CN104178539B - A kind of method of prepare with scale specified molecular weight micromolecule hyaluronic acid - Google Patents
A kind of method of prepare with scale specified molecular weight micromolecule hyaluronic acid Download PDFInfo
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- CN104178539B CN104178539B CN201410409643.1A CN201410409643A CN104178539B CN 104178539 B CN104178539 B CN 104178539B CN 201410409643 A CN201410409643 A CN 201410409643A CN 104178539 B CN104178539 B CN 104178539B
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Abstract
The invention discloses a kind of method of prepare with scale specified molecular weight micromolecule hyaluronic acid, belong to technical field of bioengineering.The present invention uses the hyaluronidase in leech source, using water as reaction medium, hydrolyze polymer hyaluronic acid, realize the hyaluronic acid for efficiently preparing 370000Da, 80000Da, 30000Da, 9000Da, 4000Da specified molecular weight, realize and micromolecule hyaluronic acid is prepared on fermentation tank, be suitable for industrialized production and application.
Description
Technical field
The present invention relates to a kind of method of prepare with scale specified molecular weight micromolecule hyaluronic acid, belong to bioengineering skill
Art field.
Background technology
Hyaluronic acid (hyaluronic acid, abbreviation HA), is commonly called as glass uric acid, be it is a kind of by D-Glucose aldehydic acid and
For 2-Acetamido-2-deoxy-D-glucose to repeat dissacharide units, the macromolecular being alternately formed by connecting by β (1-3) and β (1-4) glycosidic bond is sour
Property stickiness polysaccharide, 1934 by Meyer et al. from ox glass marble eye first extraction obtain, be widely used in medicine, cosmetics, food
The fields such as product.Research shows that molecular weight has a significant impact to the activity of hyaluronic acid, the hyaluronic acid even table of different molecular weight
Reveal the activity completely contradicted.
Hyaluronic acid shows a variety of important physiological functions with its unique molecular structure and physicochemical property in body,
Such as lubricating joint, the permeability of vascular wall is adjusted, regulatory protein matter, Water-Electrolyte spreads and operating, promotes wound healing etc..Especially
Importantly, hyaluronic acid has special water retention, to be the material that moisture retention is best in the nature having now been found that, quilt
Referred to as preferable NMF, because HA has good moisture retention, viscoplasticity, permeability and ductility, while nothing is appointed
What immunogenicity and toxicity, is widely used in the industry fields such as cosmetics, food and medicine.
Shown according to literature research, molecular size range has a great influence to HA bioactivity, the HA of different molecular weight ranges
Show completely different physiologic function.HA (the Mr of HMW>2×106) due to preferable viscoplasticity, moisturizing
Property, suppress inflammatory reaction, the function such as lubrication, can be applied to note in high-end cosmetic industry, ophthalmologic operation adhered elastomer and articular cavity
Penetrate treatment.The HA of intermediate molecular weight is (between 1 × 105-106) there is good moisture retention, lubrication and medicament slow release effect, can be wide
It is general to be used for cosmetics, eye drops, skin burn concrescence and post-operation adhesion preventing.The HA and oligomerization hyaluronic acid of low molecule amount, performance
Go out very strong bioactivity, have and suppress tumour diffusion, promote wound healing, promote bone and angiogenesis, immunological regulation etc.
Effect, and be easy to penetrate into corium, immunocyte, the activator of cell factor.Therefore, micromolecule hyaluronic acid is protected in food
Strong, cosmetics and clinical treatment field have broad application prospects.
Preparing the method for small-molecular-weight has physical degradation methods, chemical degradation method, enzymatic isolation method.Physical predominantly heats, machine
Tool shearing force, ultraviolet, ultrasonic wave,60The methods of Co irradiations, gamma-radiation radiation, promotes HA to degrade.Physical degradation methods processing
Process is simple, and product is easily recycled.It is purple but these methods all can bring certain influence, such as heating easily HA is changed colour
Outer and ultrasonic efficiency is more low, and caused small molecule molecular weight ranges are larger, and product stability is poor.HA chemical degradation side
Method has Hydrolyze method and oxidation degradation method, and Hydrolyze method divides sour water solution (HCl) and basic hydrolysis (NaOH), the conventional oxidant of oxidative degradation
For sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2).But chemical degradation method introduces chemical reagent, and reaction condition is complicated, easily gives
HA properties have an impact and bring difficulty to the purifying of product, and produce substantial amounts of industrial wastewater.Enzymatic isolation method is that hyaluronic acid exists
In the presence of hyaluronidase, the glycosidic bond on sugar chain is broken, but the cost generally digested is very high, is not suitable for large-scale
Industrial production.
The present invention, using biological enzyme catalyst degraded HA, produces molecular weight ranges to solve the problems, such as to prepare small molecule HA
Homogeneous micromolecule hyaluronic acid.Because biological enzyme reaction condition is gentle, selectivity is high and product purity is high, work is realized
Industry metaplasia production prepares micromolecule hyaluronic acid.
The content of the invention
The invention discloses a kind of method of prepare with scale specified molecular weight micromolecule hyaluronic acid, added into water
Polymer hyaluronic acid and hyaluronidase, the small molecule that the control differential responses time prepares different molecular weight (glycan unit) are saturating
Bright matter acid, realizes industrialized production.
Encode sequence of the nucleotide sequence of the hyaluronidase preferably as shown in SEQ ID NO.1.
The molecular weight of the macromolecule HA is 104More than kDa.It is preferred that 2.212 × 1014Da。
Methods described is preferably carried out in the automatic temperature-controlled and fermentation tank of agitating device.
The reaction system of methods described preferably comprises hyaluronidase 1 × 106U/L-4×108U/L and macromolecule hyalomitome
Sour 2-100g/L, 1-13h is reacted under the conditions of 10 DEG C -65 DEG C, 400-1000rpm mixing speeds, prepare mean molecule quantity and exist
4000Da to 370000Da hyaluronic acid.
Further preferably hyaluronidase 3 × 107U/L and polymer hyaluronic acid 40g/L, at 45 DEG C, 700rpm
The hyaluronic acid that different time prepares different molecular weight is reacted under the conditions of mixing speed.
To prepare the hyaluronic acid that mean molecule quantity is 370000Da, preferably 1 hour reaction time.It is average to prepare
Molecular weight be 80000Da hyaluronic acid, preferably 2.5 hours reaction time.It is the saturating of 30000Da to prepare mean molecule quantity
Bright matter acid, preferably 3.5 hours reaction time.To prepare the hyaluronic acid that mean molecule quantity is 9000Da, reaction time preferably 7
Hour.To prepare the hyaluronic acid that mean molecule quantity is 4000Da, preferably 12.5 hours reaction time.
The present invention prepares micromolecule hyaluronic acid using the hyaluronidase HA that directly degrades, and has direct purpose.With
Other modes are compared, and the invention has very big application advantage.First, reaction condition is extremely simple, is at normal temperatures and pressures
It can implement, hyaluronic acid is dissolved in water, it is not necessary to which buffer solution, production cost are low；Secondly enzyme hydrolysis process is not required to add any
Organic reagent, produced without any pollution discarded object；Finally, due to not add salt, product is relatively simple in aqueous, easily
Reclaimed in purifying, yield is high, realizes industrialized production micromolecule hyaluronic acid.Based on applied analysis, the inventive method exists
Being commercially used for preparing single oligomerization hyaluronic acid and its redundant organism has potential and very extensive value.
Brief description of the drawings
Fig. 1 is HA the change of molecular weight tendency charts during enzymatic reaction；
Fig. 2 is HPSEC (efficient molecular-exclusion chromatography) figure of molecular weight 370000Da hyaluronic acid；
Fig. 3 is the HPSEC figures of molecular weight 80000Da hyaluronic acid；
Fig. 4 is the HPSEC figures of molecular weight 30000Da hyaluronic acid；
Fig. 5 is the HPSEC figures of molecular weight 9000Da or so hyaluronic acid；
Fig. 6 is the HPSEC figures of molecular weight 4000Da or so hyaluronic acid.
Embodiment
Hydrolysate determination method：Analyzed, selected using the high performance liquid chromatograph 1260 of Agilent company
Composition distribution RID, analyzed using gel chromatographic columnses Ultrahydrogel Linear.Mobile phase selects 0.1M sodium nitrate
Eluted, column temperature is set as 40 degrees Celsius, the μ L of sample size 40, each sample elution time 25min, uses Chinese food
The dextran that drug assay research institute produces carries out the calculating of average molecular mass using GPC softwares as standard items.
Hyaluronic acid enzyme activity unit defines：At pH5.5 and 38 DEG C, 1 μ g grapes are discharged from hyaluronic acid per hour
Enzyme amount needed for the reduced sugar of sugared reducing equivalent is an enzyme activity unit.DNS Determination of Reducing Sugars determines enzyme activity.
The expression of the hyaluronidase of embodiment 1
Full chemistry synthesizes engineered hyaluronidase gene order (SEQ ID NO.1), and digestion is connected to expression vector
PPIC9K construction recombination plasmid pPIC9K-His-HaseA3887, convert into E.coil DH5 α, extraction plasmid order-checking checking.
By recombinant plasmid, electricity is transferred in expressive host P.pastoris GS115 after SalI is linearized, and recombinant clone is verified through PCR
Correctly.
Recombinate P.pastoris GS115/pPIC9K-His-HaseA3887 clones and carry out fermented and cultured expression.Dan Ke
The grand YPD culture mediums (yeast extract 10g/L, peptone 20g/L, glucose 20g/L) for being inoculated in 5ml, 30 DEG C, 200rpm trainings
Support 16h.Transferred by 10% inoculum concentration in 100ml induced expression culture medium BMGY (yeast extract 10g/L, peptone
20g/L, 3g/LK2HPO4, 11.8g/LKH2PO4, 10 × YNB 100ml/L (13.4g/L), 500 × biotin 1mL (4 × 10- 4G/L), glycerine 10mL), 30 DEG C of 200rpm are cultivated to OD600It is worth between 4.Thalline is collected by centrifugation, changes to 100mL induction tables
Up to culture medium BMMY (yeast extract 10g/L, peptone 20g/L, 3g/LK2HPO4, 11.8g/LKH2PO4, 100 × YNB
100mL/L (13.4g/L), 500 × biotin 1mL (4 × 10-4G/L), methanol 5mL), 30 DEG C of 200rpm cultures, per 24h to training
Support and 100% methanol to final concentration of 1.0% (v/v) progress induced expression, induced expression 96h are added in base.
It is prepared by the purifying that embodiment 2 recombinates hyaluronidase
Gene shown in SEQ ID NO.1 has merged 6 continuous histidine marks after transcription and translation in hyaluronidase N-terminal
Label, according to affinity chromatography principle, the label specific can be combined with glucan nickel.Glucan nickel first uses pH6.0,50mM phosphoric acid
Salt buffer balances 30min, adds the zymotic fluid by 0.45 μm of filtering, is incubated 2h at 4 DEG C, is matched somebody with somebody successively with same buffer
Put 0,10,20,30,40,50mM imidazole buffer carry out elution impurity.Finally with 500mM imidazole buffer elution mesh
Mark albumen.The target protein of high concentration imidazoles elution is dialyzed overnight.Obtain the hyaluronidase pure protein of single band, institute
Hyaluronidase pure protein is obtained, after measured, there is hyaluronic acid enzyme activity.
The preparation of the small-molecular-weight hyaluronic acid of embodiment 3
Macromolecule hyaluronic acid is hydrolyzed with the hyaluronidase in leech source, starting molecule amount size is
2.212×1014Da.Upper tank body accumulates 1L, and 900mL water is added in fermentation tank, and adds hyaluronidase 3 × 107U and transparent
Matter acid 40g, finally adds water to 1L, is reacted under the conditions of being 700rpm in 45 DEG C, rotating speed.Sample every half an hour and boiled in boiling water
Enzyme-deactivating, sample are used 0.22 μm of membrane filtration in 10 minutes by boiling, and molecular weight determination is carried out using gel column, point drawn out
Son amount changing trend diagram.Fermentation tank can prepare the hyaluronic acid that molecular weight is 370000Da in 1 hour high-temperature inactivation；Fermentation
Tank can prepare the hyaluronic acid that molecular weight is 80000Da in 2.5 hours high-temperature inactivations；Fermentation tank went out in 3.5 hours high temperature
It is living, the hyaluronic acid that molecular weight is 30000Da can be prepared, in 7 hours high-temperature inactivations, can prepare molecular weight was fermentation tank
9000Da hyaluronic acid, fermentation tank can prepare the hyaluronic acid that molecular weight is 4000Da in 12.5 hours high-temperature inactivations.Such as
Shown in Fig. 1, HA the change of molecular weight tendency chart in tank.As shown in Fig. 2 it is molecular weight 370000Da HPSEC figures.As shown in figure 3,
It is molecular weight 80000Da HPSEC figures.As shown in figure 4, it is molecular weight 30000Da HPSEC figures.As shown in figure 5, it is molecule
Measure 9000Da HPSEC figures.As shown in fig. 6, it is molecular weight 4000Da HPSEC figures.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (4)
- A kind of 1. method for preparing specified molecular weight hyaluronic acid, it is characterised in that be that polymer hyaluronic acid is added into water And hyaluronidase, control differential responses time prepare the micromolecule hyaluronic acid of different molecular weight；Encode the hyaluronic acid The nucleotide sequence of enzyme is as shown in SEQ ID NO.1；The molecular weight of the polymer hyaluronic acid is 104More than kDa；Reactant Hyaluronidase 3 × 10 is contained in system7U/L-4×108U/L and polymer hyaluronic acid 2-40g/L, at 10 DEG C -65 DEG C, 400- 1-3.5h is reacted under the conditions of 1000rpm mixing speeds, prepares hyaluronic acid of the mean molecule quantity in 30000Da to 370000Da.
- 2. according to the method for claim 1, it is characterised in that reaction 1 hour, it is 370000Da's to prepare mean molecule quantity Hyaluronic acid.
- 3. according to the method for claim 1, it is characterised in that reaction 2.5 hours, preparing mean molecule quantity is 80000Da hyaluronic acid.
- 4. according to the method for claim 1, it is characterised in that reaction 3.5 hours, preparing mean molecule quantity is 30000Da hyaluronic acid.
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| CN105820271A (en) * | 2016-05-25 | 2016-08-03 | 苏州景卓生物技术有限公司 | Preparation and purification method of scale micromolecular sodium hyaluronate |
| CN106367459A (en) * | 2016-09-30 | 2017-02-01 | 江南大学 | Method for preparing oligomeric hyaluronic acid with different molecular weights |
| CN111040048A (en) * | 2019-12-21 | 2020-04-21 | 南京汉欣医药科技有限公司 | Ultra-low molecular weight hyaluronic acid and preparation method thereof |
| CN111249302A (en) * | 2020-03-12 | 2020-06-09 | 李鑫荣 | New application and stable manufacturing method of hyaluronic acid fragments |
| CN111893151A (en) * | 2020-08-21 | 2020-11-06 | 浙江绿创生物科技股份有限公司 | Method for continuously producing low molecular weight hyaluronic acid, low molecular weight hyaluronic acid obtained by method and application of low molecular weight hyaluronic acid |
| CN114181986A (en) * | 2021-12-16 | 2022-03-15 | 新疆阜丰生物科技有限公司 | Preparation method of low-molecular sodium hyaluronate |
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| CN103436508A (en) * | 2013-08-15 | 2013-12-11 | 江南大学 | Method for efficiently recombining and expressing hyaluronidase |
| CN103484513A (en) * | 2013-10-22 | 2014-01-01 | 江南大学 | Method for preparing small-molecule oligomeric hyaluronic acid through enzyme method |
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| CN103436508A (en) * | 2013-08-15 | 2013-12-11 | 江南大学 | Method for efficiently recombining and expressing hyaluronidase |
| CN103484513A (en) * | 2013-10-22 | 2014-01-01 | 江南大学 | Method for preparing small-molecule oligomeric hyaluronic acid through enzyme method |
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Application publication date: 20141203 Assignee: Jiangsu Hongkang Future Nutrition Technology Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2023980036259 Denomination of invention: A method for large-scale preparation of specific molecular weight small molecule hyaluronic acid Granted publication date: 20171121 License type: Common License Record date: 20230606 |
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