CN109706131A - A kind of genetic engineering bacterium that expressing high specific beta cyclodextrin glycosyl transferase and its construction method and application - Google Patents
A kind of genetic engineering bacterium that expressing high specific beta cyclodextrin glycosyl transferase and its construction method and application Download PDFInfo
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Abstract
The invention discloses a kind of genetic engineering bacterium for expressing high specific beta cyclodextrin glycosyl transferase and its construction method and applications, are related to a kind of genetic engineering bacterium.Genetic engineering bacterium of the invention is using plasmid cgt/pET-22b as template, mutant A47S is obtained after rite-directed mutagenesis, A47M, A47Y, A47R, and E.coli BL21 (the DE3)/A47S converted in host strain E.Coli BL21, E.coli BL21 (DE3)/A47M, E.coli BL21 (DE3)/A47Y, E.coli BL21 (DE3)/A47R, engineering bacteria of the invention effectively increases the product specificities of the β-CGTase enzyme from Bacillus cereus, the industrialized production for being preferably applied to beta-cyclodextrin for it provides certain basis.
Description
Technical field
The present invention relates to a kind of genetic engineering bacterium more particularly to a kind of glucose glycosyls for expressing high specific beta cyclodextrin
Transferase gene engineering bacteria and its construction method and application.
Background technique
The cyclic annular glucan that cyclodextrin (CDs) is formed by 6 to more than 100 glucose units, common are α-, β-,
γ-CD (corresponding glucosyl group number of unit is respectively 6,7,8).CDs contains unique hydrophobic cavity, can be small with hydrophobicity
Molecule forming bag mixture.This unique property enables CDs to form water-soluble inclusion complex with many insoluble drugs,
Enhance the water solubility of drug, and improve the stability and bioavilability of drug, is ideal excipient substance, is applied to water
In 35 kinds of drug products such as property parenteral solution, nasal spray and eye drops.CDs is in fields such as food, agricultural and cosmetics
It is widely used.
Cyclodextrin glycosyl transferases (CGTase) (EC 2.4.1.19) belong to the alpha-amylase family (13 of glycosyl hydrolase
Family), it is the cell ectoenzyme that α-Isosorbide-5-Nitrae key is cracked into CDs in catalytic starch or other polysaccharide.CGTase can be catalyzed four kinds
Reaction: cyclisation (α-glycosidic bond cracking in amylose or amylopectin, portion of product form CDs)；It is coupled (the ring-type of CDs
α in structure-glycosidic bond cracking, by resulting oligosaccharides to being transferred to receptor substrate)；(α-glycosidic bond of linear oligosacchardides is disconnected for disproportionation
Chain, the glucose glycosyl of generation are transferred to receptor substrate, generate the different glycation product of the degree of polymerization) and hydrolysis (hydrone work
For receptor, catalysis substrate hydrolysis forms oligosaccharide).Wherein, cyclization generates the characteristic reaction that CDs is CGTase, i.e.,
The α of CGTase catalytic starch-glycosidic bond cracking, portion of product form CDs as glycosyl acceptor.And coupling reaction and cyclisation are anti-
Answer back reaction each other.Cyclisation, coupling, disproportionation are transglycosylations, and activity is higher, and CGTase is mainly utilized in industrial production
Transglycosylation；The activity of hydrolysis is usually lower, and the hydrolysing activity for controlling CGTase is beneficial to cyclodextrin
Production.
CGTase enzyme law catalysis Starch formation CDs, product is usually the mixture of α-, β-, γ-CD, after this is to CDs
Continuous separation and purifying have an adverse effect.In order to improve the product specificities of CGTase, there are mainly two types of effective strategy, to
Organic solvent is added in reaction system or changes the structure of CGTase using direct mutagenesis.It can although adding organic solvent
To improve the specificity and total recovery of cyclodextrin, but the toxicity of solvent (such as toluene or acetone) and removed from final products
The cost of solvent significantly limits the large-scale application of cyclodextrin product, especially in food and medicine industry.With use
Organic solvent is compared, and the method for changing the structure of CGTase is more environmentally friendly and cost is lower.Therefore, rational modification CGTase is to mention
High product specificities are most important.And β-CD is since the water solubility of itself is low, purification is relatively easy, so at present in practical work
Industry using it is upper it is most commonly used be β-CD, occupy the leading position of CDs output.
Currently, Master's thesis disclosed in Ling Guoqing " Bacillus cercus β-CGTase fermentation, gene cloning and expression and its
Zymologic property research " in the bacterial strain of high yield β-CGTase a kind of disclosed, the bacterial strain be using Nitrogen ion to Bacillus cercus into
Row mutagenic and breeding obtains, uncertain larger, at high cost although its bacterial strain for having obtained high yield β-CGTase.Ling Kai
It has found and in disclosed Master's thesis " cyclodextrin glycosyl transferases molecular modification, product specificities and zymologic property research "
CGTase43 amino acids derived from Bacillus cercus play a significant role CGTase product specificities, to recombinate matter
Grain pET22b/cgt is template, carries out saturation mutation, final acquisition pair to 43 hyte propylhomoserins using the mutation method of full plasmid
The best mutant enzyme of γ-CD specificity, it is seen then that although the method that Ling Kai uses vertex mutation obtains mutant enzyme, it is needle
To the applicable of γ-CD specificity, it is not particularly suited for the β-CD (i.e. beta cyclodextrin glycosyl transferase) of industrial application, it is seen then that mesh
It is preceding to be badly in need of a kind of engineering bacteria and method that directly obtain the production for β-CD.
Summary of the invention
To solve the problems, such as in the prior art to produce β-CD, the present invention is intended to provide a kind of expression high specific β ring is pasted
The genetic engineering bacterium and its construction method and purposes of glycosyltransferase provide higher conversion ratio for the industrial production of β-CD,
It reduces its cost.The technical problem to be solved by the present invention is to the means buildings by genetic engineering can express high specific β ring
The engineering strain of dextrin glycosyl transferase (β-CGTase), and production of the enzyme for β-CD is prepared by induction.
Technical purpose to realize the present invention, first aspect present invention provide a kind of building expression high specific beta cyclodextrin
The mutant of glycosyl transferase is by the cyclodextrin glycosyl transferases gene of Bacillus cercus (Bacillus cereus)
The 47th alanine carry out rite-directed mutagenesis acquisition.
Wherein, the mutant includes: to sport the 47th in cyclodextrin glycosyl transferases gene alanine Ala
Serine Ser, is named as A47S；The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into methionine
Met is named as A47M；The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into tyrosine Tyr, is named
For A47Y；The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into arginine Arg, is named as A47R.
Technical purpose to realize the present invention, second aspect of the present invention provide a kind of building expression high specific beta cyclodextrin
The primer of the mutant of glycosyl transferase, the mutant primer including introducing single mutant A47S as shown in SED ID NO.2-3
It is right；The mutant primer pair of single mutant A47M is introduced as shown in SED ID NO.4-5；Draw as shown in SED ID NO.6-7
Enter the mutant primer pair of single mutant A47Y；The mutant primer pair of single mutant A47R is introduced as shown in SED ID NO.8-9
In it is any or a variety of.
Technical purpose to realize the present invention, third aspect present invention provide a kind of building expression high specific beta cyclodextrin
The kit of the mutant of glycosyl transferase including primer described in second aspect and makes cyclodextrin glycosyl transferases gene
In the 47th alanine Ala mutate reagent.
Wherein, the reagent includes:
Utilize the primer pair recombinant expression plasmid of the mutant of building expression high specific beta cyclodextrin glycosyl transferase
PET22b-cgt template carries out mutation synthesis, obtains the reagent I of single mutant A47S, A47M, A47Y, A47R respectively；And
PCR product is handled, the reagent II of mutant is obtained.
Wherein, the recombinant expression plasmid is to be inserted into carrier pET22b (i.e. pET22b (+)) with pET22b-cgt gene
In it is built-up, carrier pET22b be purchased from Novagen company.
Especially, the pET22b-cgt gene is to expand skill with PCR using Bacillus cercus genome as template
What art was cloned.
Further, the pET22b-cgt enzyme gene shares base 2085bp, base sequenceSuch as SEQ IDNO.1 institute
Show, the accession number of GeneBank is Accession No.KF269705.
Wherein, the reagent I is cgt/pET-22b (+) 1 μ L, Forward Primer (10 μM) 1 μ L, Reverse
1 μ L, 2 × TransStart FastPfu PCRSupermix of Primer (10 μM) 25 μ L, ddH2O22 μ L amounts to 50 μ L bodies
Long-pending reaction system.
Wherein, using the reaction condition of reagent I are as follows: 94 DEG C of initial denaturation 3min；Then carry out 30 circulation (94 DEG C of 10s,
60 DEG C of 20s, 72 DEG C of 4min)；Last 72 DEG C of heat preservations 10min.
It should be noted that it is the PCR amplification system of other volume amounts that the reagent I, which can also use total volume, this
Field technical staff can be adjusted according to actual needs, and reaction condition can also be adaptively adjusted, and above-mentioned adjustment is all
Within the scope of the present invention.
Wherein, the reagent II is DMT enzyme.
Wherein, the dosage of the DMT enzyme is 1 μ L.
It should be noted that the reagent II can also use other that can make PCR product that demethylation template occur
Reagent, dosage are adjusted according to routine test.
Technical purpose to realize the present invention, fourth aspect present invention provide a kind of building expression high specific beta cyclodextrin
The method of the mutant of glycosyl transferase, comprising: the kit pair provided using primer described in second aspect or the third aspect
Recombinant expression plasmid pET22b-cgt carries out 47 alanine mutations, respectively obtains gene order the 47th and sports ammonia
The mutant A47S of sour Ser, the mutant A47M for becoming methionine Met, the mutant A47Y for becoming tyrosine Tyr and become
The mutant A47R of arginine Arg.
Technical purpose to realize the present invention, fifth aspect present invention provide a kind of expression high specific beta cyclodextrin glycosyl
The genetic engineering bacterium of transferase, be by by the mutant of first aspect in competent escherichia coli cell bacterial strain into
Row conversion obtains.
Wherein, the competent escherichia coli cell bacterial strain is E.Coli BL21 (DE3).
Wherein, the genetic engineering bacterium of the expression high specific beta cyclodextrin glycosyl transferase includes that mutant A47S exists
E.coli BL21 (the DE3)/A47S obtained is converted in competent escherichia coli cell bacterial strain E.Coli BL21 (DE3), will be dashed forward
The modification A 47M E.coli BL21 that conversion obtains in competent escherichia coli cell bacterial strain E.Coli BL21 (DE3)
(DE3)/A47M, the conversion acquisition in competent escherichia coli cell bacterial strain E.Coli BL21 (DE3) by mutant A47Y
E.coli BL21 (DE3)/A47Y, by mutant A47R in competent escherichia coli cell bacterial strain E.Coli BL21 (DE3)
Convert E.coli BL21 (the DE3)/A47R obtained.
Above-mentioned engineering bacteria provided by the invention can make mutant enzyme A47S, A47M, A47Y, A47R keep the same of high enzyme activity
When, the ratio of beta-cyclodextrin is obviously improved in product, and beta-cyclodextrin product is more single-minded, and β-ring paste is preferably applied to for it
The industrialized production of essence provides certain basis.
Technical purpose to realize the present invention, sixth aspect present invention provide a kind of expression high specific beta cyclodextrin glycosyl
The construction method of the genetic engineering bacterium of transferase, comprising:
Mutant described in first aspect is subjected to expression conversion in competent escherichia coli cell bacterial strain, obtains weight
Group engineering bacteria；
After screening using ampicillin to obtained recombination engineering, expression high specific beta cyclodextrin is obtained
The genetic engineering bacterium of glycosyl transferase.
Technical purpose to realize the present invention, seventh aspect present invention provide a kind of by mutant described in first aspect
Or the 5th aspect described in genetic engineering bacterium be used to prepare expression high specific β-CGTase application.
Wherein, prepare the expression high specific β-CGTase the following steps are included:
The genetic engineering bacterium of high specific beta cyclodextrin glycosyl transferase will be expressed in LB culture medium with ampicillin
It is middle to be cultivated twice, obtain bacterial concentration OD600Reach 1.0~1.8 bacterium solution；
Inducer is added into bacterium solution and has carried out fermented and cultured, obtains thallus；
Buffer is added into thallus, obtains cyclodextrin glycosyl transferases crude enzyme liquid.
Wherein, express high specific beta cyclodextrin glycosyl transferase genetic engineering bacterium be selected from E.coli BL21 (DE3)/
A47S, E.coli BL21 (DE3)/A47M, E.coli BL21 (DE3)/A47Y, one in E.coli BL21 (DE3)/A47R
It is a or multiple.
Wherein, described cultivated twice in LB culture medium with ampicillin includes:
The genetic engineering bacterium culture 16-19h of high specific beta cyclodextrin glycosyl transferase will be expressed, obtains seed culture bacterium
First culture of liquid；And
The second culture that seed culture bacterium solution carries out in the culture medium of ampicillin is drawn, concentration OD is obtained600It reaches
To 1.0~1.8 bacterium solution.
Wherein, the content of ampicillin is 50~100 μ g/mL, LB trainings in the LB culture medium with ampicillin
Supporting base is 50mL.
Wherein, first and second condition of culture is revolving speed 250r/min, 30~45 DEG C of temperature.
Wherein, the inducer position IPTG inducer, is 0.1-1.0mM using concentration, and use condition is at 25-30 DEG C
Fermentation inducement 4-20h.
Preferably, the use concentration of the inducer is 0.2~0.6mmol/L, and use condition is to ferment at 25-30 DEG C
Induce 8-16h.
Wherein, the glycine-NaOH buffer volume pH value is 7.0~9.0.
Wherein, β-CGTase enzyme activity is 8000-9300U/mL in the crude enzyme liquid.
Especially, in the crude enzyme liquid β-CGTase enzyme activity be detected by iodine number method specifically includes the following steps:
After being reacted in crude enzyme liquid with glycine-NaOH buffer, soluble starch, glacial acetic acid is added and carries out eventually
Only, detection product is obtained；
Iodine solution is added into detection product and carries out color development treatment, and the equal amount of distilled water enzyme solution is not added is as to shining into
Row absorbance detection confirms enzyme activity force value.
Wherein, the dosage in the crude enzyme liquid with glycine-NaOH buffer, soluble starch, glacial acetic acid are as follows: thick enzyme
10 μ L, 0.2mol/L glycine-NaOH buffer (pH 8.5) of liquid, 200 μ L, the soluble shallow lake that 200 μ L mass concentrations are 0.2%
The 500 μ L of glacial acetic acid of powder, 0.5M.
Wherein, the reaction temperature is 40 DEG C, reaction time 10min.
Wherein, it is 0.005% that the additional amount of the iodine solution, which is 3mL mass concentration,.
Wherein, the Detection wavelength of absorbance is 700nm.
Wherein, the determination method of the enzyme activity force value is that an enzyme-activity unit is defined as making the enzyme of absorbance decline 10%
Amount.
It should be noted that β-CGTase enzyme activity can be used disclosed in Yu-Heng D etc. in crude enzyme liquid
“Studies on Ferment Rule of the Cyclodextrin Glucanotransferase High-
Method in performance Strain Mutated by Ion Beam " is realized.
Technical purpose to realize the present invention, eighth aspect present invention provide a kind of by mutant described in first aspect
Or the 5th aspect described in genetic engineering bacterium preparation expression high specific beta cyclodextrin glycosyl transferase genetic engineering bacterium use
In preparing the application in β-CD.
Wherein, the application is the genetic engineering bacterium by the expression high specific beta cyclodextrin glycosyl transferase with starch
It ferments for substrate, prepares β-CD.
In particular, the step of application above-mentioned high specific beta cyclodextrin glycosyl transferase crude enzyme liquid enzymatic clarification β-CD
It is as follows:
Expression high specific beta cyclodextrin glycosyl transferase is added to soluble starch and is dissolved in Glycine-NaOH
In the starch substrates obtained after buffer heating, catalysis reaction is carried out in the environment of organic solvent, obtains β-CD.
Wherein, the soluble starch and Glycine-NaOH buffer use ratio are 5g soluble starch,
50ml Glycine-NaOH buffer.
Wherein, soluble starch is commercially available any soluble starch.
Wherein, the starch that the dosage of the expression high specific beta cyclodextrin glycosyl transferase is 600U~800U/g is used
Amount.
Wherein, the operating condition of the catalysis reaction is under 50~55 DEG C of temperature, pH value 8.0~8.5.
Wherein, the organic solvent is in the organic solvents such as ethyl alcohol, n-butanol, isobutanol, the tert-butyl alcohol, sec-butyl alcohol
It is one or more.
Especially, the additive amount of the organic solvent accounts for the percent by volume 5%~40% of overall reaction system.
Wherein, catalytic reaction condition is in 50~55 DEG C of temperature, pH value 8.0~8.5.
The present invention constructs expression high specific beta cyclodextrin glycosyltransferase gene engineering by the means of genetic engineering
Bacterium, and optimize original enzyme process processed；High specific beta cyclodextrin glycosyltransferase gene engineering bacteria of the present invention
Be on the basis of retaining former beta cyclodextrin glycosyltransferase gene engineering bacteria high enzyme activity, it is fixed by design of primers, genetic engineering
What the genetic engineering means such as point mutation and host strain conversion obtained, the yield of β-CD is higher in product, and β-CD is accounted in gross product
Ratio it is bigger, i.e., product specificities are stronger；Compared with the production technology of former beta cyclodextrin glycosyltransferase gene engineering bacteria, make
The more efficient of β-CD is prepared with the enzyme, the yield of β-CD is higher and product is more single-minded.
Detailed description of the invention
Fig. 1 is the chemical structure of cyclodextrin；
Fig. 2 is point mutation schematic illustration (quoting from Quan Shi King Company)；
Fig. 3 is enzymatic clarification cyclodextrin schematic diagram；
Fig. 4 is the agarose gel electrophoresis figure of embodiment 1；
Fig. 5 is the sequencing result comparison chart of embodiment 1；
Fig. 6 is the experimental result picture of test example 1, wherein A is influence of the cultivation temperature to β-CGTase enzyme activity；B is
OD600Influence to β-CGTase enzyme activity；C is influence of the IPTG concentration to β-CGTase enzyme activity；D is induction time to β-
The influence of CGTase enzyme activity；
Fig. 7 is the experimental result picture of test example 2, wherein A is influence of the temperature to β-CGTase enzyme activity；B.pH is to β-
The influence of CGTase enzyme activity；C. influence of the temperature to β-CGTase enzyme stability；Shadow of the D.pH to β-CGTase enzyme stability
It rings；
Fig. 8 is the experimental result picture of test example 3；
Fig. 9 is the experimental result picture of test example 4.
Specific embodiment
Present invention will be further explained below with reference to specific examples.But these embodiments be only limitted to illustrate the present invention without
For limiting the scope of the invention.The experimental method of specific experiment condition is not specified in the following example, usually according to conventional strip
Part, or according to condition proposed by manufacturer, the ingredient in specific source is not specified, unless otherwise instructed, pass through commercially available acquisition.
The acquisition of 1 mutant of embodiment
1, the acquisition of β-CGTase target gene
The present invention with Master's thesis disclosed in Ling Guoqing " Bacillus cercus β-CGTase fermentation, gene cloning and expression and
Its zymologic property research " disclosed in β-CGTase bacterial strain be purpose gene obtain source, the bacterial strainGeneBank is logged in It number is Accession No.KF269705.1, the specific steps are as follows:
1.1, the glycerol stocks bacterium solution that above-mentioned Bacillus cercus is taken out from -80 DEG C of refrigerators, is connect by 1% inoculum concentration
Kind is incubated overnight into LB liquid medium；
1.2, the bacterium solution for taking 4mL culture 12h, is centrifuged 1min, retains precipitating thallus；
1.3,250uL RB solution (A containing RNase) is added, and is sufficiently vibrated with vortex oscillator, keep Escherichia coli outstanding
It floats in RB, until without macroscopic fungus block；
1.4,250uL LB solution is added, slowly overturning 5-6 times cracks Escherichia coli；
1.5,350uL NB solution is added, it is slowly 5-6 times reverse, until generating agglutination block；
1.6, it is centrifuged 10min under room temperature, liquid-transfering gun is drawn supernatant and is added in adsorption column, and 2min adsorption column film is centrifuged
Center is Plasmid DNA；
1.7, the WB solution that 650 μ L contain ethyl alcohol is added, 12,000r/min centrifugation 1min abandon filtrate；
1.8, centrifugal column is placed in clean centrifuge tube, the Elution that preheating is advanced to 60 DEG C is added at center membrane
Buffer solution；
1.9,10,000r/min is centrifuged 1min eluted dna, obtains the DNA of β-CGTase target gene, and by β-
The DNA of CGTase target gene is saved in -20 DEG C.
It needs, the acquisition of the object of the invention gene can also be according to Bacillus cercusShown in GeneBank Gene order, and(such as method of PCR) by conventional method, artificial synthesized β-CGTase target gene cgt.
2, the acquisition of recombinant expression plasmid pET22b-cgt
Recombinant expression plasmid provided by the invention can be obtained by following steps:
β-CGTaset gene the cgt that step 1 obtains is inserted into carrier pET22b (+) and is constructed, is recombinated
Expression plasmid pET22b-cgt construction method is referred to " recombination cyclodextrin glycosyltransferase temperature control disclosed in all peaceful grades
Content in the building of type engineering bacteria and its optimization of condition of culture ", wherein carrier pET22b (+) is purchased from Novagen company.
3, rite-directed mutagenesis
47 alanine in recombinant expression plasmid pET22b-cgt carry out rite-directed mutagenesis processing, to be distinguished
Gene order the 47th is obtained to sport the mutant A47S of serine Ser, the mutant A47M for becoming methionine Met, become
The mutant A47Y of the tyrosine Tyr and mutant A47R for becoming arginine Arg, the specific operation method is as follows:
Using recombinant expression plasmid pET22b-cgt as template, the mutant primer of each mutant is introduced, utilizes One_step PCR skill
Art carries out mutation synthesis, wherein mutant primer is respectively as follows:
The mutant primer for introducing single mutant A47S is:
Forward primer: 5'-TTCTCAGACGGAAATCCAAGCAACAACCCATCTGGG-3', the base sequence of underscore
For the codon of mutating acid Ser,
Reverse primer: 5'-GCTTGGATTTCCGTCTGAGAACCGGTCGGTCACAAT-3', the base sequence of underscore
For the complementary base pair of the codon of mutating acid Ser；
The mutant primer for introducing single mutant A47M is:
Forward primer: 5'-TTCTCAGACGGAAATCCAATGAACAACCCATCTGGG-3', the base sequence of underscore
For the codon of mutating acid Met,
Reverse primer: 5'-CATTGGATTTCCGTCTGAGAACCGGTCGGTCACAAT-3', the base sequence of underscore
For the complementary base pair of the codon of mutating acid Met；
The mutant primer for introducing single mutant A47Y is:
Forward primer: 5'-TTCTCAGACGGAAATCCATATAACAACCCATCTGGG-3', the base sequence of underscore
For the codon of mutating acid Tyr,
Reverse primer: 5'-ATATGGATTTCCGTCTGAGAACCGGTCGGTCACAAT-3', the base sequence of underscore
For the complementary base pair of the codon of mutating acid Tyr.
The mutant primer for introducing single mutant A47R is:
Forward primer: 5'-TTCTCAGACGGAAATCCACGTAACAACCCATCTGGG-3', the base sequence of underscore
For the codon of mutating acid Arg,
Reverse primer: 5'-ACGTGGATTTCCGTCTGAGAACCGGTCGGTCACAAT-3', the base sequence of underscore
For the complementary base pair of the codon of mutating acid Arg.
The PCR amplification system of each mutant is plasmid cgt/pET-22b (+) 1 μ L, Forward Primer (10 μM) 1 μ
1 μ L, 2 × TransStart FastPfu PCRSupermix of L, Reverse Primer (10 μM) 25 μ L, ddH2O22 μ L, always
Count the reaction system of 50 μ L volumes.
The PCR amplification program of each mutant is equal are as follows: 94 DEG C of initial denaturation 3min；Then carry out 30 circulation (94 DEG C of 10s, 60
DEG C 20s, 72 DEG C of 4min)；Last 72 DEG C of heat preservations 10min.
PCR product through agarose gel electrophoresis (gel electrophoresis result such as Fig. 4) verify its size it is errorless after, to PCR product
In plus 1 μ L DMT enzyme, mix, 37 DEG C be incubated for one hour, removal methylation template, by the sequence of products therefrom carry out sequencing test
It demonstrate,proves (sequencing result is as shown in Figure 5), it is the expression high specific beta cyclodextrin glycosyl transferase constructed that correct product, which is sequenced,
Mutant A47S, A47M, A47Y and A47R.
The acquisition of 2 genetic engineering bacterium of embodiment
1, the plasmid of Escherichia coli is transformed into clone bacterium
1.1 take out E.coli DMT competent cell from -80 DEG C of refrigerators, melt in ice bath, are divided into six groups；
1.2 after competence thawing, 5 μ L A47S is separately added into each group, the PCR of A47M, A47Y, A47R mutant expands
Increase production object into E.coli DMT competent cell, flicks mixing, ice-water bath half an hour；
1.3 heat shocks: 42 DEG C of water-bath heat shock 45s are then immediately placed in 2min in ice bath, during which avoid acutely shaking；
1.4 activation bacterium solutions: under gnotobasis, the LB liquid medium that resistance is not added is preheated to 37 DEG C, in each group respectively
250uL LB liquid medium is added, cultivates 1h under 200rpm, 37 DEG C of shaking tables；
1.5 bacterium solutions are resuspended: the bacterium solution 4000r/min after activation being centrifuged 2min, removes supernatant culture medium, takes lower layer heavy
Shallow lake is resuspended；
1.6 spread plates: from the bacterium solution even spread taken in each group after activating in right amount to the solid containing ampicillin
On LB plate, it is placed in 37 DEG C of biochemical cultivation cases and is incubated overnight.
1.7 preservation of bacteria strain: from picking individual colonies on each plate, it is placed in the 50ml liquid LB culture containing ampicillin
In base, and activated under 37 DEG C of shaking tables.Next day draws 500uL seed culture fluid and 500uL glycerol according to the ratio preservation of 1:1
Clone has the clone bacterium strain of A47S, A47M, A47Y, A47R mutant respectively.
2, the plasmid of Escherichia coli is transformed into expression bacterium experiment
2.1 extract matter from the seed culture fluid for cloning the clone bacterium for having A47S, A47M, A47Y, A47R mutant respectively
Grain；
E.coli BL21 (DE3) competent cell is taken out from -80 DEG C of refrigerators, is melted in ice bath, is divided into six groups；
2.2 after competence thawing, and the plasmid of 5 μ LA47S, A47M, A47Y, A47R are separately added into each group to E.coli
In BL21 (DE3) competent cell, mixing, ice-water bath half an hour are flicked；
2.3 heat shocks: 42 DEG C of water-bath heat shock 45s are then immediately placed in 2min in ice bath, during which avoid acutely shaking；
2.4 activation bacterium solutions: under gnotobasis, the LB liquid medium that resistance is not added is preheated to 37 DEG C, respectively takes 250uL
Liquid LB is added thereto, and cultivates 1h under 200r/min, 37 DEG C of shaking tables；
2.5 bacterium solutions are resuspended: the bacterium solution 4000r/min after activation being centrifuged 2min, removes supernatant culture medium, takes lower layer heavy
Shallow lake is resuspended；
2.6 spread plates: take the bacterium solution even spread after activating in right amount to the solid LB plate containing ampicillin
On, it is placed in 37 DEG C and is incubated overnight.
2.7 preservation of bacteria strain: from picking individual colonies on each plate, it is placed in the 50ml liquid LB culture containing ampicillin
In base, and activated under 37 DEG C of shaking tables.Next day draws 500uL seed culture fluid and the glycerol of 500uL divides according to the ratio of 1:1
Strain of the other preservation with high specific β-CGTase is to get the genetic engineering for expressing high specific beta cyclodextrin glycosyl transferase
Bacterium E.coli BL21 (DE3)/A47S, E.coli BL21 (DE3)/A47M, E.coli BL21 (DE3)/A47Y, E.coli
BL21(DE3)/A47R。
The acquisition of 3 β-CGTase enzyme of embodiment
By genetically engineered E.coli BL21 (DE3)/A47S, E.coli BL21 (DE3)/A47M, E.coli BL21
(DE3)/A47Y, E.coli BL21 (DE3)/A47R are inoculated into the 50mL LB containing 50~100 μ g/mL ampicillins respectively
In culture medium, 16-19h is cultivated at revolving speed 250r/min, 35~40 DEG C, is drawn 50 μ L seed culture bacterium solutions and is forwarded to containing 50
In the 50mL LB culture medium of~100 μ g/mL ampicillins, cultivated at revolving speed 250r/min, 35~40 DEG C dense to bacterium solution
Spend OD600The IPTG inducer that concentration is 0.2mmol/L is added when reaching 1.8, fermentation inducement 16h at 25 DEG C is centrifugated
To thallus, the glycine-NaOH buffer that pH value is 7.0~9.0, ultrasonication, centrifuge separation, supernatant are added into thallus
Liquid is crude enzyme liquid, and the size of β-CGTase enzyme activity is detected by iodine number method, reaches 9241U/mL.
It should be noted that being 15-35 DEG C, OD in fermentation temperature600Value is 0.2-2.2, IPTG concentration value in 0.1-
Technical purpose of the invention may be implemented in any value of 1.0mM, induction time within the scope of 4-20h, such as temperature is
16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 DEG C etc.；Such as OD600Value is
0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、 1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1
Deng；Such as IPTG concentration value is 0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9；Such as induction time is 5,6,7,8,9,
10,11,12,13,14,15,16,17,18,19 technical purpose of the invention can be achieved.
The preparation of 4 β-CD of embodiment
1, zymolyte reaction solution is prepared: being weighed 5g soluble starch and is dissolved in 50mL 0.2M glycine-NaOH buffer
In, different volumes can be prepared according to the ratio, by starch liquor heating to starch gelatinization, obtain gelatinization of starch solution；
2, after gelatinization of starch solution is cooled to room temperature, ring is added according to the ratio of 600U~800U/g starch in enzyme concentration
Dextrin glycosyl transferase；
3, the mixed liquor for obtaining step 2 be placed in reaction temperature be 55 DEG C, pH value be 8.5 reaction system under urge
Change reaction 28h, the tert-butyl alcohol of 25% percent by volume of overall reaction system is added to control the yield of β-CD, organic solvent
Additive amount accounts for the percent by volume 5%~40% of overall reaction system；
4, it takes appropriate reaction solution to boil 5min in boiling water bath, obtains supernatant, warp after 12000r/min is centrifuged 10min
Up to sample after 0.45 μm of membrane filtration.
The made sample of 20~50 μ L is taken to carry out high performance liquid chromatography detection.
5, testing result
Use Composition distribution for liquid-phase sensor, mobile phase be 65% acetonitrile/35% water, flow velocity 1.0ml/min,
Chromatographic column is X-Amide chromatographic column, and column temperature is 65 DEG C and detects to the β-CD yield in product that testing result is shown, β-CD
Yield reaches 31.15mg/ml, and content accounts for the 94% of gross product, is above the existing yield level of this field.
Inventor is obtained in order to obtain the β-CGTase enzyme of high expression and high vigor, and using β-CGTase enzyme
High β-CD yield, has done a large number of experiments research, is part test and test result below.
1 β-CGTase expression of enzymes of test example and fermentating condition test
Inventor is by the genetic engineering bacterium of the expression high specific beta cyclodextrin glycosyl transferase of acquisition in different culture temperature
Degree, OD600It ferments under conditions of value, IPTG concentration and induction time, fermentation process is same as Example 3, as a result such as
Shown in Fig. 6, result is it is found that be 15-35 DEG C, OD in temperature value according to Fig.6,600Value is that 0.2-2.2, IPTG concentration value exist
Within the scope of 4-20h, β-CGTase enzyme all has the expression quantity of 1000U/mL or more for 0.1-1.0mM, induction time, but with
Temperature value, OD600The raising of value, IPTG concentration value and induction time value, the expression of genetic engineering bacterium be in first increase, then
Reduced trend, especially in 25 DEG C of temperature or OD600When value is 1.8 or IPTG concentration is 0.2mM or induction time is 1.6h
When, expression quantity highest is above 5000U/mL or more, it is seen then that although temperature value is 15-35 DEG C, OD600Value be 0.2-2.2,
IPTG concentration value still works as temperature in the expression that β-CGTase enzyme may be implemented in 4-20h in 0.1-1.0mM, induction time
25 DEG C or OD600When value is 1.8 or IPTG concentration is 0.2mM or when induction time is 1.6h, and the expression quantity of β-CGTase enzyme is most
It is high.
The property development test of 2 recombinant beta-CGTase enzyme of test example
β-CGTase the enzyme that embodiment 3 is obtained carries out optimum temperature, pH and enzyme activity, enzyme stability experiment, experiment
For method using conventional method, concrete operations are as follows:
1, the optimum temperature experiment of β-CGTase
The optimum temperature of β-CGTase: β-CGTase is incubated for 10 points under the different temperatures within the scope of 40 DEG C -100 DEG C
Zhong Hou determines the optimum temperature of β-CGTase by measuring remaining enzyme activity at each temperature.
2, the Optimal pH experiment of β-CGTase
0.2mol/L glycine-NaOH buffer (pH8.5) is replaced with following buffer respectively, is surveyed using iodine number method
Enzyme activity is measured to determine the Optimal pH of β-CGTase: 0.1mol/L citric acid/sodium buffer (pH4.0-6.0), 0.05mol/L phosphorus
Phthalate buffer (pH7.0-8.0), 0.2mol/L glycine-NaOH buffer (pH9.0-11.0).
3, enzyme stability is tested
3.1 temperature stability
By 0.01mL enzyme and 0.2mL 0.2mol/L glycine-NaOH buffer (pH8.5) at (40 DEG C -90 of different temperatures
DEG C) under be incubated for 30 minutes after calculate remaining enzyme activity to measure the temperature stability of β-CGTase.
3.2 pH stability
Remaining enzyme activity is measured after the 0.01mL enzyme purified is incubated for 30 minutes in following buffer respectively to judge β-
The pH stability of CGTase: 0.1mol/L citric acid/sodium buffer (pH4.0-6.0), 0.05mol/L phosphate buffer
(pH7.0-8.0), 0.2mol/L glycine-NaOH buffer (pH9.0-11.0).
4, enzyme activity is tested
The enzyme activity measurement of β-CGTase uses iodine number method, and will be 55 DEG C in temperature, the enzyme activity number of pH is 8.5 lower measurement
Value is denoted as 100%, calculates opposite enzyme activity, remaining enzyme activity with this.
5, experimental result
Testing result as shown in fig. 7, result according to figure 7 it is found that by β-CGTase in 40 DEG C -100 DEG C, pH value
To be incubated within the scope of 5.0-11.0 after ten minutes, remaining enzyme activity at each temperature is measured to determine the optimum temperature of β-CGTase
And optimal pH, as a result as shown in Figure 7 A, 7 B, temperature value is 40-100 DEG C, pH value is β-within the scope of 5.0-11.0
CGTase enzyme all has 10% or more remaining enzyme activity, but with temperature value, the raising of pH value, the residual enzyme of β-CGTase
Work is in first to increase, then reduced trend, with respect to enzyme activity highest, is denoted as especially when 55 DEG C of temperature or pH value are 8.5
100%, it is seen then that temperature value is 55 DEG C, pH value be 8.5 be respectively β-CGTase optimum temperature and optimal pH.
By β-CGTase after 40 DEG C -100 DEG C, pH value are to be incubated for 30 minutes within the scope of 5.0-11.0, measurement is at each temperature
Remaining enzyme activity to determine the temperature stability and pH stability of β-CGTase, as a result as shown in Fig. 5 C, Fig. 5 D, in temperature value
It is within the scope of 5.0-11.0 for 40-85 DEG C, pH value, β-CGTase enzyme all has 10% or more remaining enzyme activity, but with temperature
The raising of angle value, pH value, the remaining enzyme activity of β-CGTase are in first to increase, then reduced trend is especially in temperature value
40-70 DEG C or pH value are within the scope of 6.0-9.0, and β-CGTase enzyme all has 80% or more remaining enzyme activity, it is seen then that temperature value
It is the temperature stabilizing range and pH stability range that 6.5-9.0 is respectively β-CGTase in 40-70 DEG C, pH value.
3 β-CGTase enzyme law catalysis of test example synthesizes the temperature optimization test of β-CD
At pH8.5 and different temperatures (40-70 DEG C), 800u/g starch is added into 0.3mol/L gelatinization of starch solution
Enzyme reaction, by measuring β-CD yield at each temperature to determine that β-CGTase enzyme law catalysis synthesizes the optimum temperature of β-CD,
Experimental results are shown in figure 8, result according to figure 8 it is found that when temperature value is 40-70 DEG C, pH value is 8.5, β-CD's
Yield has 10% or more, but with temperature value, the raising of pH value, and the yield of β-CD is in first increasing, then reduced trend,
Especially at 55 DEG C of temperature, the yield highest of β-CD has reached 27.5%, it is seen then that it is β-CGTase enzyme that temperature value, which is 55 DEG C,
Method catalyzes and synthesizes the optimum temperature of β-CD.
The pH Optimum Experiment of 4 β-CGTase enzyme law catalysis of test example synthesis β-CD
At different pH (6.0-10.0) and 55 DEG C of temperature, 800u/g is added into 0.3mol/L gelatinization of starch solution and forms sediment
The enzyme reaction of powder, experimental result as shown in figure 9, result according to Fig. 9 it is found that pH is 6.0-10.0, temperature is 55 DEG C
When, the yield of β-CD has 15% or more, but with temperature value, the raising of pH value, the yield of β-CD is in first to increase, then drop
Low trend, especially when pH is 8.5, the yield highest of β-CD has reached 28.4%, it is seen then that it is β-that pH, which is 8.5,
The Optimal pH of CGTase enzyme law catalysis synthesis β-CD.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
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gatcttcata agtattgtgg tggtgactgg caaggaatta taaacaagat gaacgacggt 240
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gcgctgcacc cgagcggata cacctcatat cacggatatt gggcaagaga ttacaaaaaa 360
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aaaggcatta agattattat ggactttaca ccaaatcatt catcacctgc gctcgaaacg 480
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gacagcattt accgaaactt atacgattta gccgactatg atttgaataa caaggtcgtt 660
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acttataaac ctgtttttac cttcggtgag tggtttttag gaacaggaga agtcgatcca 840
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Claims (10)
1. a kind of mutant of building expression high specific beta cyclodextrin glycosyl transferase, which is characterized in that it is by wax-like gemma
47th alanine of the cyclodextrin glycosyl transferases gene of bacillus (Bacillus cereus) carries out rite-directed mutagenesis acquisition, packet
It includes:
The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into serine Ser, is named as A47S；
The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into methionine Met, is named as A47M；
The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into tyrosine Tyr, is named as A47Y；
The 47th in cyclodextrin glycosyl transferases gene alanine Ala is sported into arginine Arg, is named as A47R.
2. it is a kind of building expression high specific beta cyclodextrin glycosyl transferase mutant primer comprising:
The mutant primer pair of single mutant A47S is introduced as shown in SED ID NO.2-3；
The mutant primer pair of single mutant A47M is introduced as shown in SED ID NO.4-5；
The mutant primer pair of single mutant A47Y is introduced as shown in SED ID NO.6-7；
The mutant primer pair of single mutant A47R is introduced as shown in SED ID NO.8-9.
3. a kind of kit of the mutant of building expression high specific beta cyclodextrin glycosyl transferase, which is characterized in that including power
Benefit require 2 described in primer and the reagent for making the 47th in cyclodextrin glycosyl transferases gene alanine Ala mutate.
4. a kind of method of the mutant of building expression high specific beta cyclodextrin glycosyl transferase characterized by comprising
Recombinant expression plasmid pET22b-cgt is carried out using kit described in primer as claimed in claim 2 or claim 3
47 alanine mutations respectively obtain gene order the 47th and sport the mutant A47S of serine Ser, become methionine
The mutant A47M of Met, the mutant A47Y for becoming tyrosine Tyr and the mutant A47R for becoming arginine Arg.
5. a kind of genetic engineering bacterium for expressing high specific beta cyclodextrin glycosyl transferase, which is characterized in that it is by wanting right
Mutant described in asking 1 is converted to obtain in competent escherichia coli cell bacterial strain；
Preferably, the competent escherichia coli cell bacterial strain is E.Coli BL21 (DE3).
6. a kind of construction method for the genetic engineering bacterium for expressing high specific beta cyclodextrin glycosyl transferase, which is characterized in that packet
It includes:
Mutant described in claim 1 is subjected to expression conversion in competent escherichia coli cell bacterial strain, obtains recombination work
Journey bacterium；
After screening using ampicillin to obtained recombination engineering, expression high specific beta cyclodextrin glycosyl is obtained
The genetic engineering bacterium of transferase.
7. a kind of be used to prepare expression height by mutant described in claim 1 or by genetic engineering bacterium described in claim 5
The application of specific beta-CGTase.
8. the use as claimed in claim 7, which is characterized in that preparing the expression high specific β-CGTase includes following step
It is rapid:
The genetic engineering bacterium for expressing high specific beta cyclodextrin glycosyl transferase is carried out in LB culture medium with ampicillin
It cultivates twice, obtains bacterial concentration OD600Reach 1.0~1.8 bacterium solution；
Inducer is added into bacterium solution and has carried out fermented and cultured, obtains thallus；
Buffer is added into thallus, obtains cyclodextrin glycosyl transferases crude enzyme liquid.
9. it is a kind of by mutant described in claim 1 or by mutant described in claim 5 prepare expression high specific
β-CGTase is used to prepare the application in beta-cyclodextrin；
Wherein, the genetic engineering bacterium of the expression high specific beta cyclodextrin glycosyl transferase ferments by substrate of starch, makes
Standby β-CD.
10. application as claimed in claim 9, which is characterized in that the base of the expression high specific beta cyclodextrin glycosyl transferase
Because engineering bacteria ferments by substrate of starch, prepare β-C D the step of it is as follows:
Soluble starch is dissolved in Glycine-NaOH buffer, the starch substrates after heating, after being gelatinized；
After being cooled to room temperature starch substrates, above-mentioned expression high specific beta cyclodextrin glycosyl transferase is added, in organic solvent
Catalysis reaction is carried out in environment, obtains β-CD.
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| CN114107244A (en) * | 2021-12-13 | 2022-03-01 | 浙江渚隆生物科技有限公司 | Cyclodextrin glycosyltransferase mutant, coding gene and application thereof |
| CN114107242A (en) * | 2021-11-12 | 2022-03-01 | 江南大学 | Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase |
| WO2023000618A1 (en) * | 2021-07-22 | 2023-01-26 | 江南大学 | Bacillus xiaoxiensis and application thereof |
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| CN104004724A (en) * | 2012-12-10 | 2014-08-27 | 江南大学 | Cyclodextrin glycosyl transferase for improving maltodextrin substrate specificity and preparing method of cyclodextrin glycosyl transferase |
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| WO2023000618A1 (en) * | 2021-07-22 | 2023-01-26 | 江南大学 | Bacillus xiaoxiensis and application thereof |
| CN114107242A (en) * | 2021-11-12 | 2022-03-01 | 江南大学 | Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase |
| CN114107242B (en) * | 2021-11-12 | 2022-07-05 | 江南大学 | Method for improving soluble expression quantity of beta-cyclodextrin glucosyltransferase |
| CN114107244A (en) * | 2021-12-13 | 2022-03-01 | 浙江渚隆生物科技有限公司 | Cyclodextrin glycosyltransferase mutant, coding gene and application thereof |
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