CN106282211A - A kind of method of recombination bacillus coli resting cell synthesis D psicose - Google Patents
A kind of method of recombination bacillus coli resting cell synthesis D psicose Download PDFInfo
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- CN106282211A CN106282211A CN201610821862.XA CN201610821862A CN106282211A CN 106282211 A CN106282211 A CN 106282211A CN 201610821862 A CN201610821862 A CN 201610821862A CN 106282211 A CN106282211 A CN 106282211A
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- psicose
- dpe
- bacillus coli
- recombination bacillus
- epimerase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- 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
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
The method that the invention discloses a kind of recombination bacillus coli resting cell synthesis D psicose, belongs to biological technical field field.D psicose is a kind of very important rare sugar, is with a wide range of applications in fields such as food, medicine, health cares.D psicose 3 epimerase of the present invention has good pH toleration, and Co2+Substantially increase the heat stability of the full cell of DPE.Under the suitableeest reaction condition, resting cell synthesizes D psicose in a large number, and conversion ratio is 34%.Relative to pure enzymatic conversion, resting cell synthesis D psicose have technique simple, without enzyme is carried out isolated and purified, need not numerous and diverse purge process thus cost-effective.
Description
Technical field
The method that the present invention relates to a kind of recombination bacillus coli resting cell synthesis D-Psicose, belongs to biotechnology
Field.
Background technology
Rare sugar refers to exist in nature but the few class list carbohydrates and their derivative of content.Rare sugar is generally of
The sugariness of sucrose, but body not energy metabolism or metabolism are little, can be as the substitute of high heat sugar.Rare sugar is food, doctor
The field such as medicine, health care is with a wide range of applications.
D-Psicose is the epimer of D-Fructose C-3 position, is the low-down rare sugar of content in nature.D-Ah
The advantages such as Lip river ketose has that sugariness is high, dissolubility is good, low-calorie and hypoglycemic reaction, it is possible to as optimal cane sugar substitution
One of product.In food, add D-Psicose, its degree of gelation can not only be improved, it is also possible to food protein, Mei Lade occurs
Reaction improves its local flavor.2011, D-Psicose, can be in food and the conduct of meals field by U.S. FDA certification safety
Additive uses.In medicines and health protection field, D-Psicose can reduce body fat by suppression lipase and alpha-glucosidase
Accumulation and suppression blood sugar concentration rising.In meals, add D-Psicose can reduce post-prandial glycemia reaction, improve pancreas
The sensitivity of island element and glucose tolerance.Additionally, D-Psicose can more efficiently scavenging capacity oxygen-derived free radicals, in treatment
The relevant diseases such as nervous tissue's degeneration and atherosclerosis have potential function.
D-Psicose is few at nature content and is difficult to be synthesized by chemistry route.Owing to biotransformation method has instead
The advantages such as mild condition, by-product are few, purification step is simple, environmental protection are answered to be increasingly becoming the main of D-Psicose synthesis
Direction.The bioconversion of D-Psicose is broadly divided into resting cell and utilizes pure enzyme or to carrying out after pure enzyme immobilization turning
Change.Synthetic method currently mainly uses with D-Fructose as substrate, at D-Psicose 3-epimerase (D-Psicose 3-
Epimerase, is abbreviated as DPE) effect under synthesize D-Psicose.Currently with resting cell synthesis D-Psicose
Reporting fewer, relative to pure enzymatic conversion, resting cell synthesis D-Psicose technique is simple, more dives for commercial Application
Power.
Summary of the invention
First purpose of the present invention is to provide a kind of D-Psicose-3-epimerase gene, its nucleotides sequence
Row are as shown in SEQ ID NO.1.
Second object of the present invention is to provide the carrier containing described gene or cell line.
Third object of the present invention is to provide the D-Psicose-3-epimerase of described gene code.
Fourth object of the present invention is to provide a kind of recombination bacillus coli, is to be with escherichia coli Rosetta (DE3)
Host, expresses D-Psicose-3-epimerase gene shown in SEQ ID NO.1.
5th purpose of the present invention is to provide the construction method of described recombination bacillus coli, be by D-Psicose-
3-epimerase gene dpe with pET28a is connected, construction recombination plasmid pET28a-dpe, then recombiant plasmid proceeds to large intestine bar
Bacterium Rosetta (DE3).
6th purpose of the present invention is the method providing a kind of resting cell synthesis D-Psicose, is with D-fruit
Sugar is substrate, adds described recombination bacillus coli wet thallus, 60～70 DEG C of reaction 0.5-6h.
In one embodiment of the invention, by the 37 DEG C of cultivations in LB culture medium of described recombination bacillus coli, extremely
OD600When being 0.6～1.0, add final concentration of 0.1-1.0mM IPTG, 16-37 DEG C of induction 5～20h, collect wet thallus.
In one embodiment of the invention, described induction is to induce 10～20h at 16 DEG C.
In one embodiment of the invention, the buffer dilution of described wet thallus pH 7.5, and 4 DEG C of preservations.
In one embodiment of the invention, described wet thallus concentration is 2.5～30mg/mL.
In one embodiment of the invention, the described pH value of solution containing D-Fructose is 7～10, and adds 0.3-0.7mM
Metal ion；Described metal ion includes Co2+, Mn2+。
The present invention also provides for described D-Psicose-3-epimerase in food, medicine, the application of field of health care products,
Described application includes: preparation food additive, prepares the food of auxiliary hyperglycemic blood fat reducing, medicine or health product, and preparation
Product for scavenging capacity oxygen-derived free radicals.
Beneficial effect: the psicose-3-epimerase that the present invention provides has good temperature stability, at pH7
～the conversion ratio converting production D-Psicose under conditions of 10 reaches more than 20%.The present invention use resting cell produce D-Ah
Lip river ketose, conversion ratio is up to 34%.Relative to the pure enzyme transforming process of D-Psicose, resting cell synthesis D-Psicose without
Need isolated and purified enzyme, there is the features such as simple, the low cost of technique, be more suitable for large-scale application.
Accompanying drawing explanation
Fig. 1 is that SDS-PAGE detects DPE enzyme expression in escherichia coli；1: albumen marker；2: complete before induction
Cell；3: the full cell of induction 16h；4: cell lysate supernatant；5: cell pyrolysis liquid precipitates；
Fig. 2 is that DPE enzyme at different temperatures is lived；
Fig. 3 is the metal ion impact on the stability of DPE；A, adds Co2+；B, is not added with Co2+；
Fig. 4 is the conversion ratio that resting cell produces D-Psicose.
Detailed description of the invention
Enzyme activity determination method: add appropriate enzyme liquid in reaction system, after 65 DEG C of reaction 20min, high-temperature boiling 5min
To terminate reaction.At 15000rpm, reaction system is centrifuged 15min, and supernatant crosses the moisture film of 0.22 μM, passes through high-efficient liquid phase color
Spectrum detects whether the generation of D-Psicose product.
The definition that enzyme is lived: at 65 DEG C, enzyme required for the D-Psicose of generation 1 μm ol per minute under conditions of pH 8.5
Amount be defined as 1U；Enzyme relatively is lived and is defined as the percentage ratio that maximum enzyme is lived.
HPLC testing conditions: use chromatographic column HPX-87H (300 × 7.8mm), using 5mM sulphuric acid as flowing phase, flow velocity
0.5mL/min, column oven temperature 60 C, detects with Composition distribution.
The structure of embodiment 1 recombination bacillus coli
The dpe genetic fragment obtained with codon optimization, as template, expands dpe gene, it is thus achieved that such as SEQ ID NO.1
Shown sequence.The primer of amplification dpe gene is as follows:
Forward primer: 5-GCGCGGATCCATGAAGCACGGTATCTATTA-3(BamHI)
Downstream primer: 5-CCGGAAGCTTGGAGTGTTTGTGACATTCTA-3(HindIII)
PCR condition: 94 DEG C of degeneration 2min；98 DEG C of degeneration 30s；50 DEG C of annealing 30s, 68 DEG C extend 2min.Use BamHI and
The dpe gene 37 DEG C that pET-28a and PCR is expanded by two kinds of restricted enzyme of HindIII carries out enzyme action, with T4 ligase 16 DEG C
It is attached.To connect product and convert bacillus coli DH 5 alpha, and screen with the flat board with kalamycin resistance, picking converts
Son carries out plasmid extraction, digestion verification and gene sequencing.Ground recombiant plasmid pET28a-dpe will be successfully constructed and proceed to escherichia coli
In Rosetta (DE3), picking positive transformant, obtain recombination bacillus coli.
Prepared by the full cell of embodiment 2 recombination bacillus coli
By recombination bacillus coli Rosetta (DE3)/pET28a-dpe containing final concentration of 50 μ g/mL kanamycin
In LB culture medium, 37 DEG C of cultivations, treat OD600When about 0.8, add final concentration of 0.1mM IPTG induction, 16 DEG C of induction 20h, use
SDS-PAGE detects DPE enzyme expression in escherichia coli, and result is as shown in Figure 1.Thalline is collected after having induced, it is thus achieved that
Sufficient E. coli whole cell.
The determination of embodiment 3DPE optimum temperature
Detection DPE activity at different temperatures, as shown in Figure 2.DPE has preferable temperature stability at 60～70 DEG C, when
Temperature is 65 DEG C, and the DPE activity of resting cell is the highest, and when 45 DEG C, activity can reach more than 50%, under the high temperature of 70 DEG C
The enzyme still possessing about 90% is lived.
The temperature stability of embodiment 4DPE
By collect wet thallus (60 DEG C, 65 DEG C, 70 DEG C) at different temperatures place different time (0.5,1,2,4,8h),
Taking out wet thallus at set intervals, measure relative enzyme and live, result is as shown in table 1, and DPE has preferable stability at 60 DEG C, puts
Putting 1h, relative enzyme work can maintain 53.9%.Along with the rising of temperature, DPE heat stability is gradually lowered, 80 DEG C of placements
After 30min, enzyme is lived and is almost completely lost.
Under table 1 different temperatures, the relative enzyme of DPE lives (%)
Embodiment 5 metal ion improves DPE enzyme and lives
In order to measure the impact that resting cell DPE enzyme is lived by metal ion, in the presence of different metal ion, with 50mM
Tris-HCl (pH 8.5) is as buffer, and measuring concentration is that the wet thallus of 2.5mg/mL relative enzyme at 65 DEG C is lived.By table 2
Understanding, when being added without any metal ion in reaction system, DPE is active.When adding EDTA chela in reaction system
When closing all of metal ion, DPE loses activity.Co is added in reaction system2+And Mn2+, enzyme is lived and is significantly improved, and divides
Do not improve 157.19 and 126.06.
The impact on D-Psicose output of the table 2 each metal ion species
By the wet thallus collected in different temperatures (55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C) at Co2+(0.5mM) (with not in the presence of
Add Co2+For comparison), place different time (0.5,1,2,4,8h), take out wet thallus, measure relative enzyme and live.From the figure 3, it may be seen that add
Enter Co2+After ion, the heat stability of DPE is very significantly improved.When 65 DEG C, add Co2+, DPE relative activity after reaction 6h
Remain at more than 30% (Fig. 3-A)；It is added without Co2+, reaction 1h DPE substantially loses enzyme and lives (Fig. 3-B).
Resting cell synthesis D-Psicose under the different cell concentration of embodiment 6
Different cell concentrations (1mg/mL, 2.5mg/mL, 5mg/mL, 10mg/mL, 20mg/mL, 30mg/mL) is used to exist
65℃、pH 7.5、0.5mM Co2+Under the conditions of react 20min measure D-Psicose conversion ratio.Result is as shown in table 3, works as bacterium
During bulk concentration 2.5～30mg/mL, conversion ratio is more than 23%.
The impact on resting cell of table 3 cell concentration
Resting cell synthesis D-Psicose under the different pH of embodiment 7
Under condition of different pH, measure 2.5mg/mL wet thallus at 65 DEG C, 0.5mM Co2+In the presence of DPE relative enzyme live,
And calculate the conversion ratio of Synthesis D-Psicose.Result is as shown in table 4, and the optimum pH of DPE is 8.5, and this enzyme has necessarily
Degrees, under weak acid or weak base, the enzyme still possessing more than 50% is lived, and DPE is in the range of 7～10 at pH,
Conversion ratio is higher than 20%.
The table 4pH impact on DPE resting cell synthesis D-Psicose
Embodiment 8 resting cell synthesis D-Psicose
With 100g/L D-Fructose as substrate, the full cell concentration of wet thallus is 25mg/mL, at 65 DEG C, and pH 8.5,0.5mM
Co2+10mL reaction system in, add appropriate wet thallus, 65 DEG C of reactions, substrate D-fruit in high-performance liquid chromatogram determination supernatant
The consumption of sugar and the production of product D-Psicose, as shown in Figure 4, when 40 minutes, reaction reaches poised state, D-to result
The conversion ratio of psicose reaches 34%.
Although the present invention is open the most as above with preferred embodiment, but 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 with being as the criterion that claims are defined.
Claims (10)
1. a D-Psicose-3-epimerase gene, it is characterised in that nucleotide sequence is as shown in SEQ ID NO.1.
2. contain carrier or the cell line of gene described in claim 1.
3. D-Psicose-3-the epimerase of gene code described in claim 1.
4. a recombination bacillus coli, it is characterised in that with escherichia coli Rosetta (DE3) as host, expresses SEQ ID
The epimerase gene of D-Psicose-3-shown in NO.1.
5. the construction method of recombination bacillus coli described in claim 4, it is characterised in that by D-Psicose-3-epimerism
Enzyme gene dpe with pET28a is connected, and recombiant plasmid is proceeded to escherichia coli Rosetta by construction recombination plasmid pET28a-dpe
(DE3)。
6. the method for a resting cell synthesis D-Psicose, it is characterised in that with D-Fructose as substrate, add right and want
Ask the wet thallus of recombination bacillus coli described in 4,60～70 DEG C of reaction 0.5-6h.
Method the most according to claim 6, it is characterised in that by the 37 DEG C of trainings in LB culture medium of described recombination bacillus coli
Support, to OD600When being 0.6～1.0, add final concentration of 0.1-1.0mM IPTG, 16-37 DEG C of induction 5～20h, collect wet bacterium
Body.
Method the most according to claim 6, it is characterised in that the described pH value of solution containing D-Fructose is 7～10, and adds
0.3-0.7mM metal ion；Described metal ion includes Co2+, Mn2+。
Method the most according to claim 6, it is characterised in that described wet thallus concentration is 2.5～30mg/mL.
10. D-Psicose-3-epimerase described in claim 3 is in food, medicine, the application of field of health care products.
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Cited By (10)
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| CN108588149A (en) * | 2017-10-29 | 2018-09-28 | 中国科学院天津工业生物技术研究所 | A kind of algol syrup and preparation method thereof |
| CN109306347A (en) * | 2017-12-28 | 2019-02-05 | 吉林中粮生化有限公司 | A kind of novel D-Psicose 3- epimerase and its application |
| CN111004795A (en) * | 2019-11-20 | 2020-04-14 | 大连工业大学 | Method for improving heterologous expression of D-psicose-3 epimerase |
| CN111518853A (en) * | 2020-05-08 | 2020-08-11 | 东台市浩瑞生物科技有限公司 | Method for synthesizing D-psicose by whole-cell transformation |
| CN112458107A (en) * | 2020-12-29 | 2021-03-09 | 青岛龙鼎生物技术有限公司 | Method for catalytically producing D-psicose by using strain containing NAD (P) -dependent alcohol dehydrogenase |
| CN113025603A (en) * | 2021-03-04 | 2021-06-25 | 江南大学 | Method for improving storage stability of D-psicose 3-epimerase |
| CN113801833A (en) * | 2021-09-08 | 2021-12-17 | 福州大学 | Recombinant bacterium single-cell factory for producing D-psicose and construction and application thereof |
| CN114350727A (en) * | 2022-01-18 | 2022-04-15 | 广西大学 | Method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system |
| CN116064619A (en) * | 2022-09-26 | 2023-05-05 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for D-psicose conversion synthesis |
| CN111518853B (en) * | 2020-05-08 | 2024-04-30 | 东台市浩瑞生物科技有限公司 | Method for synthesizing D-psicose by whole cell transformation |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588149A (en) * | 2017-10-29 | 2018-09-28 | 中国科学院天津工业生物技术研究所 | A kind of algol syrup and preparation method thereof |
| CN108588149B (en) * | 2017-10-29 | 2022-06-10 | 中国科学院天津工业生物技术研究所 | A fructus crataegi syrup and its preparation method |
| CN109306347A (en) * | 2017-12-28 | 2019-02-05 | 吉林中粮生化有限公司 | A kind of novel D-Psicose 3- epimerase and its application |
| CN111004795A (en) * | 2019-11-20 | 2020-04-14 | 大连工业大学 | Method for improving heterologous expression of D-psicose-3 epimerase |
| CN111518853A (en) * | 2020-05-08 | 2020-08-11 | 东台市浩瑞生物科技有限公司 | Method for synthesizing D-psicose by whole-cell transformation |
| CN111518853B (en) * | 2020-05-08 | 2024-04-30 | 东台市浩瑞生物科技有限公司 | Method for synthesizing D-psicose by whole cell transformation |
| CN112458107A (en) * | 2020-12-29 | 2021-03-09 | 青岛龙鼎生物技术有限公司 | Method for catalytically producing D-psicose by using strain containing NAD (P) -dependent alcohol dehydrogenase |
| CN113025603B (en) * | 2021-03-04 | 2023-07-18 | 江南大学 | Method for improving storage stability of D-psicose 3-epimerase |
| CN113025603A (en) * | 2021-03-04 | 2021-06-25 | 江南大学 | Method for improving storage stability of D-psicose 3-epimerase |
| CN113801833A (en) * | 2021-09-08 | 2021-12-17 | 福州大学 | Recombinant bacterium single-cell factory for producing D-psicose and construction and application thereof |
| CN113801833B (en) * | 2021-09-08 | 2023-04-11 | 福州大学 | Recombinant bacterium single-cell factory for producing D-psicose and construction and application thereof |
| CN114350727B (en) * | 2022-01-18 | 2023-10-03 | 湖南成大生物科技有限公司 | Method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system |
| CN114350727A (en) * | 2022-01-18 | 2022-04-15 | 广西大学 | Method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system |
| CN116064619A (en) * | 2022-09-26 | 2023-05-05 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for D-psicose conversion synthesis |
| CN116064619B (en) * | 2022-09-26 | 2023-11-21 | 无锡特殊食品与营养健康研究院有限公司 | Bacillus licheniformis cell capable of being stably and repeatedly used for D-psicose conversion synthesis |
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Application publication date: 20170104 |