CN103789246B - A kind of permeability glutamic acid decarboxylase enzyme engineering bacteria and preparation method thereof - Google Patents
A kind of permeability glutamic acid decarboxylase enzyme engineering bacteria and preparation method thereof Download PDFInfo
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- CN103789246B CN103789246B CN201410005932.5A CN201410005932A CN103789246B CN 103789246 B CN103789246 B CN 103789246B CN 201410005932 A CN201410005932 A CN 201410005932A CN 103789246 B CN103789246 B CN 103789246B
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Abstract
The invention discloses a kind of permeability glutamic acid decarboxylase enzyme engineering bacteria and preparation method thereof, described method includes: be suspended in water or buffer by the glutamic acid decarboxylase enzyme engineering bacteria thalline expressing glutamate decarboxylase, after 50～70 DEG C process 5～40min, collect thalline, obtain described permeability glutamic acid decarboxylase enzyme engineering bacteria；Wherein, described glutamic acid decarboxylase enzyme engineering bacteria includes host cell and proceeds to the glutamic acid decarboxylase gene of host cell, and described host cell is escherichia coli.Present invention also offers the permeability glutamic acid decarboxylase enzyme engineering bacteria that described method obtains.The method of the present invention, easy, low cost, it is easy to operation, the apparent catalysis activity of glutamate decarboxylase of thalline can be effectively improved.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of permeability glutamic acid decarboxylase enzyme engineering bacteria
And preparation method thereof.
Technical background
γ-aminobutyric acid (γ-aminobutyric acid, GABA) is important in human central nervous system
Inhibitory neurotransmitter, have blood pressure lowering, diuresis, convulsion, prevention epilepsy, improve sleep,
Antidepressant, promote hormone secretion and protect the liver the profit different physiological roles such as kidney.Therefore, GABA is at food
Product and medicine have wide using value.Additionally, GABA can be used for synthesizing as precursor substance
The chemical industry such as Biodegradable material polyamide-4 and environment-friendly plastic solvent N-ketopyrrolidine produce
Product.
At present, GABA can be prepared by the method for chemical synthesis and living things catalysis.With
Chemical synthesis is compared, and utilizes glutamate decarboxylase (Glutamate decarboxylase, GAD;EC
4.1.1.15) catalysis Pidolidone decarboxylation generates the biological preparation method of GABA owing to having raw material
Source is abundant, process is simple, transformation efficiency is high, mild condition and environmental friendliness advantage, thus more
There is commercial development prospect.
Utilizing to express has the microbial cell of GAD to prepare GABA, can avoid enzyme extraction and purification
Step cost, improves utilization rate and the stability of enzyme.GAD gene is cloned by technique for gene engineering
Escherichia coli are expressed, the catalysis activity of thalline can be effectively improved.But due to thalline
The barrier action of cell wall and cell membrane limits the transmission at intraor extracellular of substrate and product, makes bacterium
The GAD vigor of body intracellular is difficult to give full play to, and the serious apparent catalysis that have impact on engineering bacteria is lived
Power, reduces the conversion rate of substrate.Therefore, cell wall and cell membrane it are effectively improved to substrate
The catalysis activity that resistance to mass tranfer improves GAD engineering bacteria is significant for preparation GABA.
Cell Permeabilization technology (cell permeabilization) can not cause cell cracking and
The permeability of cell is improved so that small-molecule substance and one in the case of not destroying cell interior structure
A little relatively macromolecular substances are free to pass in and out cell, thus improve the apparent catalysis activity of thalline.
Use the physics such as the permeability reagent such as organic solvent or surfactant or ultrasonic, multigelation
Method prepares Permeabilized cells, although can be effectively improved the apparent catalysis activity of thalline, but also deposit
In some unfavorable factors.Addition such as chemistry permeability reagent often causes excessively splitting of cell
Solving, the interpolation of organic solvent can cause the residual of toxic reagent, simultaneously because the volatilization of organic solvent
Property, for industrialized production security protection require height, and surfactant add membership to downstream
The purification of product brings adverse effect.It is high to there is equipment investment in freeze-thaw method, Ultrasonic treatment etc., and
It is difficult to the problems such as amplification.
Summary of the invention
The invention provides a kind of method preparing permeability glutamic acid decarboxylase enzyme engineering bacteria, easy,
Low cost, it is easy to operation, can be effectively improved the apparent catalysis activity of glutamate decarboxylase of thalline.
A kind of method preparing permeability glutamic acid decarboxylase enzyme engineering bacteria, including:
The glutamic acid decarboxylase enzyme engineering bacteria thalline expressing glutamate decarboxylase is suspended in water or buffering
In liquid, after 50～70 DEG C process 5～40min, collect thalline, obtain described permeability glutamic acid and take off
Carboxylic acid engineering bacteria；
Wherein, described glutamic acid decarboxylase enzyme engineering bacteria includes host cell and proceeds to host cell
Glutamic acid decarboxylase gene, described host cell is escherichia coli.
After first glutamic acid decarboxylase enzyme engineering bacteria carries out inducing culture, make expression of GAD,
Then glutamic acid decarboxylase enzyme engineering bacteria thalline is carried out heat treatment, and controls treatment temperature and time,
The cell wall of thalline, the structure of film, mobility is made suitable change to occur, to reach to improve cell
Wall and membrane passage, the mass transfer reducing substrate and product limits, and improves the apparent catalysis of thalline
The purpose of vigor.
Described glutamic acid decarboxylase enzyme engineering bacteria is obtained by conventional technique for gene engineering, the most first
Glutamic acid decarboxylase gene is introduced expression vector, builds and obtain recombinant expression carrier, then will restructuring
Expression vector transformed host cell.Wherein, expression vector can be pET-28a, pET-29, pET-30,
PET-34, pRSET etc..
In the application, described host cell is escherichia coli, be specifically as follows (E.coli) BL21,
Escherichia coli (E.coli) BLR, escherichia coli (E.coli) Origami, escherichia coli (E.coli)
NovaBlue or escherichia coli (E.coli) Rosetta etc..
Described glutamic acid decarboxylase gene is derived from the short breast that deposit number is CGMCC NO.1306
Bacillus (Lactobacillus brevis), the glutamic acid decarboxylase gene (gadB) of this strain transcribe product
Thing has preferable heat stability, still has catalysis activity at 70 DEG C, uses this glutamate decarboxylase
The heat treatment adverse effect to enzyme activity can be reduced.
Before carrying out heat treatment, first glutamic acid decarboxylase enzyme engineering bacteria is carried out inducing culture, make paddy
Propylhomoserin decarboxylase is expressed, and the method for inducing culture can use routine techniques, is usually described paddy
Propylhomoserin decarboxylase engineering bacteria is inoculated in culture medium to carry out cultivating and obtains seed liquor, then by seed liquor
It is seeded in culture medium shake and trains to OD6000.6～0.8, after adding derivant, it is further cultured for a period of time
?.
Water or buffer can provide the environment of gentleness for thalline, and wherein the kind of buffer is the tightest
Lattice requirement, can be phosphate buffer, acetate buffer solution etc..Typically, described buffer is dense
Degree is 0.05～0.25M, and pH is 4.0～7.0, it is preferred that concentration be 0.2M, pH be 4.8.
The temperature and time processed is most important, and temperature is too low or time too short meeting causes process to reach not
To preferable effect, temperature is too high or overlong time then may cause glutamate decarboxylase to inactivate.
Preferably, the temperature of described process is 60～70 DEG C, and the time of described process is 10～30min.
Present invention also offers the permeability glutamic acid decarboxylase enzyme engineering bacteria that described method prepares.
Compared with prior art, the invention have the benefit that
(1) to improve thalline thin by being processed at a temperature of 50-70 DEG C by GAD engineering bacteria for the present invention
The mass transfer of substrate and product is limited by cell wall and cell membrane, makes the GAD activity of intracellular fully be sent out
Wave, improve the apparent GAD catalysis activity of engineering bacteria.
Compared with chemical method, first, the method for the present invention need not add any organic solvent or table
The permeability reagent such as face activating agent, not only simpler in operation, it also avoid noxious substance simultaneously
Introduce, the quality of downstream product (GABA) will not be impacted, also simplify permeability reagent
Interpolation, the most just reduce cost, secondly, the method for the present invention is easily controllable, suitable by controlling
Temperature and time, it is possible to avoid the excessive fragmentation of permeability agents on cellular.
The method of the present invention still has preferably compared with current Physical such as freeze-thaw method, Ultrasonic treatment
Advantage.Freeze-thaw method is by cell multigelation, and intracellular formation ice pellets makes the salinity of residue cytosol
Increasing and cause cell swelling and improve permeability, multigelation is relatively big to the injury of cell, easily causes born of the same parents
The leakage of endoenzyme, freeze-thaw method requires height to equipment investment simultaneously, the application only disposable by short-term
Heat treated, can overcome this defect.Equipment investment is required low.
Ultrasonic method is to utilize short time, lower powered ultrasonic Treatment to improve cell permeability, ultrasonic
The improper disintegrate easily causing cell of intensity of ripple and reduce its survival rate, and the heat of ultrasonic generation
Amount the most easily makes enzyme inactivate.The method of the present invention is gentleer, is also more prone to control, can overcome
The defect of above-mentioned supersound process, also avoids the ultrasonic injury causing human body simultaneously.
It addition, the equipment that the method for the present invention needs is simple.
Therefore, compared with the existing methods, the method for the present invention is simpler, environmental protection, be prone to control
System and operation, cost is the lowest, more easily industrial applications.
(2) method of the present invention can be effectively improved the apparent catalysis activity of glutamate decarboxylase of thalline,
With compared with the GAD engineering bacteria of permeabilized treatment, the table of permeability GAD engineering bacteria thalline
See catalysis activity and can improve 13 times, and enzyme leakage situation does not occurs.This is industrialized production GABA
Improve a kind of high vigor, cheap biocatalyzer, contribute to reducing GABA Large Scale Biology system
Standby production cost.
(3) permeability GAD engineering bacteria can be regarded as a kind of insoluble GAD enzyme source, with tradition
Method immobilized enzyme has similar purpose, and permeability GAD engineering bacteria even can be with known various solid
Surely being fixed of method of intact cell is changed.The permeability GAD engineering that the method for the present invention obtains
Bacterium can preferably keep stability after immobilization.
Detailed description of the invention
The present invention is explained further below in conjunction with detailed description of the invention.
Embodiment 1
Strain: engineering bacteria E.coli BL21 (DE3)/pET28a-gadB(can be found in " Cloning,
sequencing and expression of a glutamate decarboxylase gene from the
GABA-producing strain Lactobacillus brevis CGMCC1306,Annals of
Microbiology, 2012,62 (2): 689-698 "), wherein, glutamic acid decarboxylase gene (gadB)
It is derived from the Lactobacillus brevis (Lactobacillus brevis) that deposit number is CGMCC NO.1306.
Engineering bacteria (the E.coli of picking glutamate decarboxylase from the LB solid medium of activation
BL21 (DE3)/pET28a-gadB) monoclonal thalline accesses containing 50 μ g mL-1The LB of kanamycin
In fluid medium, 37 DEG C, 200r/min shaken cultivation overnight, obtains seed liquor.Seed liquor is pressed
Volume fraction 1% inoculum concentration is inoculated into containing kanamycin (50 μ g mL-1) LB liquid culture
In base, being placed in 37 DEG C, 200r/min cultivates to OD600Add IPTG during 0.6-0.8, make IPTG
Final concentration of 0.5 μM, 30 DEG C, 150r/min inducing culture 6h.
Embodiment 2
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), then
With acetate buffer solution (0.2M, pH4.8) suspension thalline, at 50 DEG C, shake 30min, at 10000 × g
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves (thoroughly
Property GAD engineering bacteria).
One enzyme activity unit is defined as under the conditions of 37 DEG C, and 1min generates 1 μm ol GABA(1
U=1 μm ol GABA in1min at37 DEG C) needed for enzyme amount.The Rate activity of GAD is defined as often
Enzyme activity unit (the U mg that milligram cellular dry weight is possessed-1Cells, dry weight).After measured,
The apparent catalysis activity of permeability GAD engineering bacteria thalline is 1.59U/mg, with same culture conditions
Under without the apparent catalysis activity of the GAD engineering bacteria thalline of permeabilized treatment compare raising 2.49 times
(the apparent catalysis activity without the GAD engineering bacteria thalline of permeabilized treatment is 0.64U/mg), and
And do not find enzyme leakage situation in the engineering bacteria thalline GAD determination of activity of permeability GAD.
Embodiment 3
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), uses
Acetate buffer solution (0.2M, pH4.8) suspension thalline, shakes 10min, at 10000 × g at 70 DEG C
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves.
Enzyme activity unit definition and Rate activity define with embodiment 2.After measured, permeability GAD work
The apparent catalysis activity of journey bacterium thalline be under 6.37U/mg, with same culture conditions without permeability at
The GAD apparent catalysis activity of engineering bacteria thalline of reason compares raising 9.96 times (without permeabilized treatment
The apparent catalysis activity of GAD engineering bacteria thalline be 0.64U/mg), and in permeability GAD
Engineering bacteria thalline GAD determination of activity does not find enzyme leakage situation.
Embodiment 4
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), uses
Acetate buffer solution (0.2M, pH4.8) suspension thalline, shakes 30min, at 10000 × g at 70 DEG C
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves.
Enzyme activity unit definition and Rate activity define with embodiment 2.After measured, permeability GAD work
The apparent catalysis activity of journey bacterium thalline is untreated GAD under 8.35U/mg, with same culture conditions
The apparent catalysis activity of engineering bacteria thalline compares 13.05 times of (GAD without permeabilized treatment of raising
The apparent catalysis activity of engineering bacteria thalline is 0.64U/mg), and permeability GAD engineering bacteria bacterium
The GAD determination of activity of body does not find enzyme leakage situation.
Embodiment 5
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), uses
Acetate buffer solution (0.2M, pH4.8) suspension thalline, shakes 30min, at 10000 × g at 60 DEG C
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves.
Enzyme activity unit definition and Rate activity define with embodiment 2.After measured, permeability GAD work
The apparent catalysis activity of journey bacterium thalline is untreated GAD under 6.37U/mg, with same culture conditions
The apparent catalysis activity of engineering bacteria thalline compares 9.96 times of (GAD without permeabilized treatment of raising
The apparent catalysis activity of engineering bacteria thalline is 0.64U/mg), and permeability GAD engineering bacteria bacterium
The GAD determination of activity of body does not find enzyme leakage situation.
Comparative example 1
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), uses
Acetate buffer solution (0.2M, pH4.8) suspension thalline, shakes 30min, at 10000 × g at 80 DEG C
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves.
Enzyme activity unit definition and Rate activity define with embodiment 2.After measured, permeability GAD work
The apparent catalysis activity of journey bacterium thalline is untreated GAD under 0.62U/mg, with same culture conditions
The apparent catalysis activity of engineering bacteria thalline compares reduction (the GAD engineering without permeabilized treatment on the contrary
The apparent catalysis activity of bacterium thalline is 0.64U/mg), its reason is mainly GAD under 80 DEG C of process
Inactivation.
Comparative example 2
Take 1mg(dry cell weight) engineering bacteria after embodiment 1 inducing culture, in 4 DEG C, 10000g
Centrifugal 5min collects thalline, cleans thalline 1 time with acetate buffer solution (0.2M, pH4.8), uses
Acetate buffer solution (0.2M, pH4.8) suspension thalline, shakes 30min, at 10000 × g at 37 DEG C
Under be centrifuged 1min, collect thalline, be cell wall and GAD engineering bacteria that cell leakage improves.
Enzyme activity unit definition and Rate activity define with embodiment 2.After measured, permeability GAD work
The apparent catalysis activity of journey bacterium thalline is untreated GAD under 0.60U/mg, with same culture conditions
The apparent catalysis activity of engineering bacteria thalline does not has significant change (the GAD engineering without permeabilized treatment
The apparent catalysis activity of bacterium thalline is 0.64U/mg), its reason can not make large intestine at a temperature of being mainly somebody's turn to do
The saturating property of bacilli-cell tunicle changes.
Claims (2)
1. the method preparing permeability glutamic acid decarboxylase enzyme engineering bacteria, it is characterised in that take thin
Born of the same parents' dry weight is the engineering bacteria after the inducing culture of 1mg, and in 4 DEG C, 10000g is centrifuged 5min and collects
Thalline, cleans thalline 1 time with the acetate buffer solution that concentration is 0.2M, pH4.8, then by concentration is
The acetate buffer solution suspension thalline of 0.2M, pH4.8, shakes 30min, at 10000 × g at 70 DEG C
Under be centrifuged 1min, collect thalline, obtain described permeability glutamic acid decarboxylase enzyme engineering bacteria；
Wherein, described engineering bacteria includes host cell and proceeds to the glutamate decarboxylase base of host cell
Cause；Described host cell is escherichia coli (E.coli) BL21；Described glutamic acid decarboxylase gene source
From Lactobacillus brevis (Lactobacillus brevis) CGMCC 1306；
The inducing culture process of described engineering bacteria is: from the LB solid medium of activation described in picking
The monoclonal thalline of engineering bacteria accesses containing 50 μ g mL-1In the LB fluid medium of kanamycin,
37 DEG C, 200r/min shaken cultivation overnight, obtains seed liquor；Seed liquor by volume mark 1% is inoculated
Amount is inoculated into containing 50 μ g mL-1In the LB fluid medium of kanamycin, it is placed in 37 DEG C, 200
Under the conditions of r/min, cultivate to OD600When reaching 0.6-0.8, add IPTG, make IPTG final concentration
It is 0.5 μM, at 30 DEG C, under the conditions of 150r/min, inducing culture 6h.
2. the permeability glutamic acid decarboxylase enzyme engineering bacteria that method prepares as claimed in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054569A (en) * | 2006-06-21 | 2007-10-17 | 华东师范大学 | Gene engineering bacterium, preparation and use thereof |
| CN102080090A (en) * | 2010-02-01 | 2011-06-01 | 浙江大学宁波理工学院 | Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054569A (en) * | 2006-06-21 | 2007-10-17 | 华东师范大学 | Gene engineering bacterium, preparation and use thereof |
| CN102080090A (en) * | 2010-02-01 | 2011-06-01 | 浙江大学宁波理工学院 | Cloning, expression and application of Lactobacillus brevis glutamate decarboxylase gene |
Non-Patent Citations (1)
| Title |
|---|
| Preparation of γ-Aminobutyric Acid Using E. coli Cells with High Activity of Glutamate Decarboxylase;A. YU. PLOKHOV et al;《Applied Biochemistry and Biotechnology》;20001231;第88卷;257-265 * |
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Effective date of registration: 20200923 Address after: 274600 No. 1666 East Ring Road, Juancheng County, Shandong, Heze Patentee after: SHANDONG YANGCHENG BIOTECH Co.,Ltd. Address before: 315100 No. 1, Qian Hunan Road, Zhejiang, Ningbo Patentee before: Zhejiang University of science and engineering Ningbo |