CN108048500A - The biological synthesis method of Beta-alanine - Google Patents
The biological synthesis method of Beta-alanine Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses the biological synthesis methods of beta Alanine, include the reaction of recombinant Saccharomyces cerevisiae whole-cell catalytic L asparatate Synthesis beta Alanines；The wherein described recombinant Saccharomyces cerevisiae contains exogenous L aspartic acids α decarboxylase encoding genes.The method of the invention and application have raw material cheap and easy to get, easily-controllable, route of synthesis green high-efficient easy to operate, the advantages such as biological safety is good, reaction rate is fast, conversion ratio is high, lay a good foundation for the industrialized production of beta Alanine.
Description
Technical field
The present invention relates to a kind of succinct, efficient Beta-alanine Microbe synthesis method more particularly to using recombination
The method that Engineering Yeast bacterium bioconversion synthesizes Beta-alanine, belongs to biological technical field.
Background technology
Beta-alanine, also known as 3- alanines are unique existing β type amino acid in nature.Beta-alanine medicine,
The fields such as food, beauty, feed and chemical industry are widely used, such as synthetic pantothenic acid calcium, carnosine, Pamidronate Disodium, Balsalazide
Medicine or feed and food additives.The production of Beta-alanine at present mainly has chemical synthesis and bioanalysis, chemical method application
Earlier and use is more universal, but often there are seriously polluted, side reaction (product) it is more, crystal purity is not high the shortcomings of；And it gives birth to
Object enzyme process then has many advantages, such as that substrate selective high, high conversion rate and reaction process be simple, crystalline product is easily purified, in recent years
It receives significant attention.
The bacterial strain of one plant of L-Aspartic acid-α-decarboxylase is screened in patent CN105543119A from vineyard, is classified
Te Jila bacillus PanD37 (Bacillus tequilensis PanD37) is named as, Beta-alanine yield is 16.25g/
L, far below current industrialized level.The Strain comparison of high yield L-Aspartic acid-α-decarboxylase is screened directly from Natural strains
Difficulty, therefore the active into the unique method for applying it of the enzyme is improved by the method for genetic engineering.
L-Aspartic acid-α-decarboxylase gene in Escherichia coli is cloned and carried out different in patent CN 101210230A
Source is expressed, and directly carries out using full cell catalyzing and synthesizing Beta-alanine, but its reaction is to carry out in the cell, and yield is only
2.94g/L also has from practical application with a distance from very big.104531796 A of patent CN 106755155A and CN is then broken using cell
Enzyme solution after broken carries out catalytic reaction, and catalytic efficiency is accelerated when comparing using full cell, illustrates to be closed using full cell
Into when enter cell there are larger substrate and product is diffused into extracellular resistance.From most gene work from the point of view of current report
The host strain of journey bacterium is all but Escherichia coli and unofficial bio-safety strain based on Escherichia coli, is using process
In there are the bio-safeties risk such as contaminated with endotoxins, and clasmatosis can increase and isolates and purifies cost again.Deng Si grain husks in 2015 etc.
The zymologic property of L-Aspartic acid-α-decarboxylase is had studied, the stability of L-Aspartic acid-α-decarboxylase of separate sources is not
Ideal, reaction temperature is higher, and reaction rate is faster, and enzyme activity loss is also bigger, this is unfavorable for the industrialized production of the enzyme.
The content of the invention
It is an object of the invention to provide the technical solutions of the bioconversion production Beta-alanine of complete set, are expected that by
Genetic engineering and the method for cell surface display structure recombination engineering, and rational biological fermentation engineering scheme is designed according to it,
To realize Beta-alanine green, industrialized production efficiently, inexpensive.
To achieve the above object, the present invention is connected to expression vector after allogenic gene is cloned first, then by institute's structure
The recombinant vector built is transferred to the brewing yeast cell as expressive host, makes expression of enzymes thin in yeast by the culture of yeast cells
The surface of born of the same parents.Based on this, the present invention provides the biological synthesis method of Beta-alanine, including recombinant Saccharomyces cerevisiae whole-cell catalytic L-
The reaction of asparatate Synthesis Beta-alanine；The wherein described recombinant Saccharomyces cerevisiae contain exogenous L-Aspartic acid-
α-decarboxylase encoding gene.
The method of the present invention can not only overcome the cross-film resistance of substrate and product, and the separating step of enzyme is not required, together
Shi Faxian, by the expression of enzymes after cell surface, the stability of the enzyme is improved, and enzyme activity is more stablized in catalytic process,
Not easy in inactivation.Based on this, the purpose of further aspect of the present invention, which also resides in, provides a kind of biological synthesis method of Beta-alanine, the party
The step of recombinant Saccharomyces cerevisiae of the invention described above is to the conversion reaction of substrate is included the use of in method.
The present invention successfully builds recombinant Saccharomyces cerevisiae cell, it is made to convert a substrate into β-the third ammonia as whole-cell catalyst
Acid eliminates isolating and purifying for enzyme, avoids the use of endotoxic hidden danger and antibiotic, has molar yield height, production
The features such as rate is fast, the stability of enzyme is strong is to realize efficient, environmental protection, the good selection of economization production Beta-alanine.
Description of the drawings
7 width of attached drawing of the present invention, wherein：
Fig. 1 is recombinant vector structure diagram.
Fig. 2 is the fluorescent marker of recombinant Saccharomyces cerevisiae surface display enzyme and laser co-focusing image；Wherein
A-1 is fluorescence inverted microscope verification result under white light；
A-2 is fluorescence inverted microscope verification result under 493nm excitation wavelengths；
B-1 is laser confocal microscope verification result under white light；
B-2 is laser confocal microscope verification result under 493nm excitation wavelengths.
Fig. 3 is influence result of the tests of the pH to recombinant Saccharomyces cerevisiae surface enzymatic activity.
Fig. 4 is the enzymatic comparative test result of different modes.
Fig. 5 is Enzyme activity assay result of the test under condition of different temperatures.
Fig. 6 is the result of the test that recombinant Saccharomyces cerevisiae catalyzes and synthesizes Beta-alanine under condition of different temperatures.
Fig. 7 is surface enzyme activity assays result during recombinant Saccharomyces cerevisiae recycling.
Specific embodiment
On the basis of existing technology, the present invention is first by target gene (nucleotide sequence such as SEQ ID NO.1) and table
Up to after carrier pYD1 digestions, through recombination to construct recombinant vector.Then, recombinant vector is converted into bacillus coli DH 5 alpha, specifically
Method can illustrate but be not limited to thermal shock method.Then ampicillin resistance gene screening, PCR and digestion verification above-mentioned steps are utilized
The transformant obtained, is stored in slant medium.In order to obtain required recombinant yeast cell, recombination expression need to be extracted and carried
Body, and convert into Saccharomyces cerevisiae competent cell, it is screened using deficiency and obtains positive transformant.Positive transformant is activated
After be inoculated into seed culture medium, treat thalli growth to OD620It when=1.0~5.0, is forwarded in inducing culture, obtains restructuring and make
Brewer yeast.
For principle, above-mentioned Saccharomyces cerevisiae competent cell include institute the with good grounds prior art can as convert host wine
Brewer yeast competent cell.In the specific embodiment of the invention, the expressive host is Saccharomyces Cerevisiae in S accharomyces
cerevisiae EBY100。
One of protrusion reason of the host is selected, yeast cell surface display technology is available with, makes zymoprotein and ferment
Mother cell wall-held protein merges, and cell surface is anchored in the form of native conformation, is equivalent to enzyme immobilizatio.With remaining host
Expression is compared, displaying enzyme prepare it is simple, eliminate enzyme isolate and purify and immobilization, help to reduce enzyme preparation life
Produce cost.Compared with endocellular enzyme, show cross-film resistance of the enzyme without substrate and product, and enhance the stability of enzyme.
Unless otherwise specified, it is heretofore described and inclined-plane culture based formulas it is as follows：Yeast extract 5.0g/L, tryptose
Peptone 10.0g/L, NaCl 10.0g/L, agar powder 20.0g/L, 121 DEG C of sterilizing 15min add in final concentration of 10~100 after cooling
The ampicillin of μ g/mL.
The seed culture based formulas is as follows：In 82mL ultra-pure waters, no amino yeast nitrogen 0.67g is added in, is such as
Prepare the agar powder that solid medium adds in 3.0g/L, 121 DEG C of sterilizing 15min；Amino acid mixed liquor 10mL is added after sterilizing, it is bright
Propylhomoserin (6.0g/L) 1mL, histidine (2.0g/L) 1mL, threonine (20.0g/L) 1mL and 40wt% glucose 5mL；
The Fiber differentiation based formulas is as follows：In 82mL ultra-pure waters, no amino yeast nitrogen 0.67g is added in, 121 DEG C
Sterilize 15min；Amino acid mixed liquor 10mL, leucine (6.0g/L) 1mL, histidine (2.0g/L) 1mL are added after sterilizing, ammonia of reviving
Sour (20.0g/L) 1mL and 40wt% galactolipins 5mL；
Described in the preparation of above-mentioned seed and inducing culture and amino acid mixed liquor in contain following component：Arginine
0.2g/L, aspartic acid 1.0g/L, glutamic acid 1.0g/L, isoleucine 0.3g/L, lysine 0.3g/L, valine 1.5g/L,
Methionine 0.2g/L, phenylalanine 0.5g/L, serine 3.75g/L, tyrosine 0.3g/L and adenine 0.4g/L.
On this basis, the present invention further provides a kind of biological synthesis method of Beta-alanine, this method is to use
The reaction of the recombinant Saccharomyces cerevisiae whole-cell catalytic ASPARTIC ACID Synthesis Beta-alanine of the invention described above；It is wherein described
Recombinant Saccharomyces cerevisiae contain exogenous L-Aspartic acid-α-decarboxylase encoding gene.It is wherein described and exogenous L- asparagus ferns
Propylhomoserin-α-decarboxylase encoding gene has the nucleotide sequence described in SEQ ID NO.1, the wine brewing ferment as foreign gene carrier
Female preferably saccharomyces cerevisiae Saccharomyces cerevisiae EBY100.
The biosynthesis of Beta-alanine is the technology of a relative maturity, and the concentration of substrate in reaction system is imitated according to reaction
Rate is set most beneficial for production, and reaction substrate concentration is set without bound, but highest response object concentration determines to a certain extent
In solubility of the reaction substrate in system, and with the increase of reaction substrate concentration, exist to a certain degree to changing effect
Inhibition.In the specific embodiment of the present invention, the solubility a maximum of about of 37mM of L-Aspartic acid, but we in production can be with
According to the standard setting reactant inventory higher than this, such as the 100mM used, there is portion in reaction system original at this time
It is insoluble solid state to divide, but with reaction can progressively dissolve, and has no effect on the progress of conversion reaction.Therefore, this feeds intake
Method can not only ensure being smoothed out for reaction, but also can reduce operating cost.As enforceable mode of operation, in of the invention, institute
Concentration of the concentration of substrate stated in initial reaction system may be configured as 100~1000mM.
On the other hand, in the method for synthesis Beta-alanine of the present invention, conversion reaction system pH is 6.0~8.0, excellent
6.5~7.5 are selected, which can be phosphate buffer or water, preferably phosphate buffer solution.The conversion reaction
Temperature is 30~60 DEG C, preferably 50~60 DEG C, most preferably 55 ± 2 DEG C.Certain model caused by the measurement of permission or control error
Temperature fluctuation in enclosing is also acceptable.
In another aspect, in the method for synthesis Beta-alanine of the present invention, recombinate and make in the conversion reaction system
Brewer yeast dosage is OD620=1.0~5.0.
According to optimization as a result, the more specific embodiment of the biological synthesis method of the Beta-alanine of the invention described above
One of, the method that can be described as including operations described below step：Recombinant Saccharomyces cerevisiae is added in into substrate L-Aspartic acid (L-Asp)
It is reacted in solution, 50~60 DEG C of reaction temperature, pH value of reaction system is 6.5~7.5, and β-the third ammonia is measured by sampling per hour
Acid, until L-Aspartic acid is totally converted, separating thallus simultaneously collects reaction solution.
The biological synthesis method of above-mentioned Beta-alanine, due to the full cell that is used in the system that is synthesized in catalytic reaction into
Row production, reaction, which proceeds to appropriate degree, to terminate reaction by physical separation recombinant Saccharomyces cerevisiae, and separate
Recombinant Saccharomyces cerevisiae can continue to be added in the reaction system of bioconversion as cell catalyst to use, and realize Xun Huan profit
With.
To sum up, from the effective engineering bacteria beginning is built, the biological synthesis method of Beta-alanine of the present invention can describe
For following complete technical solution：
(1) structure restructuring thalline；
Cloned foreign L-Aspartic acid-α-decarboxylase encoding gene includes the external source using expression vector pYD1 structures
Property gene recombinant vector, and recombinant vector is converted into bacillus coli DH 5 alpha using thermal shock method；
(2) transformant obtained using ampicillin resistance gene screening, PCR and digestion verification step (1), is preserved
(in slant medium)；
(3) recombinant vector is extracted from transformant obtained by step (2), and is converted to saccharomyces cerevisiae EBY100 competent cells
In, it is screened using deficiency and obtains positive transformant；Seed culture medium is inoculated into after positive transformant is activated, treats thalli growth
It during to appropriate biomass, is forwarded in inducing culture, obtains recombinant Saccharomyces cerevisiae；
(4) biosynthesis of Beta-alanine：The ASPARTIC ACID aqueous solution that concentration is 0.1~1mol/L is prepared, is added in
The culture of step (3) is to OD620=1.0~5.0, it is 6.5~7.5 to adjust pH, and system is reacted under the conditions of 50~60 DEG C, instead
Reaction solution separating thallus is collected after answering；
(5) step (4) separating obtained thalline is added in new reaction system, Xun Huan production Beta-alanine.
Following non-limiting examples are not construed as being described further to present disclosure to the present invention
Any type of restriction.
Embodiment 1
The structure of surface display recombinant Saccharomyces cerevisiae and culture：
First, with 168 genome (GenBank of bacillus subtilis：CP019663.1 it is) template amplification panD genes, leads to
It crosses using restriction enzyme BamH I and Xho I, PCR product and expression vector pYD1 digestions is connected overnight after purification, so
It is converted afterwards into bacillus coli DH 5 alpha, screens transformant using ampicillin resistance gene, and PCR and enzyme are carried out to it
Verification and DNA sequencing are cut, plating medium is stored in after verification is errorless.The results are shown in Figure 1 for recombinant vector.Secondly, extraction weight
Group expression vector, and convert into saccharomyces cerevisiae EBY100 competent cells, it is screened using deficiency and obtains positive transformant, it will
Seed culture medium is inoculated into after positive transformant activation, treats thalli growth to OD620When=1.0~5.0, Fiber differentiation is forwarded to
In base, recombinant Saccharomyces cerevisiae is obtained.Finally, with containing 1mg/mL bovine serum albumin(BSA)s (BSA) and 1 μ g antibody (anti-V5-
FITC recombinant yeast cell is resuspended in phosphate buffer), after being placed in 30min on ice, cell is collected by centrifugation, and is delayed with phosphate
Cell is resuspended after cleaning twice in fliud flushing, is placed under fluorescence inverted microscope and laser confocal microscope and observes.As a result such as Fig. 2 institutes
Show, it is seen that：When biological enzyme is showed in cell surface, exposed V5 epitopes can occur with free fluorescein labelled antibody
Specific binding, the fluorescence signal detected under 493nm excitation wavelengths as specifically bind the fluorescence signal of generation, demonstrate,prove
The bright biological enzyme is successfully showed in cell surface.
Recombinant Saccharomyces cerevisiae is inoculated into seed culture medium, 30 DEG C, thalline is activated under conditions of 180rpm.Turn again afterwards
It is connected in seed culture medium and is enlarged culture, 30 DEG C, 180rpm shaken cultivations.Biomass to be grown to suitable, inoculates
Into 1.0L inducing cultures, 20 DEG C, 150rpm ventilation culture cells.16~put tank afterwards for 24 hours, 4 DEG C are collected by centrifugation cell.
Embodiment 2
Differential responses pH catalyzes and synthesizes surface display recombinant Saccharomyces cerevisiae the influence of Beta-alanine：
The culture of surface display recombinant Saccharomyces cerevisiae such as embodiment 1 is identical, and cell is collected by centrifugation and carries out catalytic reaction.Research
Differential responses pH 5 (acetate buffer), 6/7/8/9 (phosphate buffer) are catalyzed surface display recombinant Saccharomyces cerevisiae and close
Into the influence of Beta-alanine.2.66g L-Aspartic acids are dissolved in 90mL buffer solutions, with 6M NaOH solutions and 10% salt
Acid maintains above-mentioned pH, and recombinant Saccharomyces cerevisiae is added in reaction system, makes biomass OD in reaction solution620=1.0~5.0, control
Temperature is 37 DEG C, Beta-alanine is measured by sampling per hour, until L-Aspartic acid is totally converted, centrifuging and taking supernatant utilizes height
Effect liquid phase chromatogram method measures the concentration of Beta-alanine, it is found that the enzyme is respectively provided with greater activity in the range of pH 5-9, reaction is most
Suitable pH is 7.0, and the results are shown in Figure 3.
Embodiment 3
Surface display recombinant Saccharomyces cerevisiae catalyzes and synthesizes β-the third ammonia when L-Aspartic acid concentration is 0.2/0.4/0.6/0.8M
The research of acid：
The culture of surface display recombinant Saccharomyces cerevisiae such as embodiment 1 is identical, and cell is collected by centrifugation and carries out catalytic reaction.Research
Different molar concentration (0.2/0.4/0.6/0.8M) L-Aspartic acid solution surface display recombinant Saccharomyces cerevisiae is catalyzed and synthesized β-
The influence of alanine.Recombinant Saccharomyces cerevisiae is added in the reaction system (pH=7.0) of above-mentioned different concentration of substrate, makes reaction
Biomass OD in liquid620=1.0~5.0, pH=7.0 is maintained with 10% hydrochloric acid solution, controlled at 37 DEG C, is sampled per hour
Beta-alanine is measured, until L-Aspartic acid is totally converted, using the concentration of high effective liquid chromatography for measuring Beta-alanine, as a result
As shown in table 1.
1. surface display recombinant Saccharomyces cerevisiae of table catalyzes and synthesizes the result of Beta-alanine
| Concentration of substrate (M) | Reaction time (h) | The concentration (g/L) of Beta-alanine |
| 0.2 | 1 | 17.8 |
| 0.4 | 2 | 35.6 |
| 0.6 | 4 | 53.5 |
| 0.8 | 5 | 71.3 |
Embodiment 4
The enzymatic contrast test of different modes
The present embodiment investigates area of various forms of L-Aspartic acid-α-decarboxylases in Beta-alanine experiment is catalyzed and synthesized
Not, the enzyme system of used catalytic test is respectively：
(1) enzyme A：Surface display recombinant Saccharomyces cerevisiae
With reference to embodiment 1, recombinant Saccharomyces cerevisiae is collected by centrifugation, is enzyme A.
(2) enzyme B：Recombination bacillus coli
The structure of recombination bacillus coli is as follows with cultivating：Recombinant vector in embodiment 1 is converted to e. coli bl21
(DE3) in, screening obtains the recombination bacillus coli M that can catalyze and synthesize Beta-alanine.Recombination bacillus coli M is seeded to LB liquid
In culture medium, 37 DEG C, overnight shaking culture under conditions of 200rpm.It is inoculated into 1% inoculum concentration in 1.5L fermentation mediums,
37 DEG C, 200rpm ventilations culture cell to OD600After=0.6~0.8, after adding in derivant IPTG overnight inductions, bacterium is collected by centrifugation
Body is enzyme B.
(3) enzyme C：Recombination bacillus coli lysate
10mL recombination bacillus colis M is taken to carry out ultrasonic disruption, and (power 200W, work stop 4 seconds for 4 seconds, and total time is
8min), 10000rpm centrifuges 1min, takes supernatant, is enzyme C.
(4) enzyme D：Non- surface display recombinant Saccharomyces cerevisiae
The structure of non-surface display recombination yeast and culture are as follows：First, the recombinant vector in embodiment 1 is converted to wine
In brewer yeast BY4742 competent cells, screened using deficiency and obtain recombinant Saccharomyces cerevisiae N.Recombinant Saccharomyces cerevisiae N is inoculated with
Into seed culture medium, 30 DEG C, thalline is activated under conditions of 180rpm.It is forwarded to again in seed culture medium afterwards and is enlarged training
It supports, 30 DEG C, 180rpm shaken cultivations.Biomass to be grown to suitable is inoculated in 1.0L inducing cultures, 20 DEG C,
150rpm ventilation culture cells.16~put tank afterwards for 24 hours, 4 DEG C are collected by centrifugation cell, are enzyme D.
(5) enzyme E：Non- surface display recombination yeast lysate
10mL recombinant Saccharomyces cerevisiaes N is taken to carry out ultrasonic disruption, and (power 500W, work stop 5 seconds for 5 seconds, and total time is
8min), 10000rpm centrifuges 1min, takes supernatant, is enzyme E.
0.2M L-Aspartic acids solution 6M NaOH solutions are adjusted into pH=7.0, each seed ginseng survey enzyme is added to reaction
In system, make the identical (OD of biomass in reaction solution600=1.0~5.0) pH=7.0, control temperature, are maintained with 10% hydrochloric acid solution
It spends for 37 DEG C, reacts 60min, Beta-alanine is measured by sampling per 20min, utilizes the dense of high effective liquid chromatography for measuring Beta-alanine
Degree, the results are shown in Figure 4.
0.4M L-Aspartic acids substrate solution 6M NaOH solutions are adjusted into pH=7.0, each seed ginseng survey enzyme is added to
In reaction system, make the identical (OD of biomass in reaction solution600=1.0~5.0) pH=7.0, control, are maintained with 10% hydrochloric acid solution
Temperature processed is 37,45 and 55 DEG C, and Beta-alanine is measured by sampling per 20min, utilizes high effective liquid chromatography for measuring Beta-alanine
Concentration, the curve changed over time according to Beta-alanine yield calculates instantaneous enzyme activity, as a result as Fig. 5 shows.
Catalytic reaction efficiency is carried out substantially not as good as the catalytic efficiency of its lysate using full cell, mainly due to L- asparagus ferns
Propylhomoserin-α-decarboxylase is endocellular enzyme, and substrate enters cell and all there are larger resistances when product is diffused into extracellular；Using cell
Lysate participates in reaction, high catalytic efficiency, but can increase production cost, and the stability of resolvase is undesirable；By biological enzyme exhibition
Yeast cell surface is shown in, the influence of substrate and product cross-film resistance can either be overcome, improves reaction rate, the stability of enzyme
It improves a lot, in turn avoids the separation of smudge cells and enzyme, reduce production cost, improve economic benefit.
Embodiment 5
The catalysis activity stability of surface display recombinant Saccharomyces cerevisiae and crude enzyme liquid compares：
Preparing for surface display recombinant Saccharomyces cerevisiae (enzyme A) and crude enzyme liquid (enzyme E) is same as Example 5.By 1.0M L- days
Winter propylhomoserin substrate solution adjusts pH=7.0 with 6M NaOH solutions, and surface display recombinant Saccharomyces cerevisiae and crude enzyme liquid are added to instead
It answers in system, pH=7.0 is maintained with 10% hydrochloric acid solution, controlled at 37,45 and 55 DEG C, β-the third ammonia is measured by sampling per 1h
Acid, using the concentration of high effective liquid chromatography for measuring Beta-alanine, the results are shown in Figure 6.
When using high concentration substrate, with the rise of temperature, the conversion ratio drop of the catalysis generation Beta-alanine of crude enzyme liquid
Low, this is because as temperature increases, the enzyme activity loss of crude enzyme liquid is accelerated, and causes the catalytic efficiency in long-time reaction process big
Width declines.And in same substrate concentration, with the rise of temperature, generation β-the third is catalyzed using surface display recombinant yeast cell
The conversion ratio of propylhomoserin is higher and catalytic efficiency greatly improves, this is because the enzyme, after surface display, thermal stability improves,
Unobvious are inactivated under high-temperature condition, and as reaction temperature improves, mass-and heat-transfer efficiency is accelerated, so as to which catalytic efficiency is promoted.It should
Embodiment illustrates that surface display recombinant Saccharomyces cerevisiae more suitable for industrialized production, had both avoided separation of smudge cells and enzyme etc.
Step, and production efficiency is improved, production cost is reduced, improves economic benefit.
Embodiment 6
The recycling of surface display recombinant Saccharomyces cerevisiae efficiently synthesizes Beta-alanine：
The culture of surface display recombinant Saccharomyces cerevisiae is same as Example 1, and cell is collected by centrifugation and carries out catalytic reaction.It will
2.66g L-Aspartic acids are dissolved in 90mL 0.02M phosphate buffers, and pH=7.0 is adjusted with 6M NaOH solutions,
Recombinant Saccharomyces cerevisiae is added in reaction system, makes biomass OD in reaction solution620=5.0, maintain pH with 10% hydrochloric acid solution
=7.0, controlled at 55 DEG C, Beta-alanine is measured by sampling per hour, until L-Aspartic acid is totally converted；Then, use is compacted
It is dynamic pump out or centrifuge discard reaction supernatant after, again according to above-mentioned reaction condition be repeated several times react；Finally, using efficient
The concentration of liquid chromatography for measuring Beta-alanine, the results are shown in Figure 7.During recycling thalline, cell there are partial loss,
Therefore enzymatic activity is more stable during recycled for multiple times recombinant Saccharomyces cerevisiae catalyzes and synthesizes Beta-alanine, is repeated
It uses, to reduce industrial production cost, solves the problems, such as enzyme recycling is difficult, utilization rate is low etc., more meet the requirement of industrialized production.
Sequence table
<110>Dalian Yi Nuo Biological Co., Ltd.
<120>The biological synthesis method of Beta-alanine
<130> N/A
<141> 2017-12-25
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Claims (8)
1. the biological synthesis method of Beta-alanine, including recombinant Saccharomyces cerevisiae whole-cell catalytic ASPARTIC ACID Synthesis β-
The reaction of alanine；The wherein described recombinant Saccharomyces cerevisiae contains exogenous L-Aspartic acid-α-decarboxylase encoding gene.
2. according to the method described in claim 1, it is characterized in that, the recombinant Saccharomyces cerevisiae is containing exogenous L- asparagus ferns
The Saccharomyces Cerevisiae in S accharomyces cerevisiae EBY100 of propylhomoserin-α-decarboxylase encoding gene.
3. according to the method described in claim 1, it is characterized in that, the concentration of substrate ASPARTIC ACID is in the reaction
0.1~1mol/L.
4. according to the method described in claim 1, it is characterized in that, 30~60 DEG C of the reaction temperature.
5. according to the method described in claim 4, it is characterized in that, 50~60 DEG C of the reaction temperature.
6. according to the method described in claim 1, it is characterized in that, the pH value of reaction system is 6.0~8.0.
7. according to the method described in claim 1, it is characterized in that, separating reaction solution after further including reaction and the step of thalline,
Gained thalline is used for new reaction system.
8. method described in claim 1, includes the following steps：
(1) structure restructuring thalline；
Cloned foreign L-Aspartic acid-α-decarboxylase encoding gene includes the exogenous base using expression vector pYD1 structures
The recombinant vector of cause, and converted recombinant vector into bacillus coli DH 5 alpha using thermal shock method；
(2) transformant obtained using ampicillin resistance gene screening, PCR and digestion verification step (1), is preserved；
(3) recombinant vector is extracted from transformant obtained by step (2), and is converted into saccharomyces cerevisiae EBY100 competent cells,
It is screened using deficiency and obtains positive transformant；Seed culture medium is inoculated into after positive transformant is activated, treats thalli growth extremely
It during appropriate biomass, is forwarded in inducing culture, obtains recombinant Saccharomyces cerevisiae；
(4) biosynthesis of Beta-alanine：The ASPARTIC ACID aqueous solution that concentration is 0.1~1mol/L is prepared, adds in step
(3) culture is to OD620=1.0~5.0, it is 6.5~7.5 to adjust pH, and system is reacted under the conditions of 50~60 DEG C, reaction knot
Reaction solution separating thallus is collected after beam；
(5) step (4) separating obtained thalline is added in new reaction system, Xun Huan production Beta-alanine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109609536A (en) * | 2019-01-16 | 2019-04-12 | 常熟理工学院 | A kind of method of full cell one-step synthesis N-BETA-Alanyl-L-histidine |
| CN109652484A (en) * | 2019-01-16 | 2019-04-19 | 常熟理工学院 | A kind of method that full cell high-efficient catalyzes and synthesizes N-BETA-Alanyl-L-histidine |
| CN113249239A (en) * | 2021-05-12 | 2021-08-13 | 天津大学 | Saccharomyces cerevisiae for high yield of crocetin and construction method and application thereof |
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| CN106754447A (en) * | 2016-12-30 | 2017-05-31 | 大连医诺生物有限公司 | Recombinant Saccharomyces cerevisiae and its application in glutamine dipeptide is synthesized |
| CN107338258A (en) * | 2017-01-09 | 2017-11-10 | 鲁东大学 | The method for producing the engineering bacteria structure and its production beta Alanine of beta Alanine |
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| CN106754447A (en) * | 2016-12-30 | 2017-05-31 | 大连医诺生物有限公司 | Recombinant Saccharomyces cerevisiae and its application in glutamine dipeptide is synthesized |
| CN107338258A (en) * | 2017-01-09 | 2017-11-10 | 鲁东大学 | The method for producing the engineering bacteria structure and its production beta Alanine of beta Alanine |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609536A (en) * | 2019-01-16 | 2019-04-12 | 常熟理工学院 | A kind of method of full cell one-step synthesis N-BETA-Alanyl-L-histidine |
| CN109652484A (en) * | 2019-01-16 | 2019-04-19 | 常熟理工学院 | A kind of method that full cell high-efficient catalyzes and synthesizes N-BETA-Alanyl-L-histidine |
| CN109652484B (en) * | 2019-01-16 | 2020-12-29 | 常熟理工学院 | Method for efficiently catalytically synthesizing L-carnosine by whole cells |
| CN113249239A (en) * | 2021-05-12 | 2021-08-13 | 天津大学 | Saccharomyces cerevisiae for high yield of crocetin and construction method and application thereof |
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