CN108359664A - The preparation method and its application method of the starch-containing invertase cell of immobilization - Google Patents
The preparation method and its application method of the starch-containing invertase cell of immobilization Download PDFInfo
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- CN108359664A CN108359664A CN201810089198.3A CN201810089198A CN108359664A CN 108359664 A CN108359664 A CN 108359664A CN 201810089198 A CN201810089198 A CN 201810089198A CN 108359664 A CN108359664 A CN 108359664A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of preparation method and its application method of the starch-containing invertase cell of immobilization；Include the following steps：(1) buffer solution and distilled water are added into starch-containing invertase thalline, bacteria suspension is made, carrier is then added, stirs evenly；(2) sodium alginate and polyvinyl alcohol is added, stirs evenly, obtains mixed solution；(3) mixed solution is instilled into CaCl2Boric acid solution in, cure 5~15h, obtain the starch-containing invertase cell of ball shape fixation；Preparation method provided by the present invention has immobilization material at low cost, it is easy to operate, the advantages such as reusability is good, the starch-containing invertase cell prepared produces α ursin for catalysing sucrose and quinhydrones, after 20 batches of continuous conversion, immobilized cell enzyme activity is maintained as 50% or more free cell enzyme activity, has good industrial applications foreground.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of preparation side of the starch-containing invertase cell of immobilization
Method and its application method.
Background technology
Alpha-arbutin is the epimer of β-ursin, the entitled 4- hydroxyphenyl-α-D- glucopyranosides of chemistry.It grinds
Study carefully and show that alpha-arbutin is more stable compared with property for β-ursin, there is preferably anti-inflammatory, reparation and white-skinned face function, frequently as
Brightening agent is applied in high-end skin care item and cosmetics.
Currently, the main preparation methods of alpha-arbutin are free cell catalysis methods, main problem is that：Using free
Cell catalysis produces alpha-arbutin product subsequent treatment process complexity, and equipment investment and operating cost are larger；Catalyst is to environment
It is resistant to force difference, and cannot be recycled, the cost of production is improved.Such as patent CN201210508199.X, describe profit
With the method for xanthomonas campestris Synthesis alpha-arbutin, transformation efficiency is low, alpha-arbutin content about 10g/ in conversion fluid
L.For another example patent CN201610130457.3 is described and is catalyzed and synthesized alpha-arbutin using zymoprotein and what is recrystallized repeatedly carries
Pure technique is needed to produce zymoprotein and be reacted, and when purifying needs to recrystallize repeatedly using pure water, and production cost is high, conversion effect
Rate is low.
Invention content
The primary purpose of the present invention is that providing a kind of starch-containing invertase of immobilization improving cell activity and stability
The preparation method of cell.
To achieve the above object, the technical solution adopted by the present invention is：A kind of system of the starch-containing invertase cell of immobilization
Preparation Method includes the following steps：
(1) buffer solution and distilled water are added into starch-containing invertase thalline, bacteria suspension is made, carrier is then added, stirs
It mixes uniformly；
(2) sodium alginate and polyvinyl alcohol is added, stirs evenly, obtains mixed solution；
(3) mixed solution is instilled into CaCl2Boric acid solution in, cure 5~15h, obtain the starch-containing sugarcane of ball shape fixation
Carbohydrase cell.
The advantageous effect of above-mentioned technology hair is：Preparation method provided by the present invention has immobilization material at low cost,
Easy to operate, the starch-containing invertase cell of the advantages such as reusability is good, preparation produces α-bear for catalysing sucrose and quinhydrones
Fruit glycosides, after continuously converting 20 batches, immobilized cell enzyme activity is maintained as 50% or more free cell enzyme activity, has good work
Industry application prospect.
It is another object of the present invention to provide a kind of users using the starch-containing invertase cell of above-mentioned immobilization
Method can improve the yield of alpha-arbutin, shorten the production cycle.
Steps are as follows：The starch-containing invertase cell of ball shape fixation is added into sucrose and quinhydrones, mixed liquor is 28~35
3~6h is converted at DEG C, obtains alpha-arbutin；
Each substance is a concentration of in the mixed liquor：180~300g/L of sucrose, 5~20g/L of quinhydrones；Ball shape fixation contains
260~300g/L of amylosucrase cell.
Description of the drawings
Fig. 1 is that immobilized cell (being prepared by embodiment 2) and free cell (being prepared by embodiment 1) repeat to make
With batch yield comparison diagram.
Specific implementation mode
The present invention further disclosed below by way of 3 embodiments, and in following embodiment, diatomite molecular formula is SiO2,
Molecular weight is 60.08,19.6 μm of median, and proportion 0.32 can absorb water of 4 times more than itself；It is dissolved in concentrated base and hydrofluoric acid,
Not soluble in water, sour or diluted alkaline is purchased from Aladdin company.Perlite, chemical composition SiO2：70~75%, Al2O3：12~16%,
Na2O：1.0%~4.0%, K2O：1.0%~4.0%, appearance white particle, proportion 0.09, fusing point：1280~1350 DEG C, purchase
From Hangzhou Jin great Lv industrial technologies Co., Ltd.Activated carbon, molecular formula C, molecular weight 12.01, proportion 1.8, to organic color
Element and nitrogenous base have the adsorption capacity of high power capacity, are purchased from Aladdin company.
Embodiment 1：The preparation and performance measurement of starch-containing invertase thalline
The recombination engineering bacteria of starch-containing saccharase gene is seeded to slant medium, 37 DEG C of culture 12h are obtained oblique
Face thalline；Slant medium group becomes：10g/L peptones, 10g/L sodium chloride, 5g/L yeast powders, 20g/L agar, solvent are
Water, pH are natural；
Inclined-plane thalline is seeded to seed culture medium, 37 DEG C of culture 12h obtain seed liquor；Seed culture medium forms：10g/
L peptones, 10g/L sodium chloride, 5g/L yeast powders, solvent are water, and pH is natural；
Seed liquor is seeded to fermentation medium with 3.3% inoculum concentration of volumetric concentration, 37 DEG C, cultivate under the conditions of 150rpm
Then 2h is added 10g/L lactose inducements, 28 DEG C, 12h is induced under the conditions of 150rpm, zymotic fluid is taken to centrifuge, collect wet thallus,
Obtain starch-containing invertase thalline；
Fermentation medium forms：Potassium dihydrogen phosphate 1%, dipotassium hydrogen phosphate 1%, ammonium sulfate 0.5%, magnesium sulfate 0.1%, one
Water citric acid 0.1%, fumaric acid 0.1%, sodium glutamate 0.1%, ferric citrate 0.05%, sodium dihydrogen phosphate 0.1%, phosphoric acid
Disodium hydrogen 0.1%, glycerine 1%, ferrous sulfate heptahydrate 0.005%, anhydrous calcium chloride 0.005%, cupric sulfate pentahydrate 0.001%,
CoCL2 6H2O 0.001%, white vitriol 0.001%, manganese sulfate monohydrate 0.005%, Sodium Molybdate Dihydrate 0.005%, six water
Nickel chloride 0.005%, boric acid 0.005%, antifoaming agent 0.002%.
It should be noted that the recombination engineering bacteria of above-mentioned starch-containing saccharase gene is according to Chinese invention patent Shen
Please the construction methods of genetic engineering bacterium of the disclosed production alpha-arbutins of publication No. CN106148256A obtain.
Free cell enzyme activity and repeat performance measure：
It takes the wet thallus of collection in 10ml pH7.0 phosphate buffers, makes the final concentration of 10OD of thalline, 2.4g is added
Sucrose and 0.132g quinhydrones, 30 DEG C of reaction 10min, sampling, HPLC detect the concentration of product alpha-arbutin, are defined and counted according to enzyme activity
Calculate free cell enzyme activity.
Take free cell prepared by step (1) that 100mL phosphate buffers (pH=7.0,100mM) are housed in 250mL
In three-necked flask, 24g substrates sucrose and 1.32g quinhydrones is added in cell concentration 10OD, and 30 DEG C, 200rpm speeds of agitator react
4h, after vacuum filtration is separated by solid-liquid separation, conversion fluid detects production concentration with HPLC, calculates yield.Free cell through washing three times
After washing, next group conversion is carried out, calculates per a batch of yield, calculates enzyme activity.
Enzyme activity defines：30 DEG C, under the conditions of pH=7.0, unit OD cells are catalyzed the alpha-arbutin institute for generating 1mmol per hour
The enzyme amount needed is defined as 1U.
HPLC conditions：Pillar：WondaSil C18 reversed-phase columns, 4.6*150mm.Sample size：10uL.Column temperature：35℃.Flowing
Phase：5% methanol aqueous solution.Flow velocity：1.0mL/min.Detection wavelength：280nm.α-Arbutin appearance times：4.8min；
Hydroquinone appearance times：6.0min；
According to the above method, gained free cell performance is as follows：The enzyme activity of free cell containing sucrose phosphorylase is 29.5U,
Enzyme activity is 11.5% after operating continuously 3 batches under 240g/L sucrose and 13.2g/L quinhydrones concentration, as a result such as attached drawing 1.
Embodiment 2：The preparation of the starch-containing invertase cell of immobilization
(1) the Tris-HCl solution (pH of 0.05mol/L is added in the starch-containing invertase thalline in Example 1 thereto
=7.0,100mM) and distilled water, 100ml bacteria suspensions are made, bacteria concentration is OD 20, and 40g diatomite is then added, and stirring is equal
It is even；
(2) 100ml 6wt% sodium alginates and 20wt% polyvinyl alcohol is added, stirs evenly, obtains mixed solution, mixes
Starch-containing invertase thalline 10OD, carrier 20g/L, sodium alginate 3wt%, polyvinyl alcohol 10wt% in solution；
(3) mixed solution is instilled to the CaCl of 10g/L with the rate of addition of 100 drops/min210g/L boric acid solution
In (pH 7.0), cure 10h at 25 DEG C, obtain the starch-containing invertase cell of ball shape fixation of a diameter of 2~5mm, cell exists
It is stored at 4 DEG C.
Embodiment 3：The synthesis and performance measurement of alpha-arbutin
The starch-containing invertase cell 3.4g of ball shape fixation made from Example 2 is in 10ml pH7.0 phosphate buffers
In, 2.4g sucrose and 0.132g quinhydrones, 30 DEG C of reaction 10min, sampling, the concentration of HPLC detection product alpha-arbutins, root is added
It is defined according to enzyme activity and calculates immobilized cell enzyme activity；Immobilized cell enzyme activity test method is the same as embodiment 1.Immobilized cell enzyme activity is removed
The immobilized cell enzyme activity rate of recovery is calculated with free cell enzyme activity.
The starch-containing invertase cell 34g of ball shape fixation made from Example 2 is slow equipped with 100mL phosphate in 250mL
In the three-necked flask of fliud flushing (pH=7.0,100mM), addition 24g sucrose and 1.32g quinhydrones, 30 DEG C, 200rpm speeds of agitator, instead
After answering 4h, vacuum filtration to be separated by solid-liquid separation, conversion fluid detects production concentration with HPLC, calculates yield.Immobilized cell is through three
After secondary washing, next group conversion is carried out, is calculated per a batch of yield.
The result shows that：Immobilized cell performance is as follows：Immobilized cell enzyme activity is 27U, and the enzyme activity rate of recovery is 91.5%, even
20 batch enzyme activity of continuous operation retain 53.0%, as a result such as attached drawing 1.
Embodiment 4：The preparation of the starch-containing invertase cell of immobilization
Preparation method with embodiment 2, the difference is that, carrier is activated carbon in step (1), alginic acid in step (2)
Na concn is 4wt%, polyvinyl alcohol concentration 16wt%.
After measured, gained immobilized cell performance is as follows：Immobilized cell enzyme activity is 26.11U, and the enzyme activity rate of recovery is
88.5%, 20 batch enzyme activity of continuous operation retain 52.0%.
Embodiment 5：The preparation of the starch-containing invertase cell of immobilization
Preparation method with embodiment 2, the difference is that, carrier is perlite in step (1), alginic acid in step (2)
Na concn is 8wt%, polyvinyl alcohol concentration 12.4wt%.
After measured, gained immobilized cell performance is as follows：Immobilized cell enzyme activity is 24.6U, and the enzyme activity rate of recovery is
83.5%, 20 batch enzyme activity of continuous operation retain 50.5%.
Embodiment 6：The preparation of the starch-containing invertase cell of immobilization
Preparation method with embodiment 2, the difference is that, carrier is perlite in step (1), CaCl in step (3)2It is dense
Degree is 8g/L, boric acid concentration 15g/L.
After measured, gained immobilized cell performance is as follows：Immobilized cell enzyme activity is 23.6U, and the enzyme activity rate of recovery is 80%,
It operates continuously 20 batch enzyme activity and retains 50%.
Claims (9)
1. a kind of preparation method of the starch-containing invertase cell of immobilization, which is characterized in that include the following steps：
(1) buffer solution and distilled water are added into starch-containing invertase thalline, bacteria suspension is made, carrier is then added, stirring is equal
It is even；
(2) sodium alginate and polyvinyl alcohol is added, stirs evenly, obtains mixed solution；
(3) mixed solution is instilled into CaCl2Boric acid solution in, cure 5~15h, it is thin to obtain the starch-containing invertase of ball shape fixation
Born of the same parents.
2. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that described containing shallow lake
Powder invertase thalline is by the recombination engineering containing amylosucrase encoding gene through inclined-plane culture, seed culture, fermented and cultured
After obtain.
3. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(1) the Tris-HCl solution that the buffer solution in is 0.01~1mol/L.
4. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(1) carrier in is one kind in diatomite, activated carbon, perlite.
5. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(2) each substance is a concentration of in mixed solution：Starch-containing invertase thalline 8~12OD, 15~25g/L of carrier, sodium alginate 2
~5wt%, 8~15wt% of polyvinyl alcohol.
6. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(3) rate of addition of mixed solution is 70~120 drops/min in.
7. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(3) CaCl2Boric acid solution in, CaCl2A concentration of 5~15g/L, a concentration of 8~12g/L of boric acid, pH value of solution 6.5
~7.5.
8. the preparation method of the starch-containing invertase cell of immobilization according to claim 1, which is characterized in that the step
(3) the starch-containing invertase cell dia of ball shape fixation in is 2~5mm, and the enzyme activity rate of recovery is 80~91.5%.
9. a kind of method synthesizing alpha-arbutin using the starch-containing invertase cell of immobilization, which is characterized in that steps are as follows：To
The starch-containing invertase cell of ball shape fixation is added in sucrose and quinhydrones, mixed liquor converts 3~6h at 28~35 DEG C, obtains α-
Ursin；
Each substance is a concentration of in the mixed liquor：180~300g/L of sucrose, 5~20g/L of quinhydrones；Ball shape fixation is starch-containing
260~300g/L of invertase cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004135A (en) * | 2019-03-18 | 2019-07-12 | 合肥华恒生物工程有限公司 | A kind of method of ball shape fixation hydrolase containing D-pantoyl lactone cell and preparation method thereof and hydrolysis D-pantoyl lactone |
| CN112725327A (en) * | 2021-02-26 | 2021-04-30 | 华东理工大学 | Preparation method and application of cell immobilization carrier |
| CN114058660A (en) * | 2021-10-15 | 2022-02-18 | 安徽华恒生物科技股份有限公司 | Immobilized enzyme conversion synthesis method and application of alpha-arbutin |
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| CN110004135A (en) * | 2019-03-18 | 2019-07-12 | 合肥华恒生物工程有限公司 | A kind of method of ball shape fixation hydrolase containing D-pantoyl lactone cell and preparation method thereof and hydrolysis D-pantoyl lactone |
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| CN114058660A (en) * | 2021-10-15 | 2022-02-18 | 安徽华恒生物科技股份有限公司 | Immobilized enzyme conversion synthesis method and application of alpha-arbutin |
| CN114058660B (en) * | 2021-10-15 | 2023-06-27 | 安徽华恒生物科技股份有限公司 | Immobilized enzyme conversion synthesis method of alpha-arbutin and application |
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