight incubation as seed liquor.It is forwarded to 3l by 10% inoculum concentration to send out
Fermentation tank (liquid amount is 1.5l) culture 16h.Fermentation condition: rotating speed 200rpm, ventilation 1.0vvm, 37 DEG C of temperature, ph7.0.
As derivant, 2h adds the xylose of 20g/l after inoculation.
Take a sample every 2h in sweat, measure thalli growth od600Value, the sds adding final concentration 0.5% extremely sends out
In zymotic fluid, incubated at room 5min, 10000rpm is centrifuged 10min, collects supernatant.Supernatant adopts the dehydrated alcohol of 2 times of volumes
Precipitate hyaluronic acid, incubated at room 1h, 10000rpm is centrifuged 10min, and precipitation is filled using the nacl solution of isopyknic 1mol/l
Divide dissolving, after suitably being diluted, ha content is detected using bitter-muir sulfuric acid carbazole method.Result shows, ha yield is being sent out
Ferment 14h reaches highest, is 3.65g/l.
Add hyaluronidase in embodiment 2 recombined bacillus subtilis sweat during 8h (do < 5%) to carry out in batches
Fermentation
With embodiment 1, difference is that 8h adds final concentration of 5 × 10 after inoculation to specific embodiment3U/ml's is saturating
Bright matter acid enzyme.Fermentation 14h ha yield reaches 4.78g/l (as shown in Fig. 2-a), compares embodiment 1 and improves 30.9%.
In embodiment 3 recombined bacillus subtilis sweat, 8h (do < 5%) adds hyaluronidase and carries out feed supplement and send out
Ferment
With embodiment 1, difference is that 8h adds final concentration of 5 × 10 after inoculation to specific embodiment3U/ml's is saturating
Bright matter acid enzyme.Carry out sucrose feed supplement in 8h, maintenance sucrose concentration 5-10g/l feed regimes such as table 1:
Table 1 feed regimes
This sweat be added enzyme and mend sucrose after, result as shown in accompanying drawing 2-b, thalline od600Value continues to increase,
Ha yield reaches 7.25g/l in 20h simultaneously.
In embodiment 4 recombined bacillus subtilis sweat, 4h (do < 30%) adds hyaluronidase and carries out feed supplement
Fermentation.
With embodiment 3, difference is that 4h adds final concentration of 5 × 10 after inoculation to specific embodiment3U/ml's is saturating
Bright matter acid enzyme, and carry out sucrose feed supplement in 8h.As shown in accompanying drawing 2-c, ha yield reaches up to 7.36g/ in 20h to result
l.In embodiment 5 recombined bacillus subtilis sweat, 4h adds hyaluronidase and carries out fed-batch fermentation.
With embodiment 1, difference is that the initial sucrose concentration of fermentation medium is 15g/l to specific embodiment, operates by table 2
Condition carries out feed supplement, and adds final concentration of 1 × 10 in 4h4The hyaluronidase of u/ml.
This sweat passes through to add hyaluronidase in 4h, and controls carbon source concentration, in thalline rapid growth stage,
The accumulation of ha improves rapidly, reaches 9.46g/l in 12h, and result is as shown in Fig. 2-d.
Small molecule ha in above-described embodiment 2-5, after ethanol purification, is measured using efficient liquid phase gel exclusion chromatography and divides
Son amount size, result, as shown in figure 3, mean molecule quantity all concentrates between 60000-70000 dalton, meanwhile, extends enzyme activity
Response time, it is possible to obtain hyaluronic acid product oligosaccharides more than ha tetrose.
Table 2 feed regimes
In embodiment 6 recombined bacillus subtilis sweat, 3.5h (do=35%) adds hyaluronidase and carries out point
Batch fermentation.
With embodiment 1, difference is that 3.5h (now do=35%) adds final concentration after inoculation to specific embodiment
For 5 × 103U/ml hyaluronic acid enzyme liquid.The yield about 4g/l of ha after fermentation 14h.
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
Claims (9)
1. a kind of method preparing different molecular weight oligomerization hyaluronic acid it is characterised in that be in fermentation process to
Add 10-10 in fermentation liquid5U/ml hyaluronidase；Described microorganism is the genetic engineering bacterium producing high molecular weight hyaluronic acid,
Including recombined bacillus subtilis, restructuring Corynebacterium glutamicum, recombination bacillus coli；Fermentation medium 50mmol/l ph
7.0 phosphate buffered saline, its composition includes: yeast extract 20-30g/l, peptone 1.5-3g/l, mgso41.2
～2.0g/l, sucrose 20-40g/l；Using xylose as derivant, the ph controlling fermentation liquid is 6.0-8.0 to described sweat；Send out
Ferment temperature 25-40 DEG C；Described interpolation hyaluronidase is to be added in fermentation system dissolved oxygen≤30%.
2. method according to claim 1 is it is characterised in that described microorganism is recombined bacillus subtilis e168t/
pp43-du-pbms.
3. method according to claim 1 is it is characterised in that described hyaluronidase is Hirudo hyaluronidase.
4. method according to claim 1 is it is characterised in that the described fermentation medium phosphate of 50mm ph 7.0
Buffer, its composition includes: yeast extract 20g/l, peptone 2g/l, mgso41.5g/l, sucrose 20-40g/l.
5. method according to claim 1 is it is characterised in that the xylose of 2h interpolation 20g/l is as induction after inoculation
Agent.
6. it is characterised in that carrying out feed supplement in described sweat, described feed supplement is control to method according to claim 1
Sucrose concentration processed is 4～6g/l.
7. method according to claim 1 is it is characterised in that the ph of described fermentation processes fermentation liquid is 7.0.
8. method according to claim 1 is it is characterised in that described fermentation processes temperature is 37 DEG C.
9. application in cosmetics, food and field of medicaments for the method described in claim 1.
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Cited By (5)
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| CN108251346A (en) * | 2018-01-15 | 2018-07-06 | 清华大学 | A kind of recombination Corynebacterium glutamicum for expressing hyaluronidase and its application |
| CN109536550A (en) * | 2018-12-06 | 2019-03-29 | 上海景峰制药有限公司 | A kind of Sodium Hyaluronate and preparation method thereof |
| CN110923173A (en) * | 2019-12-26 | 2020-03-27 | 江南大学 | Enterobacter and application thereof |
| CN112553272A (en) * | 2020-12-24 | 2021-03-26 | 华熙生物科技股份有限公司 | Method for improving hyaluronic acid yield |
| WO2021129353A1 (en) * | 2019-12-27 | 2021-07-01 | 江苏诚信药业有限公司 | Hyaluronic acid hydrolaze and encoding sequence thereof, and method for preparing oligomeric hyaluronate by means of using same |
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| CN112553272B (en) * | 2020-12-24 | 2022-08-09 | 华熙生物科技股份有限公司 | Method for improving hyaluronic acid yield |
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