CN105838729B - Novel high vigor allinnase of one kind and preparation method thereof - Google Patents
Novel high vigor allinnase of one kind and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of novel high vigor allinnases and preparation method thereof, its technical solution is the wild type allinnase gene that garlic bulb is derived from using random mutation technological transformation, obtain the allinnase mutant gene of high vigor, i.e. novel high vigor allinnase gene, then by novel high vigor allinnase gene respectively in bacillus subtilis expression system, expression in pichia yeast expression system (including Pichia pastoris dissociate expression system and Pichia pastoris surface display system), obtain producing the recombinant bacterial strain of high vigor allinnase, after fermentation expression, the specific enzyme activity for detecting high vigor allinnase improves 50% compared with wild type allinnase.
Description
Technical field
The invention belongs to bioengineering field, it is related to mentioning by the specific enzyme activity that fallibility round pcr lactam enzyme by directional anagenesis in vitro constructs
High novel alliin enzyme mutant, relates in particular to the random mutation and recombinant DNA technology of gene, especially a kind of new
High vigor allinnase of type and preparation method thereof.
Technical background
Allinnase is also known as S-allyl-L-cysteine sulfoxide lyase, Alkylcysteine sulfoxide lyase etc. (E.C.4.4.1.4), 1949 by Stoll and
Seeback first discovery.It has the forms such as dimer, tripolymer, the tetramer.Different vegetable protein polymerized forms is different
, if the allinnase of garlic (Alliium sativum) is dimer, and onion (Alliium cepa) is then poly- for four
Body.
Foreign scholar obtains the gene of coding allinnase from onion, leek, ramps and garlic, but reported
Gene order, protein molecular quality and biochemical property have many disagreements.The garlic bulb allinnase base of the clones such as Rabinkov
It is the full-length gene order of prematurity enzyme (precursor alliinase) before post-translational cleavage because being up to 2200bp.The garlic ammonia
Sour enzyme exists only in garlic bulb and leaf, may be not present in garlic root, but has very high enzymatic activity in its root, thus pushes away
Contain the isodynamic enzyme of allinnase in disconnected garlic root.2000, Lancaster etc. purified a kind of new-type garlic ammonia from onion root
Sour enzyme has obtained the cDNA of coding onion root allinnase albumen, has elaborated its structure and function.
1998, Weik etc. had studied allinnase and recombinantly expresses in Escherichia coli, saccharomyces cerevisiae and Pichia pastoris, obtained
Obtained recombination allinnase.2010, allinnase gene is obtained to celebrate the clones such as diligent, and table has been carried out in Escherichia coli
It reaches, obtains recombinant protein inclusion body, through protein renaturation, recombinate the report that allinnase Rate activity is higher than Weik.But with through planting
The sample that object chemical method is directly extracted from garlic is compared to still undesirable.2010, Wu Xiaoli etc. was logged according to GenBank
Allinnase primers, by RT-PCR technology, clone has logged in China Zhejiang garlic bulb alliin for the first time at home
Enzyme gene.3 bases more than the garlic alliin enzyme sequence that Zhejiang allinnase ratio GenBank is logged in have 9 sites to occur prominent
Become.And target gene is successfully building up on pPICZ α C yeast expression vector.Recombinating allinnase has enzymatic activity, enzyme
Rate activity is (82.09 ± 3.89) U/mg, lower than the natural allinnase of extraction.
Effect of the garlic in terms of medicine and health care has obtained universal approval, thus the effect of allinnase just seems outstanding
It is important.The garlic tablet and capsule produced both at home and abroad at present reaches 30 kinds or more, extracts alliin and garlic respectively from garlic
Propylhomoserin enzyme utilizes alliin and allinnase production complex capsule or injection.Therefore, carry out DNA molecular recombinant technique structure
It builds allinnase expression system production allinnase, and improves enzymatic activity and stability grinding in terms of these using random mutation
Study carefully, just becomes the effective approach for improving garlic similar drug drug effect, and will be for the exploitation and application of garlic similar drug and health care product
Broader space is provided.
Enzyme molecule lactam enzyme by directional anagenesis in vitro belongs to the nonideal explosives of protein, is the new strategy of protein engineering.Using point
Sub- biological means create the diversity of molecule in molecular level, in conjunction with sensitive screening technique, are preferably dashed forward rapidly
Variant.It is not required to understand the factors such as space structure, active site, the catalyst mechanism of protein in advance, but artificially creates special
Different evolution conditions simulate natural evolution mechanism, and enzyme gene is transformed in vitro, obtain the structure enzyme with certain expected features.
Wherein, fallibility PCR refers to while amplifying target genes, does not have 3 ' → 5 ' proofreading functions using Taq enzyme, changes simultaneously anti-
Answer Mn in system2+、Mg2+With the concentration of various dNTP, base mispairing is randomly incorporated into target gene with certain frequency, is caused
Random mutation occurs for target gene.However, the gene through being once mutated generally be difficult to obtain it is satisfied as a result, thus develop again
Continuous error-prone PCR (Sequential Error-prone PCR) strategy.Will the obtained product of a PCR amplification as next
The template of secondary PCR amplification continuously repeatedly carries out fallibility, and the micromutation obtained each time is made constantly to be accumulated and be generated important
Beneficial mutation.Therefore, there is exclusive advantage.
Bacillus subtilis belongs to gram-positive bacteria.Bacillus subtilis expression system has the advantage that 1, can be efficient
Secrete various protein in ground；2, use of many bacillus subtilises in fermentation industry has quite long history, without pathogenic
Property, any endotoxin is not generated；3, the research of bacillus micro-organism genetic background is fully aware of, and has growth fast
Speed, the advantages of to nutrition without particular/special requirement；4 codon-bias are unobvious；5 fermentations are simple, and bacillus subtilis is aerobic bacteria,
Without anaerobic fermentation equipment, to culture medium without particular/special requirement, after fermentation, it is easily separated fermentation liquid and microorganism, i.e.,
The purification and recovery stage of destination protein can be entered；6 have resistance, can produce a variety of heat resistance enzyme preparations.
Pichia pastoris is a kind of unicellular lower eukaryotes, and condition of culture is common, and growth and breeding speed is rapid.Finish red ferment
When female expression system is used for expressing gene engineering product, it can be mass produced, effectively reduce production cost.Pichia pastoris table
There is certain post translational processing ability up to system, the exogenous proteins of harvest have folding processing and glycosyl to a certain extent
Change modification, property is more stable compared with the protein of prokaryotic expression, and there are two types of express shape for pichia yeast expression system tool
Formula, including Pichia pastoris dissociate expression system and Pichia pastoris surface display system.Pichia pastoris dissociates expression system table
The foreign gene reached has certain post translational processing ability, and the exogenous proteins of harvest have folding processing to a certain extent
With it is glycosylation modified, property is more stable compared with the protein of prokaryotic expression, and certain yeast expression systems have outer point
Expressed exogenous proteins can be secreted into extracellularly, be easy to purify by secretion signal sequences, and the exhibition of Pichia pastoris surface
Show system in addition to the folding processing with the post translational processing ability of foreign gene and albumen and appropriateness are glycosylated, through this
The whole-cell catalyst that system obtains can also reuse to reduce production cost.Pichia yeast expression system has become existing
It is the more satisfactory tool of expression alien gene for the most important tool of molecular biology research and model.
Summary of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of novel high vigor allinnase mutation
Body gene, the present invention use fallibility round pcr, carry out random mutation to wild type allinnase, obtain novel high vigor garlic ammonia
Sour enzyme (Glu225Val, Glu267Phe), the specific enzyme activity of the specific enzyme activity of novel high vigor allinnase than wild type allinnase
Improve 50%.
The purpose of the present invention is what is completed using following technical scheme:
A kind of novel high vigor allinnase mutant gene, gene order are SEQ ID NO:5.
A kind of construction method of novel high vigor allinnase mutant gene, by garlic bulb wild type allinnase base
Because carry out random mutation, the 674th bit base A → T, the 799th bit base G → T, the 800th bit base A → T, the 801st bit base A →
C obtains novel high vigor allinnase mutant gene；The garlic bulb wild type allinnase gene order is SEQ ID
NO:3.
A kind of novel high vigor allinnase mutant, passes through nucleotide sequence coded wild type as shown in sequence 3
In allinnase amino acid sequence, the amino acid Glu that the 225th amino acid Glu replaces with Val and the 267th is replaced with
Phe obtains the amino acid sequence as shown in sequence 6.
A kind of preparation method of novel high vigor allinnase mutant, includes the following steps:
(1) by the wild type allinnase gene of fallibility PCR random mutation garlic bulb, the 674th bit base A → T, the
799 bit base G → T, the 800th bit base A → T, the 801st bit base A → C obtain novel high vigor allinnase mutant and compile
Code gene；
By the novel high vigor allinnase mutant code gene digestion, be connected to expression or display carrier,
It obtains carrying high vigor allinnase mutant code gene recombined vector；
(3) recombinant vector is converted to host cell, obtain recombinant bacterial strain；
(4) the recombinant bacterial strain is expressed, and purifying obtains the novel high vigor allinnase as shown in SEQ ID NO:6.
A kind of expression vector and host cell containing said gene.
Moreover, the expression vector is pBSA43, the host cell is bacillus subtilis WB600.
Moreover, the expression vector is pPIC9K, the host cell is Pichia pastoris GS115.
Moreover, the display carrier is pPIC9K-Flo, the host cell is Pichia pastoris GS115.
Unique distinction following points of the invention:
1, the present invention in novel high vigor allinnase gene in bacillus subtilis expression system and Pichia anomala expression
It is expressed in system, respectively obtains the novel high vigor allinnase recombinant bacterial strain of bacillus subtilis and Pichia pastoris is novel
High vigor allinnase recombinant bacterial strain (including the free expression recombinant bacterial strain of the novel high vigor allinnase of Pichia pastoris and finish red ferment
The novel high vigor allinnase recombinant bacterial strain of mother cell surface display).After recombinant bacterial strain fermentation, it can be obtained newly by handling accordingly
The high vigor allinnase catalyst of type.
2, the present invention uses fallibility round pcr, carries out random mutation to wild type allinnase, obtains novel high vigor garlic
Propylhomoserin enzyme (Glu225Val, Glu267Phe), the ratio enzyme of the specific enzyme activity of novel high vigor allinnase than wild type allinnase
Work improves 50%.
3, the specific enzyme activity of allinnase after the novel high vigor allinnase recombinant bacterium of bacillus subtilis ferments in the present invention
It can reach (105.34 ± 1.73) U/mg, the specific enzyme activity of the free novel high vigor allinnase recombinant bacterium of expression of Pichia pastoris is reachable
(173.72 ± 2.89) U/mg, the specific enzyme activity of the novel high vigor allinnase whole-cell catalyst of Pichia pastoris surface display
Up to (196.47 ± 4.93) U/ (g stem cell).
4, the present invention uses yeast surface display system, and allinnase is showed in yeast surface, yeast cells both conducts
The producer of enzyme, and the carrier in immobilised enzymes can be served as, the operation such as purifying and fixation of enzyme is eliminated, production ring is simplified
Section, reduces production cost.
Detailed description of the invention
Fig. 1 is the PCR amplification electropherogram of allinnase maturation peptide gene of the present invention, in which: M is DNA Marker, and 1 is garlic
Propylhomoserin enzyme maturation peptide gene；
Fig. 2 is recombinant plasmid pBSA43-alliinasem digestion verification figure of the present invention, in which: M is DNA Marker, and 1 is
PBSA43-alliinasem is through BamHI and HindIII double digestion；
Fig. 3 is recombinant plasmid pPIC9K-alliinasem digestion verification figure of the present invention, in which: M is DNA Marker, and 1 is
PPIC9K-alliinasem is through EcoRI and NotI double digestion；
Fig. 4 is recombinant plasmid pPIC9K-Flo-alliinasem digestion verification figure of the present invention, in which: M DNA
Marker, 1 is pPIC9K-Flo-alliinasem through SnaBI and EcoRI double digestion；
Fig. 5 is pyruvate standard curve；
Fig. 6 is with FeSO4Standard curve is drawn for standard substance；
Specific embodiment
Technology contents of the invention are described further below with reference to embodiment, but the present invention is not limited solely to these implementations
Example cannot be limited the scope of protection of the present invention with following embodiments.
The purpose technology path that the present invention realizes is as follows
It takes fresh garlic bulb to grind in liquid nitrogen, extraction and the progress of total serum IgE is carried out according to Trizol reagent specification
RT-PCR.Using the first chain of cDNA of synthesis as template, design primer expands wild type allinnase gene order (such as SEQ ID
Shown in NO:3).Random mutation is carried out by fallibility PCR after it is connect with pET28a carrier, obtains novel high vigor alliin
Enzyme gene (as shown in SEQ ID NO:5) constructs recombinant vector and in bacillus subtilis WB600 and Pichia pastoris GS115
Successful expression obtains the recombinant bacterial strain for producing novel high vigor allinnase, further obtains high vigor by fermenting extraction process
Novel allinnase.
It uses and such as gives a definition in the present invention:
1, the nomenclature of amino acid and DNA nucleic acid sequence
Using the generally acknowledged IUPAC nomenclature of amino acid residue, with three-letter codes form.DNA nucleic acid sequence is using generally acknowledged
IUPAC nomenclature.
2, the mark of novel alliin enzyme mutant
The amino being mutated in novel alliin enzyme mutant is indicated using " amino acid of Original amino acid position replacement "
Acid.Such as Glu225Val, indicate that the amino acid of position 225 is substituted for Val by the Glu of parent's allinnase, the number of position is corresponding
The amino acid sequence number of allinnase in SEQ ID NO:4.
In the present invention, alliinase indicates original amino acid (such as SEQ ID NO:4 of wild type allinnase
It is shown), alliinasem indicates the amino acid sequence variants of novel allinnase (as shown in SEQ ID NO:6).
Host cell for expressing the novel alliin enzyme mutant is bacillus subtilis WB600, and expression carries
Body is pBSA43；
Host cell for expressing the novel alliin enzyme mutant is Pichia pastoris GS115, and expression vector is
pPIC9K；
Host cell for expressing the novel alliin enzyme mutant is Pichia pastoris GS115, and display carrier is
pPIC9K-Flo；
Embodiment 1
The acquisition of wild type allinnase maturation peptide gene
1, it takes fresh garlic bulb to grind in liquid nitrogen, goes forward side by side according to the extraction that Trizol reagent specification carries out total serum IgE
Row RT-PCR.
2, using the first chain of the cDNA of synthesis as template, according to the allinnase sequence of Genbank sequence number S73324.1 login
Column design pair of primers in its ORF frame upstream and downstream, introduce restriction enzyme site BamH I, Hind III respectively.The design present invention
Allinnase maturation peptide gene amplimer it is as follows:
Upstream primer P1 (SEQ ID NO.1): 5 '-CGCGGATCCATGATCTGCCTAGTGATTTTGACATG-3’
Downstream primer P2 (SEQ ID NO.2): 5 '-CCCAAGCTTTTAAATGAAAGGACGACGGGAG-3’
Its reaction condition expanded are as follows:
Amplification condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 60 DEG C of annealing 40s, 72 DEG C of extension 1min30s reactions
30 circulations；72 DEG C of extension 10min.Pcr amplification product obtains the band (figure of 1422bp through 0.8% agarose gel electrophoresis
1) PCR product, is recycled with miniprep dna QIAquick Gel Extraction Kit, obtains wild type allinnase maturation peptide gene of the invention
Alliinase is shown in sequence 3 after sequencing.
Embodiment 2
The acquisition of novel high vigor allinnase gene.
1, recombinant plasmid is transferred in bacillus coli DH 5 alpha, is verified through BamH I, Hind III's double digestion, wild type garlic ammonia
Phytase gene is successfully cloned on pET28a carrier.
2, random mutation
(1) random mutation is carried out based on fallibility round pcr, constructs novel high vigor allinnase, design primer is as follows:
Upstream primer P1 (SEQ ID NO.1): 5 '-CGCGGATCCATGATCTGCCTAGTGATTTTGACATG-3’
Downstream primer P2 (SEQ ID NO.2): 5 '-CCCAAGCTTTTAAATGAAAGGACGACGGGAG-3’
In fallibility PCR reaction system, using P1 and P2 as upstream and downstream primer, with wild type allinnase maturation peptide gene
For template, fallibility PCR is carried out.
Its reaction condition expanded are as follows:
Amplification condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 60 DEG C of annealing 40s, 72 DEG C of extension 1min30s reactions
30 circulations；72 DEG C of extension 10min.
(2) novel high vigor allinnase mutant gene (alliinasem) is cloned into expression vector pET28a,
It converts e. coli bl21 (DE3), it is thin to be inoculated in 96 holes of every hole containing 200 μ L LB liquid mediums (Kan containing 30 μ g/mL)
In born of the same parents' culture plate, 200r/min shaking table culture, works as OD under the conditions of 37 DEG C600Reach 0.6, it is (dense eventually that IPTG is added into each hole
Spend 1mmol/L), 16h is induced at 16 DEG C, culture is carried out to following 3 kinds of processing modes: (1) multigelation 3 times (- 80 respectively
DEG C, 15min；37 DEG C, 15min；0 DEG C, 15min)；(2) lysozyme (200 μ g/ml, 30min) is added；(3) (100 DEG C are heat-treated
Boiling water bath, 20min), it is centrifuged 10min at 4000r/min then to remove precipitating, supernatant is transferred in another piece of 96 orifice plates
Detect its enzyme activity.
(3) enzyme activity detects
1. enzyme activity defines
Allinnase 1 molecule alliin of every catalysis reacts and can generate 2 molecule pyruvic acid, therefore can be by measuring acetone
The concentration of acid calculates enzyme activity.Therefore enzyme activity of the present invention is defined as: under the conditions of 35 DEG C, allinnase is catalyzed the anti-of alliin
It answers, generates l μ g pyruvic acid per minute and be defined as 1 unit of activity (U).
2. the drafting of concentrations of pyruvate and absorbance value standard curve
Pyruvic acid can be acted on 2,4-dinitrophenylhydrazine, generate pyruvic acid -2,4- dinitrophenylhydrazone, and the latter is molten in alkalinity
It is in cherry red in liquid, light absorption value is measured at wavelength 520nm.The acetone acid solution for configuring various concentration respectively takes 1mL, and 1mL is added
0.1%2,4- dinitrophenylhydrazine sufficiently shakes up, and adds 2mL1.5mol/LNaOH and shakes up, and stands 10min colour developing, ultraviolet with 752
Spectrophotometer measures light absorption value at 520nm.Draw the standard curve of concentrations of pyruvate and absorbance value.
Pyruvate standard curve is as shown in figure 5, acquiring regression equation is Y=0.0244X+0.0167, related coefficient R2
=0.9990.
3. the measuring method and step of novel allinnase enzyme activity
Enzymatic activity is measured with acetone acid system.Prepare the alliin substrate of 3mmol/L.Tunning is collected, the bottom 1.0mL is added
Object reacts 5min at 30 DEG C, and 1.5mL10% trichloroacetic acid is added and terminates reaction.Add 0.5mL0.1%2,4- dinitrobenzene
Callosity mixes well, and 7mL0.5mol/LNaOH solution is added and shakes up colour developing, 520nm wavelength measures light absorption value.It is calculated according to absorption value
Pyruvic acid total concentration and enzyme activity out.With allinnase albumen quality in Folin- phenol method measurement solution.It finds out in institute's sample
The Rate activity of allinnase.
4. the measurement of novel allinnase enzyme activity
The specific enzyme activity of novel high vigor allinnase, the ratio of allinnase after mutation after measuring original allinnase and being mutated
Enzyme activity is than improving 50% before mutation.
Specific enzyme activity definition: the unit of activity number of had enzyme in the protein of Unit Weight, generally with U/mg protein come
It indicates.
(4) sequencing
Be sequenced (Beijing Hua Da bio-engineering corporation) the result shows that, at this time amplification obtain Glu225Val, Glu267Phe
Novel high vigor allinnase gene alliinasem, is shown in sequence 5.
Embodiment 3
The building of the novel high vigor allinnase recombinant bacterium of bacillus subtilis
1, the building of expression vector pBSA43
PBSA43 is cloned into one strong using bacillus coli-bacillus subtilis shuttle cloning vector pBE2 as skeleton
Bacillus constitutive promoter P43, and recombinant protein can be made directly to be secreted into levansucrase signal in culture medium
Sequence sacB and obtain.It has AmprGene, can be in Escherichia coli using amicillin resistance as selection markers；
Also there is Km simultaneouslyrGene can be marked in bacillus subtilis, bacillus licheniformis using kalamycin resistance as screening
Note.
2, the building of novel high vigor allinnase expression vector pBSA43-alliinasem
Will through fallibility PCR construct obtain novel high vigor allinnase gene after BamHI and HindIII double digestion with
The bacillus subtilis expression vector pBSA43 connection of same double digestion, construction recombination plasmid pBSA43-alliinasem.It will
PBSA43-alliinasem converts bacillus coli DH 5 alpha competent cell, selects positive transformant, extracts plasmid progress digestion and tests
It demonstrate,proves and is sequenced, determine that building obtains recombinant bacterial strain JM109/pBSA43-alliinasem.
3, expression vector pBSA43-alliinasem converts bacillus subtilis WB600
1 μ L (50ng/ μ L) pBSA43-alliinasem is added in 60 μ L competent cells to mix and be transferred to ice-cold
In electrotransformation cup (1mm), after ice bath 1-1.5min, shock by electricity primary (25 μ F, 200 Ω, 4.5-5.0ms).After electric shock finishes, stand
1mL recovery medium (LB+0.5mol/L sorbierite+0.38mol/L mannitol) is added.37 DEG C of shaking table shake culture 3h it
Afterwards, on LB plate by the coating of recovery object, 37 DEG C of culture 24-36h, picking positive transformant obtain bacillus subtilis recombination
Bacterial strain WB600/pBSA43-alliinasem.
Embodiment 4
The building of the free expression recombinant bacterium of the novel high vigor allinnase of Pichia pastoris
1, the building of novel high vigor allinnase Expression vector pPIC9K-alliinasem
By the novel high vigor allinnase gene obtained through fallibility PCR building after EcoRI and NotI double digestion and together
The yeast expression vector pPIC9K of sample double digestion is attached with ligase, and connection product is converted e. coli jm109
In (DH5 α) competent cell, through Amp resistance screening, positive transformant is selected；Positive transformant plasmid is extracted, shakes pipe through 37 DEG C
Plasmid is extracted after culture, and carries out single, double digestion preliminary identification, and the correct recombinant plasmid of digestion verification is named as
pPIC9K-alliinasem；The correct positive colony of digestion verification is sent to Beijing Hua Da Gene science limited liability company and is surveyed
Sequence, it is final to determine that building obtains correct recombinant bacterial strain JM109/pPIC9K- to further ensure that the correctness of target gene
alliinasem。
2, the sieve of the building of novel high vigor allinnase recombinant bacterial strain and novel high vigor allinnase height expression bacterial strain
Choosing
(1) preparation of recombinant expression plasmid pPIC9K-alliinasem is linearized
Recombinant plasmid is before electrotransformation Pichia pastoris GS115, the recombinant expression plasmid pPIC9K- will first build
Alliinasem is linearized to improve integration efficiency of the recombinant plasmid on Pichia chromosome.Conversion needs line every time
Property Plasmid DNA 5-20 μ g, and plasmid is purer, transformation efficiency is higher.It can be restricted interior with this 2 kinds of Sac I and Sal I respectively
Enzyme cutting carries out linearized enzyme digestion.After digestion is complete, linearisation digestion products are recycled with miniprep dna QIAquick Gel Extraction Kit.
(2) linearization plasmid pPIC9K-alliinasem electrotransformation Pichia pastoris GS115, positive transformant identification and
The screening of novel high vigor allinnase superior strain
1. the linearized DNA of 80 μ L competent cells and 5-20 μ g is added in the centrifuge tube of 1.5mL pre-cooling, mix
It is even, reaction solution is transferred in the conversion cup of preparatory ice bath；
2. ice bath is equipped with the conversion cup 5min of conversion fluid, according to the parameter that electric rotary device is recommended, carries out Pichia pastoris electricity and turn
Change:
3. after pulse, the sorbitol solution for the 1mol/L that 1mL is pre-chilled being added into conversion cup immediately, conversion fluid is transferred to
In one new 1.5mL centrifuge tube；
4. 30 DEG C of stationary culture l-2h, absorption Pichia pastoris GS115 electricity turns 200 μ L of liquid and is coated on MD culture medium；
5. 30 DEG C of cultures are until transformant occurs；
6. picking transformant single colonie is dissolved in 10 μ L deionized waters, 2 μ L bacterium solutions are taken, Lyticase wall breaking enzyme is added,
30 DEG C of reaction l0min, reaction solution are immediately placed in -80 DEG C of refrigerators and freeze l0min, crack yeast cell wall, the gene of release
Group carries out PCR as template.Pichia pastoris GS115/pPIC9K to be transferred to empty plasmid pPIC9K determines positive turn as control
Beggar.
7. on the basis of determining positive transformant, first with the high heredity of resistant panel screening of the Geneticin containing various concentration
Then the transformant of chloramphenicol resistance measures the enzyme activity of the novel allinnase of the transformant of these high geneticin resistants respectively,
To obtain the superior strain GS115/pPIC9K-alliinasem of novel high vigor allinnase.
Embodiment 5
The building of the novel high vigor allinnase recombinant bacterium of Pichia pastoris surface display
1, the building of recombinant plasmid pPIC9K-Flo-alliinasem
By fallibility PCR purified product and carrier pPIC9K-Flo after SnaBI and EcoRI double digestion, by novel high vigor
Allinnase gene is connected in carrier pPIC9K-Flo, and connection product is transformed into bacillus coli DH 5 alpha competence, is being contained
There is screening and culturing in the LB solid medium of Amp, picking positive transformant, upgrading grain after culture, double digestion is identified and is sequenced, and is ordered
Entitled pPIC9K-Flo-alliinasem.
2, the building of Pichia pastoris recombinant bacterium
Correct recombinant plasmid will be sequenced after SalI is linearized, convert Pichia pastoris GS115, MD plate with electrotransformation
Recon is screened, the novel high vigor allinnase recombinant bacterium GS115/pPIC9K-Flo- of Pichia pastoris surface display is obtained
alliinasem。
Embodiment 6
Expression and preparation of the novel high vigor allinnase in bacillus subtilis recombinant bacterium
Bacillus subtilis recombinant bacterial strain WB600/pBSA43-alliinasem is inoculated in LB liquid medium (containing card
Receive mycin, 30 μ g/mL) in, 37 DEG C, 200r/min overnight incubation, by the switching of 1% inoculum concentration in 50mL fresh culture,
200r/min cultivates 48h, can prepare novel high vigor allinnase crude enzyme liquid, is then precipitated using salt fractionation method novel
High vigor allinnase collects protein precipitation, after dissolution, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, it is cold
It is lyophilized and dry the novel pure enzyme enzyme powder of high vigor allinnase is made.
Embodiment 7
Expression and preparation of the novel high vigor allinnase in the free expression recombinant bacterium of Pichia pastoris
The free novel high vigor allinnase recombinant bacterium of expression of the Pichia pastoris being incubated on YPD solid plate is seeded to
In YPD fluid nutrient medium, 30 DEG C, 250r/min culture for 24 hours.It is transferred in fresh BMGY culture medium with 1% inoculum concentration, 30 DEG C
For 24 hours, then 6000r/min is centrifuged 5min and obtains thallus for culture, is transferred in BMMY culture medium.30 DEG C, 250r/min, every for 24 hours
Methanol is mended, so that its final concentration is maintained at 0.5%V/V, the crude enzyme liquid of novel alliin enzyme mutant can be obtained after culture 120h, so
Novel high vigor allinnase is precipitated using salt fractionation method afterwards, collects protein precipitation, after dissolution, desalination of dialysing, then through from
After sub- displacement chromatography, gel chromatography, the novel pure enzyme enzyme powder of high vigor allinnase is made in freeze-drying.
Embodiment 8
The preparation of the novel high vigor allinnase whole-cell catalyst of Pichia pastoris surface display
The novel high vigor allinnase recombinant bacterium of the Pichia pastoris surface display being incubated on YPD solid plate is connect
Kind into YPD fluid nutrient medium, 30 DEG C, 250r/min culture for 24 hours, be transferred in fresh BMGY culture medium with 1% inoculum concentration,
For 24 hours, then 6000r/min is centrifuged 5min and obtains thallus for 30 DEG C of cultures, is transferred in BMMY culture medium, 30 DEG C, 250r/min culture
120h makes its final concentration be maintained at 0.5%V/V every mending methanol for 24 hours, is then centrifuged for collection and takes thallus, washes 1-2 with distilled water
It is secondary, protective agent is added, it is entirely thin that the novel high vigor allinnase of Pichia pastoris surface display is made by vacuum freeze drying
Born of the same parents' catalyst.
Embodiment 9
Recombinant bacterium enzyme activity determination
Enzyme activity is measured after obtain three plants of recombinant bacteriums are fermented, wherein the novel high vigor allinnase weight of bacillus subtilis
The specific enzyme activity of novel allinnase can reach (105.34 ± 1.73) U/mg in group bacterium fermentation post-fermentation liquid, and Pichia pastoris dissociates table
Up to the specific enzyme activity in novel high vigor allinnase recombinant bacterium fermentation post-fermentation liquid up to (173.72 ± 2.89) U/mg, finish red ferment
The specific enzyme activity of the novel high vigor allinnase whole-cell catalyst of mother cell surface display is up to (196.47 ± 4.93) U/ (g
Stem cell).And the specific enzyme activity of allinnase is reachable in bacillus subtilis wild type allinnase recombinant bacterium fermentation post-fermentation liquid
To (70.23 ± 2.13) U/mg, the free ratio enzyme expressed in wild type allinnase recombinant bacterium fermentation post-fermentation liquid of Pichia pastoris
Reachable (115.82 ± 2.46) U/mg living, the ratio enzyme of Pichia pastoris surface display wild type allinnase whole-cell catalyst
Reachable (130.98 ± 3.93) U/ (g stem cell) living.
Embodiment 10
Novel allinnase Antioxidative Activity Determination
1, the measurement of DPPH free radical scavenging ability
Referring to the method for Brand-WilliamsW..It operates and is carried out under oxygen free condition and aerobic conditions respectively below: being used
Ultrapure water mixing alliin and recombination allinnase crude enzyme liquid, it is (final concentration of to be made alliin/allinnase mixture solution
10mmol/Lalliin, allinnase crude enzyme liquid total protein content are 0.275mg/mL), enzymic catalytic reaction is not in 37 DEG C of water-baths
The same time takes l mL sample and 4mL concentration for the DPPH ethanol solution (volume fraction 95%) of 0.12mmol/L, mixes, room
It is protected from light 30min under temperature, is centrifuged 5min if any being deposited under the conditions of 5500r/min.It is 95% ethanol solution with volume fraction
Make reference, measures light absorption value in 517nm.Each sample liquid is calculated according to the following formula to the clearance rate of DPPH free radical:
Clearance rate/%=[1- (Ai-Aj)/Ac] × 100%
In formula: Ai: for the light absorption value for adding DPPH solution after sample liquid；
Aj: for the light absorption value of sample liquid；
Ac: the light absorption value of DPPH solution when not add sample liquid.
2, the measurement (measurement of FRAP value) of total antioxidant capacity
Each test solution is taken, pipettor takes 100 μ L after appropriate dilution, and FRAP working solution 3mL is added, and mixes reaction
20min reads absorbance at 593nm.With FeSO4Standard curve (as shown in Figure 6) is drawn for standard substance, acquires recurrence side
Journey are as follows:
Y=0.3584x-0.0043, coefficient R2=0.9986
The total antioxidant capacity of sample is indicated with FRAP value: 1FRAP unit is equivalent to 1mmol/L FeSO4, i.e. sample
Total antioxidant capacity is equivalent to FeSO4Mmol/L number.
Claims (2)
1. a kind of preparation method of novel high vigor allinnase mutant, characterized by the following steps:
(1) by the wild type allinnase gene of fallibility PCR random mutation garlic bulb, the 674th bit base A → T, the 799th
Bases G → T, the 800th bit base A → T, the 801st bit base A → C obtain novel high vigor allinnase mutant code base
Cause；
By the novel high vigor allinnase mutant code gene digestion, be connected to expression or display carrier, obtain
Carry high vigor allinnase mutant code gene recombined vector；
(3) recombinant vector is converted to host cell, obtain recombinant bacterial strain；
(4) the recombinant bacterial strain is expressed, and purifying obtains the novel high vigor allinnase as shown in SEQ ID NO:6.
2. a kind of construction method of novel high vigor allinnase mutant gene, it is characterised in that: by garlic bulb wild type
Allinnase gene progress random mutation, the 674th bit base A → T, the 799th bit base G → T, the 800th bit base A → T, the
801 bit base A → C obtain novel high vigor allinnase mutant gene；The garlic bulb wild type allinnase gene
Sequence is SEQ ID NO:3.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255200A (en) * | 2008-04-09 | 2008-09-03 | 暨南大学 | Technique for producing toasted garlic polysaccharides and allinase by using toasted garlic as raw material |
| CN102372779A (en) * | 2011-10-25 | 2012-03-14 | 李江伟 | Camel single domain antibody with allinase activity as well as preparation method and application thereof |
| CN104073479A (en) * | 2014-06-27 | 2014-10-01 | 齐鲁工业大学 | Method for purifying alliinase by double-water-phase separation |
| CN104928273A (en) * | 2015-06-24 | 2015-09-23 | 中国农业科学院生物技术研究所 | Garlic allinase, encoding gene thereof and application of garlic allinase |
-
2016
- 2016-05-18 CN CN201610330256.8A patent/CN105838729B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255200A (en) * | 2008-04-09 | 2008-09-03 | 暨南大学 | Technique for producing toasted garlic polysaccharides and allinase by using toasted garlic as raw material |
| CN102372779A (en) * | 2011-10-25 | 2012-03-14 | 李江伟 | Camel single domain antibody with allinase activity as well as preparation method and application thereof |
| CN104073479A (en) * | 2014-06-27 | 2014-10-01 | 齐鲁工业大学 | Method for purifying alliinase by double-water-phase separation |
| CN104928273A (en) * | 2015-06-24 | 2015-09-23 | 中国农业科学院生物技术研究所 | Garlic allinase, encoding gene thereof and application of garlic allinase |
Non-Patent Citations (3)
| Title |
|---|
| alliinase[Allium sativum];Genbank:ACN78838.1;《NCBI》;20090322;全文 |
| alliinase[Allium sativum=garlics,bulbs,mrna,1705nt];Genbank:S73324.1;《NCBI》;19950228;全文 |
| Generating Mutant Libraries Using Error-Prone PCR. In: Arnold F.H., Georgiou G. (eds) Directed Evolution Library Creation.;Cirino P.C., Mayer K.M., Umeno D.;《Arnold F.H., Georgiou G. (eds) Directed Evolution Library Creation. Methods in Molecular Biology™》;20031231;摘要，第3-9页 |
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