CN105907816A - Method for producing cycloamylose with enzymic method - Google Patents
Method for producing cycloamylose with enzymic method Download PDFInfo
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- CN105907816A CN105907816A CN201610438468.8A CN201610438468A CN105907816A CN 105907816 A CN105907816 A CN 105907816A CN 201610438468 A CN201610438468 A CN 201610438468A CN 105907816 A CN105907816 A CN 105907816A
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- cyclodextrin
- unit
- alpha
- glycosyl transferase
- absolute ethyl
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
Abstract
The invention discloses a method for producing cycloamylose with an enzymic method and belongs to the technical field of cyclodextrin production. According to the method, 4-alpha-glycosyl transferase is used for producing the cycloamylose by taking nature starch as a substrate, the blank in the technical field is filled up, and a foundation is laid for large-scale biological method production of the cycloamylose; the total reaction period of the method is short, and only 12 hours are need; the method has the advantages that the production cost is low, the product purity is high, the technological process is simple, the production period is short and the like.
Description
Technical field
The method that the present invention relates to a kind of Production by Enzymes big unit cyclodextrin, belongs to cyclodextrin production technical field.
Background technology
Cyclodextrin (cyclodextrin, CD) is the cyclodextrine being formed by connecting by the α of varying number-Isosorbide-5-Nitrae glycosidic bond, it can be common that
Alpha-cyclodextrin, beta-schardinger dextrin and gamma-cyclodextrin, its degree of polymerization is respectively 6,7,8；And big unit cyclodextrin (Cycloamylose, greatly
Unit cyclodextrin), refer to the degree of polymerization from 9 to hundreds of not wait cyclodextrine, also referred to as macrocyclic-cyclodextrin.
Owing to the degree of polymerization of big unit cyclodextrin is higher, and viscosity is low, good water solubility be difficult to bring back to life, and it is on structure and properties
Be different from the α of low polymerization degree-, β-and gamma-cyclodextrin.Common cyclodextrin forms the cavity structure being similar to tubbiness, and the degree of polymerization is relatively
Little big unit cyclodextrin forms an independent ring；When big ring cyclodextrin poly right bigger time, looped situation is complicated, its size
Relatively big with architectural difference, when big ring cyclodextrin poly is right rise to a certain degree time, two ring-type hydrophobic cavity structures can be formed.
Big unit cyclodextrin has not of uniform size, the hydrophobic cavity of configurations, and these hydrophobic channels are similar to the v-shaped of amylose molecule
Helical duct.The circulus of big unit cyclodextrin can change along with the change of guest molecule, solvent, there is unstability
And polytropy, can be used for the mixture that inclusion complicated component, molecular size are different.Common α-, β-and gamma-cyclodextrin make in a large number
Used time has certain toxicity so that it is the application in association area is restricted.And current research report, the most do not find big
The toxicity of unit's cyclodextrin.Therefore, big unit cyclodextrin is considerable in field prospects such as biology, food, medicine, chemical industry.
Big unit cyclodextrin cost of manufacture is high, and expensive, its preparation method mainly includes enzymatic clarification and chemical synthesis.Wakao
Deng with maltotriose for initial reaction substrate, by adjacent two acyl bridging molecule technique for fixing, through 26 steps, synthesized polymer degree is 9
Big unit cyclodextrin, but the structural complexity of glucide inhibits the development of chemical method synthesis big unit cyclodextrin.Not yet have at present
Utilizing the report of the 4-alpha-glycosyl single degree of polymerization of transferase enzymatic clarification big unit cyclodextrin, also not having can be with big unit cyclodextrin mixt
In the single degree of polymerization big unit the specific crystalline deposit of cyclodextrin organic solvent.The success such as Terada Y the most recombinant expressed from
The 4-alpha-glycosyl transferase gene of Thermus aquaticus ATCC 33923, demonstrate,proves and with the synthesis amylose of 0.02% (w/v) is
Substrate preparation big unit cyclodextrin, conversion ratio is about 90%；Xu Yan etc. utilize this enzyme the amylomaize with 1% (w/v)
For substrate, after de-pretreatment of isoamylase, big unit cyclodextrin conversion ratio is brought up to 45.6% by 24.6%.Enzyme process at present
Preparation unit's cyclodextrin subject matter greatly is: one, the most few for preparing the report of the 4-alpha-glycosyl transferase of big unit cyclodextrin,
It is concentrated mainly on recombinant expressed new gene, and studies its zymologic property and crystal structure aspect, less about its application study,
And the recombinant expressed amount of enzyme has much room for improvement；Two, lacking the big unit corresponding production technology of cyclodextrin, substrate amylose is expensive,
Native starch is that substrate preparation big unit cyclodextrin conversion ratio is relatively low.
Summary of the invention
Technical problem solved by the invention there is provided a kind of bioanalysis and produces the technique of big unit cyclodextrin, reduces big ring and sticks with paste
The production cost of essence.
The present invention uses 4-alpha-glycosyl transferase to produce big unit cyclodextrin, and (1) carries out starch according to the concentration of 1%-10% and sizes mixing,
Stir 10-15 minute under the conditions of 70 DEG C；(2) set reaction temperature 65-70 DEG C, adjust pH5.0-7.0, with every gram of amylometer,
It is separately added into the 4-alpha-glycosyl transferase of 30-70 unit, the Pullulanase of 30-70 unit, reacts about 12 hours；(3)
Absolute ethyl alcohol intermediate processing is used to obtain big unit cyclodextrin.Described starch is farina, tapioca, cornstarch.
In one embodiment of the invention, the amino acid sequence of described 4-alpha-glycosyl transferase is as shown in SEQ ID NO.1.
In one embodiment of the invention, step (1) carries out starch according to the concentration of 1%-10% and sizes mixing, and according to every gram
The ratio of 0.25 unit of starch adds 4-alpha-glycosyl transferase, stirs 10-15 minute under the conditions of 70 DEG C.
In one embodiment of the invention, step (1) carries out starch according to the concentration of 1% and sizes mixing.
In one embodiment of the invention, step (2) sets reaction temperature 65 DEG C.
In one embodiment of the invention, step (2) adjusts pH5.5.
In one embodiment of the invention, step (2), with every gram of amylometer, is separately added into the 4-alpha-glycosyl of 50 units
Transferase, the Pullulanase of 50 units.
In one embodiment of the invention, the step that step (3) uses absolute ethyl alcohol intermediate processing to obtain big unit cyclodextrin is
After question response terminates, take enzyme reaction solution supernatant, add the absolute ethyl alcohol of 8-10 times of volume, be centrifuged and obtain absolute ethyl alcohol and big ring
The complex compound sediment that dextrin is formed, stands overnight being deposited in 60 DEG C of baking ovens of obtaining, obtains unit's cyclodextrin crystallization greatly.
In one embodiment of the invention, the preparation of described 4-alpha-glycosyl transferase is under certain condition of culture, by micro-
Biofermentation certain time, obtaining zymotic fluid, zymotic fluid obtains thalline through centrifugal, thalline redissolve after through ultrasonication obtain 4-α-
Glycosyl transferase crude enzyme liquid.
The present invention is the technique that the feature according to 4-alpha-glycosyl transferase and product big unit cyclodextrin produces big unit cyclodextrin, relatively
In prior art, have the advantage that
1) production method of the big unit cyclodextrin with native starch as substrate of a kind of low cost is provided, has filled up in this technical field
Blank, produce for the big unit large-scale bioanalysis of cyclodextrin and lay a good foundation；
2) reaction time that this reaction is total is short, it is only necessary to 12 hours；
Generally speaking, the present invention have that production cost is low, product purity is high, technological process is simple, with short production cycle etc. many excellent
Point.
Detailed description of the invention
The determination method of 4-alpha-glycosyl transferase vigor: take respectively 0.25mL 0.5% soluble starch solution, 0.05mL
1% maltose solution and the Na of 0.6mL 50mM pH 5.52HPO4-citrate buffer solution is in test tube, and 70 DEG C of water-baths preheat
10min.Adding the enzyme liquid of 0.1mL dilution, concussion mixing, 70 DEG C of reactions 10min, boiling water bath 15min terminate reaction.
10mL iodine liquid (0.2%KI+0.02%I is added in above-mentioned reaction system2), mixing, under 620nm, measure its light absorption value.
Enzyme unit definition alive: live under measurement system at enzyme, the enzyme amount needed for degraded 1mg/ml starch per minute.
The analysis employing high performance liquid chromatography chromatographic condition of big unit cyclodextrin: Hitachi's Hitachi chromatograph, SuperMultipore
PW-N (6.0mm × 150mm) gel chromatographic columns, Hitachi's L-2490 Composition distribution, flow phase 0.05mM pH 7.0
Na2HPO4-citrate buffer solution, flow velocity 0.3mL/min；Column temperature is set as 50 DEG C.When processing sample, boiling water bath 30min
Go out enzyme, and in reactant liquor, the ratio according to 50 units of every gram of starch adds the carbohydrase in Rhizopus sp. source, Thermobifida
The isoamylase in fusca source and the Pullulanase in Bacillus deramificans source, at 40 DEG C of shaking baths after fully mixing
Middle reaction 10h；After reaction terminates, boiling water bath 30min goes out enzyme, reactant liquor 12000r/min is centrifuged 10min, takes supernatant,
Analyze with HPLC after membrane filtration (0.45 μm).
The preparation of embodiment 1 4-alpha-glycosyl transferase mutant and expression
According to the TaAM amino acid sequence on NCBI, (NCBI numbers: GI:6225654), designs primer, with buy
Thermus aquaticus ATCC 33923 genome is template, obtains TaAM genetic fragment by PCR amplification.
Forward primer: 5 '-GGGTTTCATATGGAGCTTCCCCGCGCTTTC-3 ',
Reverse primer: 5 '-CGGCCGGAATTCCTAGAGCCGTTCCGTGGC-3 ',
PCR reaction system is: 5 × PS buffer 10 μ L, dNTPs Mix (2.5mM) 4 μ L, forward primer (10 μMs) 0.5 μ L,
Reverse primer (10 μMs) 0.5 μ L, template DNA 1 μ L, DNA Polymerase 0.5 μ L, addition distilled water to 50 μ L.
PCR amplification condition is: 94 DEG C of denaturations 4min；30 circulations (98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 120s) subsequently；
72 DEG C are continued to extend 10min.
For build the plasmid of coli expression carrier be pET24a (+).By pET24a (+) plasmid and TaAM gene are respectively
Carrying out Nde I and Hind III double digestion, digestion products, after glue reclaims, connects overnight with T4 ligase, connects product and converts
To escherichia coli jm109 competent cell, converted product coats the LB flat board containing 100mg/L kanamycins, through 37 DEG C
Overnight incubation, 3 single bacterium colonies of picking on flat board, access LB fluid nutrient medium, after 8h, extract plasmid checking, result is correct,
Obtain enrichment TaAM/pET24a (+) plasmid.
The gene order of the 4-alpha-glycosyl transferase according to Thermus aquaticus ATCC 33923, designs and synthesizes introducing Y54
The primer of sudden change, carries out rite-directed mutagenesis to 4-alpha-glycosyl transferase gene, measures DNA encoding sequence.Mutant gene is placed in
Suitable expression vector importing in Escherichia coli is expressed, and obtains amino acid sequence single mutation as shown in SEQ ID NO.1
4-alpha-glycosyl transferase.The rite-directed mutagenesis of single mutation Y54G: utilize fast PCR technology, with expression vector TaAM/pET24a (+)
For template.
The rite-directed mutagenesis primer introducing Y54 sudden change is:
Forward primer: 5 '-GGCCCCACGGGCGGTGGCGACTCCCCCTAC-3 ' (underscore is mutating alkali yl),
Reverse primer: 5 '-GGGGGAGTCGCCACCGCCCGTGGGGCCCAA-3 ' (underscore is mutating alkali yl),
PCR reaction system is: 5 × PS buffer 10 μ L, dNTPs Mix (2.5mM) 4 μ L, forward primer (10 μMs) 0.5 μ L,
Reverse primer (10 μMs) 0.5 μ L, template DNA 0.5 μ L, DNA Ploymerase 0.5 μ L, addition distilled water to 50 μ L.
PCR amplification condition is: 94 DEG C of denaturations 4min；30 circulations (98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 7min) subsequently；
72 DEG C are continued to extend 10min.
PCR primer digests through Dpn I, converts e. coli jm109 competence, and competent cell is at LB solid medium
After (containing 100mg/L ampicillin) overnight incubation, choose and be cloned in LB fluid nutrient medium (containing 100mg/L ampicillin)
Extract plasmid after middle cultivation, mutant plasmid is converted and expresses host e. coli BL21 (DE3) competent cell.
Recombinant bacterium is inoculated in LB fluid nutrient medium (containing 100mg/L kanamycins) growth 8h, by 5% inoculum concentration by seed
Access TB liquid fermentation medium (containing 100mg/L kanamycins).After Escherichia coli cultivate 2h at 37 DEG C, add 0.2mM
The IPTG (isopropylthio-β-D galactoside) of final concentration induces, and after 30 DEG C of shaking tables continue cultivation and fermentation 24h,
By certain volume zymotic fluid in 4 DEG C, 12000r min-1Centrifugal 10min removes supernatant, collects thalline, and bacterial sediment is with 50
mmol·L-1pH 7.0Na2HPO4-NaH2PO4Buffer solution Eddy diffusion, mixing.Thalline is crushed with ultrasonic cell disruptor
The cell membrane of suspension (condition of work of ultrasonic cell disintegration machine:Work probe, working time 5min, work 3s stop
3s, operating power is 20%), then 12000r min-1Centrifugal 10min, centrifugal rear supernatant is fermentation intracellular crude enzyme liquid,
After measured, enzyme is lived as 120U/mL.
Embodiment 2
(1) pretreatment of raw material:
Concentration according to 1% carries out farina and sizes mixing, and stirs 10-15 minute, make starch granules abundant under the conditions of 70 DEG C
Swelling；
(2) enzymatic production process:
After pretreatment, temperature is set as 65 DEG C, adjusts after pH5.5, according to the ratio of 50 units of every gram of starch be separately added into 4-α-
Glycosyl transferase and Pullulanase, fully reaction 12 hours.Boiling water bath 30min goes out enzyme, according to every gram of starch in reactant liquor
The ratio of 50 units add the carbohydrase in Rhizopus sp. source, the isoamylase in Thermobifida fusca source and
In 40 DEG C of shaking baths, 10h is reacted after the Pullulanase in Bacillus deramificans source, fully mixing；Reaction terminates
After, boiling water bath 30min goes out enzyme, reactant liquor 12000r/min is centrifuged 10min, takes supernatant, through membrane filtration (0.45 μm)
Analyze with HPLC afterwards.Result shows, the conversion ratio of big unit cyclodextrin (the big unit cyclodextrin quality/starch substrates quality of generation ×
100%) 43.2% is reached.
After question response terminates, taking enzyme reaction supernatant, add 10 times of volume absolute ethyl alcohols, 12000r/min is centrifuged 10min and obtains
The complex compound sediment that absolute ethyl alcohol and big unit cyclodextrin are formed, stands overnight being deposited in 60 DEG C of baking ovens of obtaining after abandoning supernatant,
Obtaining unit's cyclodextrin crystallization greatly, purity reaches 94%.
Embodiment 3
(1) pretreatment of raw material:
Concentration according to 5% carries out farina and sizes mixing, and adds 4-alpha-glycosyl according to the ratio of 0.25 unit of every gram of starch and turns
Move enzyme to stir 10-15 minute under the conditions of 70 DEG C, make starch granules the most swelling；
(2) enzymatic production process: with embodiment 2
Result shows, the conversion ratio of big unit cyclodextrin reaches 29.6%.
Embodiment 4
(2) pretreatment of raw material:
Concentration according to 5% carries out farina and sizes mixing, and adds 4-alpha-glycosyl transferase according to the ratio of 1 unit of every gram of starch
Stir 10-15 minute under the conditions of 70 DEG C, make starch granules the most swelling；
(2) enzymatic production process: with embodiment 2
Result shows, the conversion ratio of big unit cyclodextrin reaches 22.3%.
Embodiment 5
On the basis of embodiment 2, when step (2) system pH is adjusted to 7-9, the conversion ratio of big unit cyclodextrin declines 32.5～70%.
Embodiment 6
On the basis of embodiment 2, the consumption of 4-alpha-glycosyl transferase in step (2) is adjusted to 80 units of every gram of starch
4-alpha-glycosyl transferase, the conversion ratio of big unit cyclodextrin declines 12%.
Embodiment 7
On the basis of embodiment 2, by when in step (2), reaction temperature is adjusted to 55 DEG C, under the conversion ratio of big unit cyclodextrin
Fall 47.2%.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (7)
1. the method producing big unit cyclodextrin, it is characterised in that comprise the following steps:
(1) carry out starch according to the concentration of 1%-10% to size mixing, stir 10-15 minute under the conditions of 70 DEG C；(2) reaction is set
Temperature 65-70 DEG C, adjusts pH5.0-7.0, with every gram of amylometer, is separately added into the 4-alpha-glycosyl transferase of 30-70 unit, 30-70
The Pullulanase of individual unit, reacts about 12 hours；(3) absolute ethyl alcohol intermediate processing is used to obtain big unit cyclodextrin；Described shallow lake
Powder is farina, tapioca, cornstarch；The amino acid sequence such as SEQ ID NO.1 of described 4-alpha-glycosyl transferase
Shown in.
Method the most according to claim 1, it is characterised in that step (1) carries out starch according to the concentration of 1%-10%
Size mixing, and add 4-alpha-glycosyl transferase according to the ratio of 0.25 unit of every gram of starch, under the conditions of 70 DEG C, stir 10-15 divide
Clock.
Method the most according to claim 1, it is characterised in that step (1) carries out starch according to the concentration of 1% and sizes mixing.
Method the most according to claim 1, it is characterised in that step (2) sets reaction temperature 65 DEG C.
Method the most according to claim 1, it is characterised in that step (2) adjusts pH5.5.
Method the most according to claim 1, it is characterised in that step (2), with every gram of amylometer, is separately added into 50
The 4-alpha-glycosyl transferase of unit, the Pullulanase of 50 units.
Method the most according to claim 1, it is characterised in that step (3) uses absolute ethyl alcohol intermediate processing to obtain greatly
The step of unit's cyclodextrin is after question response terminates, and takes enzyme reaction solution supernatant, adds the absolute ethyl alcohol of 8-10 times of volume, is centrifuged and obtains
The complex compound sediment that absolute ethyl alcohol and big unit cyclodextrin are formed, stands overnight being deposited in 60 DEG C of baking ovens of obtaining, obtains unit greatly
Cyclodextrin crystallization.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998431A (en) * | 2018-07-25 | 2018-12-14 | 南京林业大学 | A method of improving 4- alpha-glycosyl transferase Soluble expression levels |
| CN110628872A (en) * | 2019-09-19 | 2019-12-31 | 华东师范大学 | Method for primarily screening bacterial strains with high 4-alpha-glycosyltransferase enzyme activity by using high-throughput flat plate |
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| CN101633900A (en) * | 2009-07-02 | 2010-01-27 | 江南大学 | Method for preparing cycloamylose 4-alpha-glycosyl transferase production |
| CN101696441A (en) * | 2009-10-19 | 2010-04-21 | 江南大学 | Method for preparing large cyclodextrin |
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| CN101633900A (en) * | 2009-07-02 | 2010-01-27 | 江南大学 | Method for preparing cycloamylose 4-alpha-glycosyl transferase production |
| CN101696441A (en) * | 2009-10-19 | 2010-04-21 | 江南大学 | Method for preparing large cyclodextrin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998431A (en) * | 2018-07-25 | 2018-12-14 | 南京林业大学 | A method of improving 4- alpha-glycosyl transferase Soluble expression levels |
| CN108998431B (en) * | 2018-07-25 | 2019-07-23 | 南京林业大学 | A method of improving 4- alpha-glycosyl transferase Soluble expression levels |
| CN110628872A (en) * | 2019-09-19 | 2019-12-31 | 华东师范大学 | Method for primarily screening bacterial strains with high 4-alpha-glycosyltransferase enzyme activity by using high-throughput flat plate |
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Application publication date: 20160831 |